IL-1ß and TGF-ß weaken the placental barrier through destruction of tight junctions: an in vivo and in vitro study.

INTRODUCTION: Chorioamnionitis is a gestational pathological condition characterized by acute inflammation of the amniochorionic membranes and placentas leading to high concentrations of IL-1ß, Il-6, Il-8 and TGF-ß in the amniotic fluid. In normal conditions, the permeability of foeto-maternal barrier is due to the assembly and maintenance of different cellular junctional domains. METHODS: In the present study, first we aimed to evaluate the protein expression (by immunohistochemistry and western blotting) and mRNA (by real time PCR) levels of the molecular components of tight junctions (Zonula occludens-1 and occludin), and of adherent junctions (VE-cadherin and ß-catenin) in placentas from chorioamnionitis compared to that in normal pregnancies. RESULTS: Western blotting results showed a significant down-regulation of occludin in placentas affected with chorioamnionitis. No differences were detected for the other proteins analysed. We evaluated whether occludin expression was regulated by IL-1ß, IL-6, IL-8 and TGF-ß by means of in vitro studies using HUVEC cultures and demonstrated a key role of IL-1ß and TGF-ß in the disappearance of occludin at cellular border. CONCLUSIONS: We conclude by suggesting a pivotal role of these two cytokines in facilitating intra-placental infection via para-cellular way due to the disassembly of tight junctions at trophoblastic and endothelial cells in placental tissues.

Alterations of maternal plasma HTRA1 level in preeclampsia complicated by IUGR.

We evaluated the presence of HtrA1 in maternal plasma of normal pregnancies and of pregnancies complicated by preeclampsia (PE) without and with Intrauterine Growth Restriction (IUGR). We demonstrate that HtrA1 maternal plasma levels show significant different concentrations in first, second and third trimester of gestation and that HtrA1 concentration increases in maternal plasma of gestations complicated by PE with IUGR compared with control maternal plasma matched for gestational age. Based on these data high maternal plasma levels of HtrA1 could be considered as a possible marker of an occurring IUGR in preeclamptic women.

Syndecan expressions in the human amnion and chorionic plate.

The syndecan family consists of four distinct membrane glycoproteins in mammals. Syndecans control cell proliferation, differentiation, adhesion and migration through participation in cell-cell interactions, anchorage of cells to the extracellular environment, and modulation of multiple growth factors. Therefore, syndecans may play a pivotal role in the regulation of cell behaviour depending on the cellular microenvironment. Here, we demonstrate that syndecan-1, syndecan-2 and syndecan-4 are expressed in fetal membrane tissue with different immunolocalizations. Syndecan-1 is expressed in the amniotic epithelium, localizing at basolateral cell surfaces. Syndecan-2 and syndecan-4, in contrast, are mostly localized in intracellular compartments, in the extravillous cytotrophoblastic cells and in some fibroblasts of the chorionic plate as well as in the amniotic epithelial cells. In the latter, syndecan-4 is mainly localized in the apical part of the cells. Our results strongly suggest a key role of syndecan-1, syndecan-2 and syndecan-4 in the determination of structural and functional characteristics of human amnion and chorionic plate. Since the solute exchanges between fetus and mother take place in fetal membranes, our data suggest that syndecans are important players in the placenta for the establishment of the fetal-maternal inter-communication.

Expression patterns of two serine protease HtrA1 forms in human placentas complicated by preeclampsia with and without intrauterine growth restriction.

Preeclampsia (PE) and intrauterine growth restriction (IUGR) are pregnancy-specific disorders that have in common abnormal placental implantation, a marked proliferation of villous cytotrophoblastic cells and focal necrosis of the syncytiotrophoblast. Several studies show an ischemic placenta with a high-resistance vasculature, which cannot deliver an adequate blood supply to the feto-placental unit. The cause of PE is a matter of debate, but recently studies in mice suggest that the primary feto-placental lesions are sufficient to initiate the disease. HtrA1, a member of the family of HtrA proteins, is a secreted multidomain protein with serine protease activity. It is expressed in first and third trimester of gestation. In specimens from the first trimester of gestation, immunostaining for HtrA1 is generally found in both layers of villous trophoblast, syncytiotrophoblast and cytotrophoblast. Cytoplasm of extravillous trophoblast and extracellular matrix of cell islands and cell columns are labeled for HtrA1. Specimens from third trimester of gestation show a more intense positivity for HtrA1 in the syncytiotrophoblast than in cytotrophoblast. The extravillous trophoblast and the decidual cells, is positive for HtrA1. The purpose of this study is to investigate the expression pattern of HtrA1 in placentas from PE without IUGR (maternal PE) and with IUGR (fetal PE) by quantitative western blotting and immunohistochemistry. By quantitative western blotting analysis we observed a significant upregulation of approximately 30 kDa HtrA1 form in PE. Differently, we detected a significant total HtrA1 down-regulation in PE-IUGR. Moreover, immunostaining for HtrA1 was positive in the villous trophoblast, in the syncytial knots and irregularly in the fetal vessel walls in PE placentas while immunostaining for HtrA1was present particularly in the syncytial knots in PE-IUGR placentas. In conclusion, we suggest that the approximately 30 kDa HtrA1 form can be correlated to maternal PE while that the significant down-regulation of total HtrA1 can be correlated to placental PE. These HtrA1 alterations could be considered as possible markers to discriminate placental PE from maternal PE.

Neurochemistry of the gustatory subgemmal plexus.

Nerve fibers present in the basal plexus of the vallate papilla of the rat tongue were analyzed using cytochemical, immunocytochemical and ultrastructural methods to investigate whether the subgemmal plexus is subdivided into neurochemical compartments and to provide a clear definition of the reciprocal spatial relationships between nitrergic, peptidergic and acetylesterase positive structures. Several neuronal fibers were detected under the chemoreceptorial epithelium. Some of these fibers were in contact with the taste buds and in some cases neuronal projections were also present between the buds or inside them; some others fibers were present below this layer but in a more peripheral area. Antibodies against CGRP, SP and CCK stained fibers just below the chemoreceptorial epithelium, whereas fibers more distally located were immunolabeled by anti VIP, NOS-1 and NF-200 antibodies. Some double staining experiments were conducted using confocal microscopy. Other sections were processed cytochemically for AChE and subsequently for NADPH-d in colocalization experiments. All the data obtained using these techniques confirmed the results obtained with single immunostaining, as did the ultrastructural results. In conclusion, the present work demonstrates that the subgemmal plexus is a bilayered structure, suggesting that the complex relationship between the two layers plays a pivotal role in taste and in the control of processes ancillary to taste, such as control of vascular or secretory mechanisms.

Immunohistochemical investigation of the nitrergic system in the taste organ of the frog, Rana esculenta.

We have studied by immunocytochemistry, the taste discs of the frog, Rana esculenta, with the aim of providing morphological and neurochemical data on the nitrergic system and of assessing the eventual presence of intrinsic neurons associated with the gustatory organs. In taste discs, antibodies against neuronal nitric oxide synthase (nNOS) revealed a positive immunoreaction in the taste receptor cell bodies and processes. The basal cells were also stained. All the fungiform papillae contained intragemmal nerve fibers showing nNOS immunoreactivity; these fiber were mainly located in the basal plexus. Immunoreactive nerve fibers were also visible at the periphery of the papilla-contacting ciliate cells, which form a ring around the taste disc. In conclusion, the findings obtained in this study suggest that the occurrence of nNOS-immunoreactivity in basal cells, taste cells and nerves might reflect a role for nitric oxide in taste mechanisms of Amphibia. The results may also sustain the physiological implication of NO as a molecule involved in the local target function of maintaining the taste bud mucosal integrity and in regulating the blood flow to the epithelium.

Hyaluronate and CD44 expression patterns in the human placenta throughout pregnancy.

Hyaluronan (HA) and CD44 are involved in several processes such as cell migration and differentiation. In the present study, we examined the expression and distribution of both hyaluronan and its cell surface receptor (CD44) in the human placenta, which is a rapidly growing and differentiating organ that plays a fundamental role in fetal life. Hyaluronan was detected by a specific biotinylated binding probe, termed b-PG. In the first half of gestation, HA was strongly expressed in the stroma of the mesenchymal villi which have been previously identified as responsible for the growth and differentation of the villous trees. The other villous types showed an intense staining only in the fetal vessel walls and in the connective tissue closely underlying the trophoblastic cover. In addition, hyaluronan positive staining was also apparent in a restricted rim of villous stroma directly apposed to extravillous cytotrophoblastic cell islands and cell columns. In full term placentas, all villi expressed HA in their stromal tissue with a more homogenous staining than in the first half of gestation. In contrast to hyaluronan, in the first trimester CD44 was restricted to some of the Hofbauer cells which may be able to internalize hyaluronan, thus playing a significant role in its removal in early pregnancy. CD44 was primarily expressed starting from the 16th week of gestation. At the end of pregnancy it was expressed in the various villous types, especially in stem villi. Moreover, the plasma membrane of some extravillous cytotrophoblastic cells in the basal plate and the large majority of the decidual cells showed a positive immunostaining for this receptor. Taken together, these data suggest that HA is strongly involved in early villous morphogenesis, whereas CD44 seem to be play an important role in tissue remodelling later in gestation.

A brief survey of the modifications in sensory-secretory organs of the neonatal rat tongue.

Recent data obtained on rats suggest that in the days immediately following birth several events take place in the circumvallate papillae of the oral cavity. A phylogenetically primitive system of solitary chemosensory cells develops and is rapidly replaced by taste buds. The lipase-secreting von Ebner gland, which is associated with taste organs, begins to develop by forming short tubules. The intrinsic nervous system of the gustatory organs rapidly completes its maturation showing fast proliferation of fibers and immunocytochemical maturation. Intraepithelial lipid accumulation is visible in the non-receptorial mucosa of the tongue, showing aspects which suggest an active lipid secretion. These data demonstrate that in the rat the structure of the sensory-secretory organs of the newborn's tongue shows a typical conformation with respect to the adult and rapidly changes its organization in the first week after the birth. At the present level of knowledge, it is difficult to link the anatomical structures to peculiar functional roles but the rather simple organization of the neonatal gustatory epithelium could be in relation to the dietary regimen. The data obtained in laboratory animals underline the necessity of studies on human newborns to update the anatomical knowledge of the oral chemoceptive system.

Postnatal development of the intrinsic nervous system in the circumvallate papilla-vonEbner gland complex.

We have studied the postnatal development of the intrinsic nervous system in the circumvallate papilla-vonEbner gland complex using NADPH-diaphorase cytochemistry, immunocytochemistry (for nitric oxide synthase-1 and alpha-internexin) and electron microscopy. In rats sacrificed in their first day post partum (1 p.p.), only isolated NADPH-diaphorase positive neurons were visible in the organ. At 2 p.p., a small group of neurons was visible at the base of the papillae and positive neurons formed short chains close to the developing glandular tubules. In the following days, the NADPH-diapharase positive cells increased in number and nerve fibres were associated to small ganglia located at the base of the papilla or in the gland. After the first week of extrauterine life, the intrinsic nervous system was similar to the intrinsic system of adult animals. An immunocytochemical positivity for nitric oxide synthase-1 appeared at 4 p.p. in neurons located in the gland and at 7 p.p. in cells located at the base of the papilla. Immunocytochemical staining for alpha-internexin showed that at 1 p.p. developing nerve fibres were present in the connective tissue of the tongue's muscle layer. At 2-3 p.p., developing nerve fibres were also present at the bases and in the core of the papilla. In the following days, the positivity for alpha-internexin was reduced and one week after birth was virtually absent. Ultrastructural examination revealed that since 1 p.p. isolated neurons can be found at the base of the papilla. In conclusion, the intrinsic nervous system originates from neurons present in the organ at the birth which, in the first days, undergo a biochemical and morphological maturation while the nerve fibres rapidly grow. These findings support the hypothesis that the intrinsic nervous system of the circumvallate papilla has a role in the maturation of the vonEbner gland.

Alpha-gustducin-immunoreactive solitary chemosensory cells in the developing chemoreceptorial epithelium of the rat vallate papilla.

The presence of solitary chemosensory cells was studied in rat vallate papillae during the first week of post-natal life by alpha-gustducin immunocytochemistry. In 1- to 3-day-old rats, isolated alpha-gustducin-immunoreactive cells were found within the epithelium of the vallate papilla. These cells, mainly located in the basal layer, were scattered among keratocytes and wrapped in alpha-gustducin-negative epithelial cells in a glia-like fashion. The alpha-gustducin-immunoreactive cells were usually round and some of them gave rise to short, large processes directed towards the lumen of the oral cavity or the basal lamina. Rarely, some cells showed an evident bipolar shape. Small taste buds containing either alpha-gustducin-immunoreactive or alpha-gustducin-negative cells appeared in the vallate papillae of 4-day-old rats in which isolated, bipolar-shaped alpha-gustducin-immunoreactive cells were also found. After the first week of post-natal life, the taste buds appeared basically similar to those of adult animals. In conclusion, the present study demonstrates that the presence of epithelial cells with characteristics of solitary chemosensory cells precedes the development of the taste buds.

The anatomy and functional role of the circumvallate papilla/von Ebner gland complex.

We suggest that the description of the reciprocal relationships between the circumvallate papilla (CP) and von Ebner glands (VEGs) reported in most textbooks of anatomy must be rewritten. In the past, the VEG was described as a gland ancillary to the taste buds, mainly involved in the washing of the vallum around the CP or in perireceptorial events. Recent data obtained in our laboratory or reported in the literature indicate that both these structures form a single functional unity which could be called circumvallata papilla/von Ebner gland (CP/VEG) complex. The CP/VEG complex seems to represent an important enzyme- and pheromone-producing system composed of a sensitive (taste buds) and an effectory (VEG) branch linked by feedback mechanisms of control. In our hypothesis, the taste buds located in the distal portion of the VEG ductal system can be considered similar to the chemoreceptory cells located in other portions of the digestive apparatus such as pancreatic and bile ducts. Therefore, the CP/VEG complex represents a rare example of chemoreceptor-secretory organ.

NADPH-diaphorase and NOS-1 positive ganglion cells are found in the rat vallate papilla/von Ebner gland complex.

The nervous system of the vallata papilla and von Ebner glands was investigated in the rat tongue. Cells involved in the production of nitric oxide were identified by immunohistochemical detection of neuronal nitric oxide synthase type-1 and by cytochemical detection of NADPH-diaphorase. The analysis of serial sections showed that a ganglion composed of about 180-190 neuronal cells was present between the vallata papilla and von Ebner glands. These cells were positive for nitric oxide synthase type-1 and NADPH-diaphorase. From the ganglion, we observed nitrergic fibres running: (a) in the lamina propria of the receptor-free mucosa; (b) just below the gustatory epithelium; (c) in the von Ebner glands; and (d) around the vascular system of the vallata papilla. Our study suggests that the nitrergic ganglion cells may mediate interactions between chemoreceptorial systems in the vallata papilla and secretory cells in the von Ebner glands and that nitric oxide could be involved in the regulation of the blood supply to the vallata papilla and in the regulation of the von Ebner glands.

Immunohistochemical evidence suggests intrinsic regulatory activity of human eccrine sweat glands.

Immunohistochemistry of normal eccrine sweat glands was performed on paraffin sections of human skin. Immunoreactivity (ir) for neuron specific enolase, S100 protein (S100), regulatory peptides, nitric oxide synthase type I (NOS-I) and choline-acetyltransferase (ChAT) was found in small nerve bundles close to sweat glands. In the glands, secretory cells were labelled with anticytokeratin antibody. Using antibodies to S100, calcitonin gene-related peptide (CGRP) and substance P (SP) a specific distribution pattern was found in secretory cells. Granulated (dark) and parietal (clear) cells were immunopositive for CGRP, and S100 and SP, respectively. Immunoreactivity was diffuse in the cytoplasm for CGRP and S100, and peripheral for SP. Myoepithelial cells were not labelled. Electron microscopy revealed electron dense granules, probably containing peptide, in granulated cells. Using antibodies to NOS-I and ChAT, ir was exclusively found in myoepithelial cells. Immunoreactivity for the atrial natriuretic peptide was absent in sweat glands. These results provide evidence for the presence of both regulatory peptides involved in vasodilation and key enzymes for the synthesis of nitric oxide and acetylcholine in the secretory coil of human sweat glands. It is suggested that human sweat glands are capable of some intrinsic regulation in addition to that carried out by their nerve supply.

Intra-epithelial lipids in the dorsoposterior area of the tongue in new-born rats.

The mucosa covering the posterior surface of the tongue in new-born rats was studied by scanning and transmission electron microscopy. The results demonstrated that in the first days of extra-uterine life in the epithelium of this mucosa there is an accumulation of lipidic material, which appeared as round or oval droplets with a mean diameter of about 0.8-1.0 microm. Their density was homogeneously low and they did not contain fibrillar or granular material. In the first day of extra-uterine life, the droplets were scarce and mainly localized in the dorsal surface of the vallate papilla. In the following days, the lipid deposit was present in large areas of the posterior surface of the tongue. At the end of the first week of extra-uterine life, as well as in adult animals, the lipids were rather scarce. A multilocular lipid deposit was also visible in superficial cells detaching from the mucosa. With magnetic resonance spectroscopy, lipids extracted from the posterior mucosa of the tongue appeared mainly saturated. Small amounts of unsaturated and polyunsaturated lipid were also detectable. These findings suggest that in the first week of extra-uterine life, specialized areas of the oral epithelium store lipid.

Expression pattern alterations of syndecans and glypican-1 in normal and pathological trophoblast.

Syndecans (syn-1, -2, -3, -4) and glypican-1 are proteoglycans expressed during development in association with changes in tissue organization and differentiation. They participate in the modulation of growth factor actions and in cell-cell and cell-matrix adhesion. The expression of syn-1, -2, -3, -4, and glypican-1 has been studied in normal human placenta and in gestational trophoblastic disease such as hydatidiform mole, invasive mole, and choriocarcinoma, using immunohistochemistry and western blots. Syndecan-3 was not expressed in normal or pathological tissues. During normal gestation, the other proteoglycans showed a specific staining pattern, which for some was modified during pregnancy. For instance, syn-1 was only expressed in syncytiotrophoblast; syn-4 was mainly localized in the villous and extravillous cytotrophoblast in the first trimester, whereas at term it was expressed in the syncytiotrophoblast. The most striking results are the altered expression patterns of syndecans and glypican-1 in pathological tissues. These proteoglycans showed a progressive decrease of immunostaining related to the increase of severity of trophoblastic disease, in particular in invasive mole and choriocarcinoma. In addition, dysregulation in the localization of the expression patterns was observed for syn-2 and -4. Because changes in syndecan expression enable cells to become more or less responsive to their micro-environment, the down-regulation and/or dysregulation of syndecans in relation to the degree of severity of trophoblastic diseases provides new insights into the progression of these pathologies.

BCL-2 expression in the human placenta and its correlation with fibrin deposits.

The human placenta performs numerous functions during its limited lifespan and its survival is a necessary prerequisite for fetal nutrition, even in unfavourable conditions. BCL-2 is a proto-oncogene implicated in the regulation of cell death and survival without affecting cell proliferation. An extracellular matrix molecule involved in the reparative and degenerative processes in the human placenta is fibrin. We have analysed by immunohistochemistry the expression of BCL-2 and correlated it with fibrin deposits in placental tissues. In first and third trimester placentas BCL-2 was expressed in the syncytiotrophoblast. Only a few mesenchymal villi (first trimester) or terminal villi (third trimester) showed no staining in the syncytiotrophoblast. Villous cytotrophoblast, mesenchymal cells of the villous cores and extravillous cytotrophoblast of cell columns and cell islands were all negative for BCL-2. BCL-2 expression was enhanced in the syncytiotrophoblast overlying subtrophoblastic fibrin deposits. However, discontinuities and/or variations in intensity of BCL-2 expression characterized not only the villi showing perivillous fibrinoid but also those villi with a massive presence of fibrinoid in their cores. These data suggest that BCL-2 may be necessary for the preservation of the placenta during gestation as well as for the reparative processes of the trophoblast.

Membrane-bounded intratubular bodies in rat taste buds.

The ultrastructural features of membrane-bounded bodies contained in the tubulo-vesicular system in the outer segment of taste bud cells are described. Each body showed a round, fusiform or oval shape, was surrounded by a trilaminar membrane and enclosed an electron dense matrix sometimes containing inclusions. These bodies were found at all ages studied. Similar structures were also found embedded in the material plugging the taste pore. Our findings suggest that these bodies could be secreted at the free surface of the cells and be involved in the concentration of divalent cations.

Solitary chemosensory cells in the developing chemoreceptorial epithelium of the vallate papilla.

The solitary chemosensory cells are considered typical of aquatic vertebrates and have never been described in the oral cavity of terrestrial vertebrates. We describe elements with ultrastructural characteristics of the solitary chemosensory cell in the gustatory epithelium of rats in the first week of extrauterine life. These elements appeared isolated, located among keratinocytes, and wrapped by glial-like elements. They showed a bipolar shape with a round cell body, a thin apical process, and a thicker basal one. Nerve fibers contacted the cell body and the processes. The cells showed small dense granules mainly located near nerve contacts. Small bundles of tonofilaments were visible in the perinuclear cytoplasm. Similar elements were not found in rats after the first week of extrauterine life. The present study demonstrates in a mammal that the development of taste buds is preceded by the presence of epithelial elements with ultrastructural characteristics of the solitary chemosensory cells described in lower vertebrates. This finding suggests that the oral chemoreception in the suckling rats may be mediated by three different pathways (i.e., gustatory system, common chemical sense, and solitary chemosensory cell system).

Cloning of choriocarcinoma cells shows that invasion correlates with expression and activation of gelatinase A.

Implantation and placental development are dependent upon trophoblast invasion of the endometrium. While the villous trophoblast does not display invasive behavior, the extravillous cytotrophoblast is highly invasive. By cloning BeWo choriocarcinoma cells, we have isolated two distinct clones that share similarities with villous and extravillous cytotrophoblasts. When cultured at the surface of a type I collagen gel, BeWo MC-1 cells were not invasive, whereas BeWo MC-2 cells rapidly invaded this matrix. When injected subcutaneously in nude mice, BeWo MC-1 cells developed a localized tumor and BeWo MC-2 cells developed larger tumors with micrometastases. Gelatinase A expression and minute amounts of gelatinase B were detected in the parental cell line and in both clones. However, the parental and the BeWo MC-2 cells secreted 5-to 10-fold more gelatinase A than the BeWo MC-1 cells. Laminin and matrigel stimulated the production of gelatinase A in BeWo MC-2 cells. Type I collagen promoted the conversion of the 72-kDa progelatinase A in an active enzyme only in parental BeWo and in BeWo MC-2 cells. These clones provide an interesting model for studying the complex mechanisms regulating implantation as well as the controlled invasiveness during implantation compared to tumor invasion.

Codistribution of basic fibroblast growth factor and heparan sulfate proteoglycan in the growth zones of the human placenta.

In order to obtain an insight into morphogenetic processes such as angiogenesis, cell proliferation, and tissue remodeling we have studied the localization of basic fibroblast growth factor (bFGF) and heparan sulfate proteoglycan (HSPG) in the human placenta by immunohistochemistry. Positive reaction product for bFGF is found mainly in the villous trophoblastic covering and for HSPG in the villous basement membranes. A codistribution of the two molecules is detectable in first trimester placental tissue, in areas previously identified as being responsible for the growth of the villous tree, i.e., in the mesenchymal villi and the cytotrophoblastic cell islands and cell columns, both consisting of extravillous trophoblast. HSPG and bFGF are codistributed in the distal half of the villous stroma in the mesenchymal villi. In cell islands and cell columns, bFGF is detectable in the cytoplasm of the extravillous cytotrophoblastic cells, whereas HSPG is localized between the extravillous cytotrophoblastic cells and in their cytoplasm. HSPG-bFGF codistribution in term placenta is confined to the walls of fetal vessels and to some extravillous cytotrophoblastic cells in the basal plate. The codistribution of bFGF and HSPG in first trimester placental tissue suggests that these two molecules play a pivotal role in the morphogenetic processes mentioned above in early stages of gestation.