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.

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.

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.

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.

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.

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.

Differentiation and proliferation patterns in human trophoblast revealed by c-erbB-2 oncogene product and EGF-R.

It is well known that growth factors and proto-oncogenes play a pivotal role in organogenesis as well as in tumor development. The human placenta is a rapidly growing organ which shares some aspects with malignant tumors. We have studied the expression of epidermal growth factor receptor (EGF-R) and the receptor encoded by the c-erbB-2 proto-oncogene in first- and third-trimester human placentas. We compared these expression patterns with that of the proliferation marker Ki-67. By immunohistochemistry, EGF-R was intensively expressed in the villous cytotrophoblast in the first trimester. The apical plasma membrane of the syncytium was weakly stained. In placental villi from the third trimester the reaction product for EGF-R was most intense in single villous cytotrophoblastic cells and along the apical plasma membrane of the syncytium, whereas the basal plasma membrane was much less stained. C-erbB-2 protein product was expressed in the first and third trimesters along the apical membrane of the syncytiotrophoblast. Concerning the extravillous trophoblast in cell islands and cell columns, EGF-R was expressed in the cells proximal to the villous stroma whereas the distal cells were c-erbB-2 positive. The Ki-67 antibody revealed the proliferative character of the villous cytotrophoblast and of the EGF-R-positive extravillous trophoblast. In contrast, most of the c-erbB-2-positive cells were Ki-67 negative. By in situ hybridization, c-erbB-2 transcripts were found in all types of villous and extravillous trophoblast, including those that did not express c-erbB-2 protein product. Our data indicate that EGF-R expression is strongly related to the proliferative trophoblast and, with advancing pregnancy, to the differentiated villous trophoblast.off contrast, expression of c-erB-2 protein product occurs only in more advanced stages of trophoblast differentiation, although transcripts of c-erbB-2 are found in both proliferative and differentiated trophoblast. In addition, the coexpression of EGF-R and c-erbB-2 protein product in the syncytiotrophoblast suggests their involvement in complex regulation of hormones and growth factors.

[Importance and limitations of the immunological monitoring of patients with malignant tumors]. / Importanza e limiti del monitoraggio immunologico nei pazienti affetti da tumori maligni.

The authors, after considering the incidence of neoplastic diseases is particularly high in subjects suffering from congenital or acquired immunologic lacks, assert the importance to perform an immunologic monitoring in the patients bearing malignant neoplasms, with the purpose to identify and calculate the direrent deficits of the sheltering organism. Then, they state the method adopted for the study of the cellulo-mediate immunity (in vivo and in vitro) and the humoral one. At last, on the basis of the results they obtained, the Authors, as a conclusion, affirm there is always in neoplastic patients a certain degree of depression of tumour immunity (especially if cellulo-mediate), and it can be put in relation to the evolutive stage of neoplasm.