Microanalysis of Organic Pigments in Ancient Textiles by Surface-Enhanced Raman Scattering on Agar Gel Matrices

We review some new methods based on surface-enhanced Raman scattering (SERS) for the nondestructive/minimally invasive identification of organic colorants in objects whose value or function precludes sampling, such as historic and archeological textiles, paintings, and drawing. We discuss in detail the methodology we developed for the selective extraction and identification of anthraquinones and indigoids in the typical concentration used in textiles by means of an ecocompatible homogeneous nanostructured agar matrix. The extraction system was modulated according to the chemical properties of the target analyte by choosing appropriate reagents for the extraction and optimizing the extraction time. The system has been found to be extremely stable, easy to use and produce, easy to store, and at the same time able to be analyzed even after long time intervals, maintaining its enhancement properties unaltered, without the detriment of the extracted compound. Highly structured SERS band intensities have been obtained from the extracted dyes adopting laser light excitations at 514.5 and 785 nm of a micro-Raman setup. This analytical method has been found to be extremely safe for the analyzed substrates, thus being a promising procedure for the selective analysis and detection of molecules at low concentration in the field of artworks conservation

Macrophage Migration Inhibitory Factor in Fetoplacental Tissues from Preeclamptic Pregnancies with or without Fetal Growth Restriction

The proinflammatory cytokine MIF (macrophage migration inhibitory factor) is involved in physiological and pathological processes in pregnancy. MIF maternal serum levels are increased in preeclampsia (PE). We hypothesize that pregnancy tissues are the source of MIF overexpression in PE. MIF protein was studied in maternal sera, placental tissues, fetal membranes, and umbilical cord of 8 control and 20 PE pregnancies: 10 with normal fetal growth (PE-AGA) and 10 with fetal growth restriction (PE-FGR). MIF levels were significantly higher in PE-AGA membranes than in controls and PE-FGR. In PE-FGR, MIF cord concentrations were higher than in PE-AGA while MIF placental levels were lower than in controls. MIF maternal serum levels were higher in PE, compared to controls, and the difference was mainly due to PE-FGR samples. These data support MIF involvement in PE pathogenesis and suggest that different pregnancy tissues contribute to MIF production in PE with and without fetoplacental compromise

Human trophoblast differentiation: possible role for trophoblast cell surface antigen 2

Human trophoblast cell surface antigen 2 (Trop2) is a 40-kDa transmembrane glycoprotein, encoded by TACSTD2 gene and identified for the first time in human trophoblast and choriocarcinoma cell lines. Trop2 has a short intracytoplasmic tail essential for the control of several pathways that regulate cellular functions such as cell- cell adhesion, cell proliferation and mobility [1]. We analysed the expression of Trop2 in human normal placentas during gestation and in placentas complicated by preeclampsia (PE). Trop2 protein expression and miR125b1 were analysed by morphological and bio-molecular techniques. Trop2 increased during gestation, i.e. from first to third trimester of gestation while it was low expressed in placental tissues collected from patients with PE. Since PE is a pathology associated with placental hypoxia, we demonstrated that Trop2 is downregulated in hypoxic conditions by in vitro model. Our study suggests a possible involvement of Trop2 in maintaining trophoblast morphology and function during placental development in normal and PE conditions

Expression of the ciliary neurotrophic factor and its receptor α in human placenta of first and third trimester of gestation

The ciliary neurotrophic factor (CNTF) is a member of the IL-6 family of cytokines along with cardiotrophin-1, IL-11, leukemia inhibitory factor, oncostatin-M and IL-6 itself. These cytokines play an important role in the regulation of cellular processes such as gene activation and cell proliferation and differentiation. CNTF is a pleiotropic cytokine which effects are mediated via CNTF receptor α (CNTFRα). CNTF increases differentiation and/or survival in neuronal cells but it also has different effects on other cell types such as muscle cells, bone cells, adipocytes, retinal cells and pancreatic β-cells (1, 2). In addition, recent studies demonstrate that CNTF plays an important role in weight control since exogenously administration of CNTF has an anorectic effect in mice (3,4). Although many studies proved that CNTF plays different roles in many cell types, its role in the development of human placenta has never been investigated. In this study we investigated the expression of CNTF and CNTFRα in human trophoblast by, immunohistochemistry, immunocytochemistry and Western Blot analysis using normal first and third trimester human placentas and HTR-8/SVneo cell lines. Interestingly, using immunohistochemistry CNTF and CNTFRα were expressed in the cytotrophoblast and syncytiotrophoblast in the first and third trimester of gestation respectively. Moreover, the immunofluorescence analyses by confocal microscopy showed that CNTF is expressed in the cytoplasm and nuclei whereas CNTFRα is mainly expressed in the cell membrane and cytoplasm of HTR-8/SVneo cell line. In this study we demonstrated that CNTF and CNTFRα are normally expressed in human placenta and they may play an important role during placental development

Placental Expression of CD100, CD72 and CD45 Is Dysregulated in Human Miscarriage

CONTEXT AND OBJECTIVE: The etiology of miscarriage is often multifactorial. One major cause, immunological rejection of the fetus, has not been clearly elucidated. Our aim was to establish whether the semaphorin CD100, its natural receptor CD72, and the glycoprotein CD45, implicated in immune mechanisms, are involved in pregnancy loss by examining their placental expression with real-time PCR, immunohistochemistry and western blotting techniques. PATIENTS: Placenta tissue from 72 Caucasian women undergoing surgical uterine evacuation due to early spontaneous pregnancy loss between the 8(th) and 12(th) week of gestation was divided into four groups based on miscarriage number. Gestational age-matched placentas from 18 healthy women without a history of miscarriage undergoing voluntary pregnancy termination were the control group. Placenta from 6 Caesarean deliveries performed at 38-40 weeks of gestation was also studied. RESULTS: CD100, CD72 and CD45 were expressed in placenta and exhibited different mRNA and protein levels in normal pregnancy and miscarriage. In particular, protein levels were highly dysregulated around 10 weeks of gestation in first and second miscarriage placentas. The CD100 soluble form was produced and immediately shed from placental tissue in all samples. CONCLUSIONS: Fetal CD100, CD72 and CD45 seem to play a role in miscarriage. The present data support the involvement of the fetal immune system in pregnancy maintenance as well as failure

miR125b1 and TROP2 in preeclampsia complicated by foetal growth restriction: a morphological and biomolecular study

Trophoblast cell surface antigen 2 (TROP2) is a transmembrane glycoprotein originally identified in human trophoblast cell lines and is highly expressed in a variety of epithelial cancers. The TROP2 gene was validated as a direct target of miR-125b1. The purpose of our study was: - to investigate the expression of TROP2 protein in normal placental tissues, in placentas affected by preeclampsia as well as in placentas with preeclampsia complicated by foetal growth restriction (IUGR); - to verify how miR-125b1 was involved in the regulation of TROP2 gene expression. TROP2 protein expression was assessed by immunohistochemistry and quantitative western blotting analyses while miR-125b1 expression was detected by quantitative real-time PCR. The studies were made in normal and pathologic placental tissues. Increasing expression of TROP2 was detected in physiological placental tissue, in according with the increasing gestational age. Probably, it means that TROP2 is related with the differentiation of the cytotrophoblast in syncytiotrophoblast, that occurs during the development of placenta. Moreover, miR-125b1 showed an unchanged expression during normal pregnancy. Higher expression of TROP2 protein was detected in placental tissues collected from patients with preeclampsia complicated by foetal growth restriction, compared with those from preeclampsia and gestational age-matched control samples. The miR-125b1 expression in samples from placentas affected by preeclampsia complicated by IUGR was detected higher than in normal placentas and in placentas affected by preeclampsia. These results suggest that miR-125b1 is not involved I the overproduction of the TROP2 mRNA although the high expression of the miRNA. Our study suggests a possible involvement of TROP2 in the differentiation of the syncytiotrophoblast from villous cytotrophoblast and a possible role of this protein in preeclampsia complicated by foetal growth restriction

Resistin modulates glucose uptake and glucose transporter-1 (GLUT-1) expression in trophoblast cells

The adipocytokine resistin impairs glucose tolerance and insulin sensitivity. Here, we examine the effect of resistin on glucose uptake in human trophoblast cells and we demonstrate that transplacental glucose transport is mediated by glucose transporter (GLUT)-1. Furthermore, we evaluate the type of signal transduction induced by resistin in GLUT-1 regulation. BeWo choriocarcinoma cells and primary cytotrophoblast cells were cultured with increasing resistin concentrations for 24 hrs. The main outcome measures include glucose transport assay using [3H]-2-deoxy glucose, GLUT-1 protein expression by Western blot analysis and GLUT-1 mRNA detection by quantitative real-time RT-PCR. Quantitative determination of phospho(p)-ERK1/2 in cell lysates was performed by an Enzyme Immunometric Assay and Western blot analysis. Our data demonstrate a direct effect of resistin on normal cytotrophoblastic and on BeWo cells: resistin modulates glucose uptake, GLUT-1 messenger ribonucleic acid (mRNA) and protein expression in placental cells. We suggest that ERK1/2 phosphorylation is involved in the GLUT-1 regulation induced by resistin. In conclusion, resistin causes activation of both the ERK1 and 2 pathway in trophoblast cells. ERK1 and 2 activation stimulated GLUT-1 synthesis and resulted in increase of placental glucose uptake. High resistin levels (50–100 ng/ml) seem able to affect glucose-uptake, presumably by decreasing the cell surface glucose transporter

Raf Kinase Inhibitor Protein (RKIP) expression and function in human myometrium and leiomyoma

Many growth factors been identified in human myometrium and leiomyoma and activate multiple signaling pathways in order to regulate major cellular processes, including proliferation and fibrosis which are linked to uterine leiomyoma development and growth. The Raf kinase inhibitor protein (RKIP) has emerging roles as regulator of multiple signaling networks including mitogen activated protein (MAP) kinase cascade, as well as interaction with glycogen synthase kinase 3 (GSK3). In our study, we aimed to investigate the presence of RKIP in human myometrium and leiomyoma as well as to determine the effect of locostatin (RKIP inhibitor) on extracellular matrix (ECM) production, proliferation and migration in human myometrial and leiomyoma cells. Myometrial and leiomyoma tissues were used to investigate the localization and the expression level of RKIP through immunohistochemistry and western blotting. Myometrial and leiomyoma cells were treated with locostatin to measure ECM expression by real time PCR, GSK3b expression by western blotting, cell migration by wound-healing assay and cell proliferation by MTT assay. We found that RKIP is expressed in human myometrial and leiomyoma tissue. Locostatin treatment resulted in the activation of the MAPK signal pathway (ERK phosphorylation), providing a powerful validation of our targeting protocol. Further, RKIP inhibition by locostatin reduces ECM components. Moreover, the inhibition of RKIP by locostatin impaired cell proliferation and migration in both leiomyoma and myometrial cells. Finally, locostatin treatment reduced GSK3β expression. Therefore, even if the activation of MAPK pathway should increase proliferation and migration, the destabilization of GSK3β leads to the reduction of proliferation and migration of myometrial and leiomyoma cells

Analysis of tight junctions in placentas affected by chorioamnionitis: in vivo and in vitro analysis

The human placenta and fetal membranes provide a barrier regulating the transfer of materials between the mother and the developing fetus throughout gestation. Chorioamnionitis is an important risk factor for preterm delivery that is associated with high perinatal morbidity and mortality. Chorioamnionitis is the term applied to infections of the placenta and membranes resulting in high concentrations of IL- 1beta, IL-6, IL-8 and TGF-beta in the amniotic fluid (D’Alquen et al., 2005). With progression of inflammation, immune cells penetrate blood vessels and infiltrate the umbilical cord, resulting in funisitis (Romero and Mazor, 1988). In normal conditions the two important physical entities in endothelial/epithelial paracellular clefts are adherens junctions and tight junctions. Tight junction governs the paracellular movement of water, solutes and immune cells, through the intercellular space creating a boundary between the apical and basolateral sides of cellular barriers (Gruenheid and Finlay, 2003). We have evaluated the localization of tight junctions studying the Zonula Occludens-1 (ZO-1) and Occludin expressions as well as the localization of adherent junctions, testing the expression of VE-cadherin and beta-catenin in placentas from normal gestations, from preterm idiopathic deliveries and from chorioamnionitis by immunohistochemistry. In addition, we have evaluated the mRNAs by real time PCR, the protein levels of these molecules by Western blot analysis in placental tissues, and to better clarify the action of some cytokines on occludin we performed in vitro analysis of HUVEC cultures. Our more striking result is the decrease of occludin expression in placentas from chorioamnionitis and an evident action of the cytokines on this molecule