The effect of ethanol and nicotine on ER stress in human placental villous explants.

Pregnant mothers continue smoking and drinking during pregnancy. To clarify the mechanisms of nicotine and ethanol toxicity during development, we have examined their effects on endoplasmic reticulum (ER) stress in human first trimester and term placental explants. First trimester and term human placental explants were treated with ethanol (2 ‰) or nicotine (15 µM), or their combination. The ER stress markers glucose regulated protein 78 (GRP78/BiP) and inositol requiring enzyme 1 α (IRE1α) were analyzed by immunoblotting. A statistically significant increase (p < 0.05) of GRP78/BiP by nicotine was noted in first trimester placental explants at 48 h, and in term placental explants at 24 h. Ethanol did not change protein expression of GRP78/BiP in either first trimester or term placental explants. IRE1α increased, although not statistically significantly, by all treatments in both first trimester and term placental explants. Thus, regardless of the known structural and functional differences in early and late placenta, both responded very similarly to the toxic compounds studied. These data support our earlier results in BeWo cells (Repo et al., 2014) implicating that nicotine induces ER stress in human placenta and may interfere with placental functions potentially disrupting fetal growth and development.

Impact of bisphenol A (BPA) on cells and tissues at the human materno-fetal interface.

Bisphenol A (BPA) is an endocrine disruptor extensively used in the production of polycarbonate plastics and epoxy resins and a component of liquid and food containers. It is a hazard in the prenatal period because of its presence in the placenta, fetal membranes, amniotic fluid, maternal and fetal blood and its ability to cross the placenta and reach the fetus. Estimation of the risk of BPA exposure during in utero life is extremely important in order to prevent complications of pregnancy and fetal growth. This review describes in vitro models of the human materno-fetal interface. It also outlines the effects of BPA at doses indicated as "physiological", namely at the concentrations found in the general population, and at "supraphysiological" and "subphysiological" doses, i.e. above and below the physiological range. This work will help clarify the discrepancies observed in studies on the effects of BPA on human reproduction and pregnancy, and it will be useful for the choice of appropriate in vitro models for future studies aimed at identifying the potential impact of BPA on specific functional processes.

Evidence for a role of TGF-beta1 in the expression and regulation of alpha-SMA in fetal growth restricted placentae.

There is evidence that alpha-smooth muscle actin (alpha-SMA) is a protein that plays a pivotal role in the production of contractile forces and it is induced by transforming growth factor-beta1 (TGF-beta1). We have analysed the expression of alpha-SMA, TGF-beta1, its receptor RI and the activator phospho-Smad2 in (a) fetal growth restriction pre-eclamptic placentae characterised by early onset and absence of end diastolic velocities in the umbilical arteries (FGR-AED) and (b) control placentae accurately matched for gestational age. The study was performed by immunohistochemical, quantitative Western blotting, ELISA, RT-PCR and in vitro analyses. We found that TGF-beta1 stimulates alpha-SMA production in chorionic villi cultured in vitro. In addition, we observed that in vivo TGF-beta1 concentration is significantly higher in FGR-AED placental samples than in control placentae and that this growth factor could have a paracrine action on villous stroma myofibroblasts expressing TGF-beta1 receptors and phospho-Smad2. Indeed, we report that alpha-SMA undergoes a redistribution in FGR-AED placental villous tree, i.e. we show that alpha-SMA is enhanced in medium and small stem villi and significantly decreased in the peripheral villi. Our data allow us to consider TGF-beta1 and alpha-SMA as key molecules related to FGR-AED placental villous tree phenotypic changes responsible for increased impedance to blood flow observable in this pathology.

History of reptile placentology, part III: Giacomini's 1891 histological monograph on lizard placentation.

By the 1890s, placental arrangements had been documented macroscopically in lizards and fishes, but placental studies on such species lagged far behind research on mammals. In 1891, the biologist Ercole Giacomini (at the University of Siena, Italy) published the first histological analysis of a reptile placenta. Focusing on a placentotrophic lizard (Chalcides chalcides) with a morphologically complex placenta, Giacomini documented the histological and cellular bases for placental nutrient transfer and gas exchange. In conjunction with a follow-up study in 1906, he demonstrated that placental structure is correlated with function and can vary dramatically between related species. Giacomini's work was highly influential in showing that placentation in lizards had converged evolutionarily on that of mammals, while establishing reptile placentology as a highly promising area for future research.

The xenoestrogens, bisphenol A and para-nonylphenol, decrease the expression of the ABCG2 transporter protein in human term placental explant cultures.

Many endogenous and xenobiotic compounds are substrates and regulators of human placental ABC transporters. ABCG2 is protecting fetus against foreign chemicals. Environmental xenoestrogens, like bisphenol A (BPA) and p-nonylphenol (p-NP), mimic natural estrogens and can affect hormonal systems. Effects of BPA, p-NP, DES (diethylstilbestrol) and estradiol (E2), on ABCG2 expression were studied using human first trimester and term placental explants. Role of estrogen receptors (ER) in the effects of chemicals was studied by ER antagonist. Term placenta expressed less ABCG2 protein. In term placentas BPA (p < 0.05), p-NP (p < 0.01) and E2 (p < 0.05) decreased the ABCG2 protein expression after 48 h exposure while after 24 h exposure, only E2 decreased the expression (p < 0.05). The chemicals did not affect ABCG2 in first trimester placentas. The ER antagonist affected differently the responses of chemicals. In conclusion, environmental xenoestrogens downregulate placental ABCG2 protein expression depending on gestational age.

First Evidence of Cardiac Stem Cells From the Left Ventricular Apical Tip in Patients With Left Ventricular Assist Device Implantation.

BACKGROUND: Recent studies have challenged the dogma that the adult heart is a postmitotic organ and raise the possibility of the existence of resident cardiac stem cells (CSCs). Our study aimed to explore if these CSCs are present in the "ventricular tip" obtained during left ventricular assist device (LVAD) implantation from patients with end-stage heart failure (HF) and the relationship with LV dysfunctional area extent. METHODS: Four consecutive patients with ischemic cardiomyopathy and end-stage HF submitted to LVAD implantation were studied. The explanted "ventricular tip" was used as a sample of apical myocardial tissue for the pathological examination. Patients underwent clinical and echocardiographic examination, both standard transthoracic echocardiography (TTE) and speckle tracking echocardiography (STE), before LVAD implantation. RESULTS: All patients presented severe apical dysfunction, with apical akinesis/diskinesis and very low levels of apical longitudinal strain (-3.5 ± 2.9%). Despite this, the presence of CSCs was demonstrated in pathological myocardial samples of "ventricular tip" in all 4 of the patients. It was found to be a mean of 6 c-kit cells in 10 fields magnification 40×. CONCLUSIONS: Cardiac stem cells can be identified in the LV apical segment of patients who have undergone LVAD implantation despite LV apical fibrosis.

Pulmonary catabolism of interferon-gamma evaluated by lung perfusion of both normal and smoke-exposed rats.

The role of the lungs in the catabolism of rat recombinant interferon-gamma, either in normal rats or in rats subjected to an acute cigarette smoking episode, was evaluated using an isolated and perfused lung preparation. After administration of interferon-gamma into the lung perfusion medium, there was no clearance of the cytokine in either control or smoke-exposed rat lungs, and only 0.1 +/- 0.2% of the total dose was recovered in the bronchoalveolar lavage fluid. When the same amount of interferon-gamma was instilled into the bronchial alveolar tree, concentrations of the cytokine in the perfusate increased progressively so that after 3 h up to 71.2 +/- 4.3 and 62 +/- 5.7% of the administered dose, as measured by ELISA test, had been transferred from the bronchial lumen to the perfusion medium of either control or smoke-exposed rat lungs, respectively, the latter values being significantly lower (p < or = 0.05) than those obtained in control lungs. Moreover, total recoveries of interferon-gamma evaluated in smoke-exposed rat lungs (78.4 +/- 8.6%) were also significantly lower than those observed in control rat lungs (91.4 +/- 11.8%). Biologic activity evaluations on the same samples gave values significantly lower than those obtained using ELISA, indicating a partial loss of biologic activity during transalveolar transit. In conclusion, it appears that the transfer of interferon-gamma is almost exclusively unidirectional from the alveolar space to the plasmatic pool, with partial degradation during transalveolar passage.

Presence of interferon in venous blood draining from gut-associated lymphoid tissue.

The question has been examined of whether interferon (IFN), produced in the microenvironment of the gut-associated lymphoid tissue (GALT) during the physiological response, is consumed locally, or whether some spills over and is drained into the general circulation. Plasma IFN levels were measured in venous blood draining from abdominal organs that are normally in contact with exogenous and endogenous interferon inducers. The results obtained from three rodent species indicate the presence of a venous-arterial and GALT venous-peripheral venous gradient, suggesting that at least some of the IFN produced in the GALT and spleen is absorbed via blood capillaries and detectable in the regional vessels. Owing to the rapid turnover of IFN, arterial or peripheral venous blood showed a basal level. The antiviral activity (AA) in rat and mouse blood appears to be attributable to IFN-gamma.

Interferon production in BCG-sensitized rabbits challenged with PPD.

Intravenous administration of PPD into BCG-primed rabbits results in the release of IFN into the circulation. Peak interferon titres occur in plasma 1 h after PPD injection, rapidly decline during the following 3 h and depend on the dose of tuberculin administered. Bilateral nephrectomy of BCG-primed rabbits challenged with PPD affects remarkably the disappearance of IFN from the circulation. Characterization of the BCG-PPD-induced IFN shows that it is heat-labile, is almost completely inactivated after dialysis at pH 2 and displays a remarkable cross-species activity, particularly on human cell lines.

Interferon induction in rabbits after intraduodenal administration of a phosphorylated glucomannan-protein fraction of the cell wall of Candida albicans.

The aim of this work was to demonstrate whether a glucomannan protein fraction (GMP) of Candida albicans cell wall could induce interferon after intraduodenal administration in normal rabbits and rabbits immunized against C. albicans. For this purpose we collected simultaneously plasma and abdominal lymph for 10 h after the administration of the inducer. We observed a peak of antiviral activity in the lymph 4 h after intraduodenal administration of 20 mg GMP dissolved in saline to 6 normal rabbits. Immunized rabbits (anti-GMP titres greater than 1024) responded earlier (peak after 2 h) and more intensely; analysis of the values of the areas under the curve indicated that the IFN response in the lymph of immunized rabbits was significantly higher (P less than 0.0025) than in normal rabbits. Antiviral activity was absent in plasma in all cases. Preliminary characterization of the IFN activity has shown it to be trypsin-sensitive, acid and heat stable, and species-specific.

Effect of tuftsin and its retro-inverso analogue on the release of interferon (IFN-gamma) and tumor necrosis factor (TNF-alpha) by human leucocytes.

The aim of this work was to demonstrate whether natural tuftsin or a retro-inverso (r.i.) analogue may induce interferon (IFN) and tumor necrosis factor (TNF) in peripheral-blood-mononuclear-cells (PBMC). For this purpose tuftsin or its analogue were added at different molar concentrations to PBMC and the supernatants were tested for IFN and TNF activity. Both cytokines were released after 12 hours incubation with r.i. tuftsin at an optimum concentration of 10(-10) M. Under the same conditions no activity was observed in the presence of natural tuftsin. In comparison to natural tuftsin the stimulatory activity of this tuftsin analogue is likely to be due to its high stability.

Glycosylation of the materno-foetal interface in the pregnant viviparous placentotrophic lizard Chalcides chalcides: a lectin histochemical study.

Glycosylation of the foeto-maternal interface of the skink Chalcides chalcides has been examined at various stages of gestation using lectin histochemistry. Specimens of incubatory chamber or placenta from early, mid-, late- and near-term pregnancy were fixed and embedded in epoxy resin. Areas of foeto-maternal apposition were probed with a panel of biotinylated lectins followed by an avidin-peroxidase revealing system to identify various classes of glycan at the interface. Both the external epithelium of unspecialized bilaminar omphalopleure, which forms by early pregnancy, and chorioallantoic membrane which develops by mid-pregnancy, were composed of two phenotypes, one of which secreted a wide range of glycans including high mannose and complex N-glycan, N-acetyl glucosamine, lactosamine and galactosamine, which became less prominent from mid-pregnancy onwards. The uterine epithelium also contained a well-developed secretory apparatus producing a similar range of glycans and there were indications that glycosylated secretions were taken up by the overlying chorioallantois. Foetal vasculature was well developed while maternal vessels appeared more contracted, and both were richly sialylated like their therian equivalents. Our findings indicate that this reptile has evolved a true epitheliochorial placenta with many aspects in common with its therian counterparts but also with unique features of its own.

Cytokines in the viviparous reproduction of squamate reptiles: interleukin-1 alpha (IL-1 alpha) and IL-1 beta in placental structures of a skink.

Placental viviparity is known in many species of squamate reptiles. Among these, some scincids have developed an epithelio-chorial chorio-allantoic placenta which in the structure of its central ridged zone is similar to those of certain therian mammalian species. A broad range of immunoregulatory peptides, cytokines, has been identified at the maternofetal interface of several species of mammals, either with invasive or non-invasive types of placenta. Thus we began to study whether interleukin-1, which is considered to play a crucial role in mammalian pregnancy, might also be involved in the viviparity of reptilian species. Placentae of Chalcides chalcides L. were processed by immunohistochemistry and incubated in a culture medium for different times. A very strong immunoreactivity for interleukin-1 alpha (IL-1 alpha) and for interleukin-1 beta (IL-1 beta) was present in the chorial epiblast and in uterine epithelial cells, with varying degree and localization in different periods of pregnancy. IL-1 beta was also released into the medium at different amounts during incubation. In light of the mammalian data, our results suggest that the role of cytokines in pregnancy may represent a significant event in the evolution of placental viviparity.

Evidence of H beta 58, a gene involved in mammalian placental development, in the three-toed skink, Chalcides chalcides (Squamata: Scincidae), a viviparous placentotrophic reptile.

The H beta 58 gene, whose disruption in mice causes reabsorption of the embryo at 9.5 days post-conception, is believed to be essential for development of the placenta. Although the H beta 58 gene is well conserved in some Amniota, nothing is known about its presence in reptiles, some species of which have developed a chorioallantoic placenta. In this work, we investigated the expression of H beta 58 mRNA and protein in the three-toed skink, Chalcides chalcides. H beta 58 protein expression was found in the uterine epithelium beginning from the peri-ovulatory stage. However, it increased strongly at the moment of placental formation, when a high level of expression of mRNA and protein was also observed in the extra-embryonic membranes. The expression of H beta 58 mRNA and protein was maintained, although to a lesser degree, in the placenta during late pregnancy. It was also present in the early embryo. Finally, cloning and sequencing of a gene fragment revealed strong homology of the reptile gene with that of mammals. The high degree of conservation of the gene in amniote vertebrates and its presence in a viviparous squamate reptile (as in mammals) indicates an important role of this gene in the chorioallantoic placenta formation and development.

11Beta-hydroxysteroid dehydrogenase expression in first trimester human trophoblasts.

This study evaluated the levels and the enzymatic characteristics of 11beta-hydroxysteroid dehydrogenase activity (11beta-HSD) of chorionic villi isolated from first trimester human placenta. The results demonstrated a predominant expression of the NAD-dependent dehydrogenase isoform (11beta-HSD2) over the NADP-dependent oxoreductase (11beta-HSD1). Thus, in tissue homogenates exogenous NAD increased the conversion of corticosterone to 11-dehydrocorticosterone of about 14-fold while NADP was ineffective. There was no conversion of 11-dehydrocorticosterone to corticosterone either with NADH or NADPH demonstrating the lack of reductase activity. In keeping with these results, RT-PCR analysis indicated a mRNA for 11beta-HSD2 in villous tissue while 11beta-HSD1 mRNA levels were undetectable. In addition, immunohistochemical staining localized the 11beta-HSD2 protein to syncytiotrophoblasts and cell columns of the chorionic villi. These results suggest roles for the trophoblast-associated 11beta-HSD2 oxidative activity in modulating the exposure of the embryo to active glucocorticoids in the early gestation and in regulating trophoblasts invasion of the uterine wall.

Pulmonary catabolism of interferons: alveolar absorption of 125I-labeled human interferon alpha is accompanied by partial loss of biological activity.

The catabolism of interferon was examined in isolated rabbit lungs which were ventilated and perfused with homologous blood. Natural human interferon-alpha (HuIFN-alpha) from lymphoblastoid Namalwa cells or recombinant DNA-derived HuIFN-alpha 2 were labeled with 125I, mixed with an excess of the respective cold interferons and added to the perfusion blood. Protein-bound and acid-soluble radioactivity, as well as antiviral activity, were measured at regular time intervals. During the first 3 h of perfusion, only very small fractions of the interferons disappeared from the perfusate, irrespective of whether lungs were inserted in the perfusion system. This indicated that catabolism of interferons in the pulmonary circulation was negligible. On the other hand, when the interferons were instilled into the bronchial-alveolar tree, absorption of antiviral activity differed from that of acid-precipitable protein-associated radioactivity. While most of the radioactivity was transferred into the perfusate, only 2% of antiviral activity of natural HuIFN-alpha and 30% of that of HuIFN-alpha 2 were recovered in the perfusate. In both cases acid-soluble radioactivity in the system reached about 10%. Since radioiodide, instilled in the bronchial-alveolar tree, was transported rapidly into the perfusate, this type of analysis did not help in locating the site(s) of degradation. Alveolar macrophages did not catabolize or inactivate interferons in vitro.

First trimester human trophoblast expresses both interferon-gamma and interferon-gamma-receptor.

Interferon-gamma (IFN-gamma) is a lymphokine, produced by activated T lymphocytes, which plays a key regulatory role in the host immunological responses. In addition, IFN-gamma is expressed by human and porcine trophoblast. As IFN-gamma exerts its biological functions through specific cell surface receptors and a great number of IFN-gamma receptors (IFN-gamma R) have been purified from human placenta, we have examined the relative distribution of IFN-gamma and IFN-gamma R in human placental tissues at different stages of pregnancy. By using immunohistochemical analysis and monoclonal antibodies, it was found that IFN-gamma expression is intense in the first trimester but almost imperceptible at term, whereas the expression of IFN-gamma R is present at both stages of pregnancy. For both lymphokine and receptor, the most intense expression was observed in villous syncytiotrophoblast and in extravillous interstitial trophoblast. From these results it appears that the expression of IFN-gamma R in trophoblast is related to the presence of the lymphokine in the early phase of gestation but not later. On this basis, it can be argued that IFN-gamma exerts its functional role via an autocrine and/or a paracrine loop mainly during the first trimester.

Progestin regulation of 11beta-hydroxysteroid dehydrogenase expression in T-47D human breast cancer cells.

This study examined the enzymatic characteristics and steroid regulation of the glucocorticoid-metabolizing enzyme 11beta-hydroxysteroid dehydrogenase (11beta-HSD) in the human breast cancer cell line T-47D. In cell homogenates, exogenous NAD significantly increased the conversion of corticosterone to 11-dehydrocorticosterone, while NADP was ineffective. There was no conversion of 11-dehydrocorticosterone to corticosterone either with NADH or NADPH demonstrating the lack of reductase activity. In keeping with these results, RT-PCR analysis indicated a mRNA for 11beta-HSD2 in T-47D cells, while 11beta-HSD1 mRNA levels were undetectable. In T-47D cells treated for 24 h with medroxyprogesterone acetate (MPA), 11beta-HSD catalytic activity was elevated 11-fold, while estrone (E(1)), estradiol (E(2)) and the synthetic glucocorticoid dexamethasone (DEX) were ineffective. The antiprogestin mifepristone (RU486) acted as a pure antagonist of the progestin-enhanced 11beta-HSD activity, but did not exert any agonistic effects of its own. In addition, RT-PCR analysis demonstrated that MPA was a potent inducer of 11beta-HSD2 gene expression, increasing the steady-state levels of 11beta-HSD2 mRNA. Taken together, these results demonstrate that 11beta-HSD2 is the 11beta-HSD isoform expressed by T-47D cells under steady-state conditions and suggest the existence of a previously undocumented mechanism of action of progestins in breast cancer cells.

Cytokines in mammalian reproduction and speculation about their possible involvement in nonmammalian viviparity.

In recent years, the secretion of immunoregulatory factors (cytokines) at the maternofetal interface in mammals has been widely documented. Although cytokine production seems to be a specific phenomenon in mammalian reproduction, the specific roles of these substances in different species are still not clear. However, a balance of different cytokine activities appears to be crucial for regulation of the establishment and survival of the semiallogeneic embryo in maternal tissues. The apparent immunological role of placental cytokines in the mechanisms of implantation and embryonic development in mammals has raised the question of whether cytokines are also involved in the reproduction of nonmammalian vertebrates. Our studies have shown that the production of cytokines by the maternofetal unit is not limited to mammalian species, but that interleukin-1 (IL-1)alpha, IL-1beta, and transforming growth factor-beta (TGF-beta) are secreted by the placenta of a viviparous squamate reptile, Chalcides chalcides. Our finding of this parallelism between reptilian and mammalian reproduction suggests that immunological mechanisms, possibly mediated by the secretion of cytokines, played an important role in the evolution of viviparity.

Effect of exercise on plasma interferon levels.

The effect of exercise on plasma interferon activity was studied on eight male subjects before and after exercise on a bicycle ergometer for 1 h at 70% of their maximal O2 consumption (VO2 max). Acid-labile interferon, alpha-type according to immunological characterization, rose significantly from a preexercise value of 3 +/- 1 to 7 +/- 2 IU/ml postexercise. Negligible changes were recorded for plasma protein, lipid, and glucose concentrations, whereas blood lactate slightly increased only at the end of exercise. According to hematocrit and plasma protein values before and after exercise, hemoconcentration did not occur. These data provide evidence that plasma interferon activity increased following a bout of submaximal exercise.