Chiara Avellini

IL-1β and TGF-β 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-1β, Il-6, Il-8 and TGF-β 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 β-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-1β, IL-6, IL-8 and TGF-β by means of in vitro studies using HUVEC cultures and demonstrated a key role of IL-1β and TGF-β 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.

High temperature requirement A1 and fibronectin: two possible players in placental tissue remodelling

High temperature requirement A1 (HtrA1) is a secreted protease involved in placental development. Fibronectin (FN) is involved in important process such as wound healing, cell adhesion and spreading, growth, migration, and differentiation. The purpose of this study was to analyse the expression patterns of HtrA1 in relationship to FN and to the key growth zones of placenta such as mesenchymal villi as well as cell islands and cell columns. We demonstrated that FN and HtrA1 are localized in the placental key growth zones suggesting a pivotal role in maintaining the balance among the molecules involved in the placental development and differentiation.

Sclerostin and Antisclerostin Antibody Serum Levels Predict the Presence of Axial Spondyloarthritis in Patients with Inflammatory Bowel Disease

Objective. The early diagnosis of inflammatory bowel disease (IBD)-associated spondyloarthritis (SpA/IBD) in patients affected by IBD represents a major topic in clinical practice; in particular, to date there are no available serum biomarkers revealing the presence of joint inflammation in these patients. Sclerostin (SOST), an antagonist of the Wnt/β-catenin pathway, and antisclerostin-immunoglobulin G (anti-SOST–IgG) have been recently studied in patients with ankylosing spondylitis (AS) as a putative marker of disease activity. Methods. SOST and anti-SOST-IgG serum levels were assayed in 125 patients with IBD, 85 with axial or peripheral SpA, and in control groups (patients with AS and rheumatoid arthritis, and healthy individuals). The diagnostic performance in discriminating the presence of SpA/IBD was assessed for both candidate biomarkers. Results. Patients affected by SpA/IBD with axial involvement displayed significantly lower levels of SOST and higher levels of anti-SOST-IgG compared to patients with only peripheral arthritis, IBD, and controls. Moreover, SOST and anti-SOST-IgG serum levels were inversely correlated and were associated with the duration of articular symptoms. Both biomarkers showed good accuracy in predicting the presence of axial SpA in patients with IBD. Conclusion. We demonstrated that in patients with IBD, SOST and anti-SOST-IgG might represent novel biomarkers to assess the presence of axial joint involvement. Moreover, the development of anti-SOST-IgG and the subsequent decrease of SOST serum levels could play a role in the pathogenesis of SpA/IBD.

The trophoblast cell surface antigen 2 and miR-125b axis in urothelial bladder cancer

Human trophoblast cell surface antigen 2 (Trop-2) is a 40-kDa transmembrane glycoprotein that was first identified as a marker of human trophoblast cells. Trop-2 acts on cell proliferation, adhesion, and migration by activating a number of intracellular signalling pathways. Elevated Trop-2 expression has been demonstrated in several types of cancer and correlated with aggressiveness and poor prognosis. Since no data are available on Trop-2 in bladder cancer (BC), the purpose of the study was to determine its levels in tissue specimens from normal individuals and patients with BC at different stages. Moreover, since according to recent evidence Trop-2 is a miR-125b target, miR-125b expression was also assessed in tissue specimens. Finally, the effect of the Trop-2/miR-125b axis on the proliferation and migration of BC cells was evaluated in vitro.The Trop-2/miR-125b axis was seen to be differentially expressed in normal urothelium, non-invasive BC and invasive BC tissue. Significant miR-125b down-regulation was associated with a significant increase in Trop-2 protein levels in BC tissue and correlated with disease severity. In vitro analysis confirmed the role of miR-125b in down-modulation of Trop-2 protein levels and showed that Trop-2/miR-125b axis affects cellular proliferation in bladder tissue.In conclusion, our findings highlight a role for the Trop-2/miR-125b axis in BC progression and suggest Trop-2 and miR-125b as diagnostic/prognostic marker candidates as well as druggable targets for innovative therapeutic approaches.Human trophoblast cell surface antigen 2 (Trop-2) is a 40-kDa transmembrane glycoprotein that was first identified as a marker of human trophoblast cells. Trop-2 acts on cell proliferation, adhesion, and migration by activating a number of intracellular signalling pathways. Elevated Trop-2 expression has been demonstrated in several types of cancer and correlated with aggressiveness and poor prognosis. Since no data are available on Trop-2 in bladder cancer (BC), the purpose of the study was to determine its levels in tissue specimens from normal individuals and patients with BC at different stages. Moreover, since according to recent evidence Trop-2 is a miR-125b target, miR-125b expression was also assessed in tissue specimens. Finally, the effect of the Trop-2/miR-125b axis on the proliferation and migration of BC cells was evaluated in vitro. The Trop-2/miR-125b axis was seen to be differentially expressed in normal urothelium, non-invasive BC and invasive BC tissue. Significant miR-125b down-regulation was associated with a significant increase in Trop-2 protein levels in BC tissue and correlated with disease severity. In vitro analysis confirmed the role of miR-125b in down-modulation of Trop-2 protein levels and showed that Trop-2/miR-125b axis affects cellular proliferation in bladder tissue. In conclusion, our findings highlight a role for the Trop-2/miR-125b axis in BC progression and suggest Trop-2 and miR-125b as diagnostic/prognostic marker candidates as well as druggable targets for innovative therapeutic approaches.

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.

OP0271 Gastrointestinal damage and microbial translocation are involved in the development of immune system activation in inflammatory bowel disease-associated spondyloarthritis

Background The altered composition of the gastrointestinal (GI) microbiota, known as dysbiosis, can induce and modulate the systemic inflammation, through microbial translocation and T-cell activation, in several immuno-mediated diseases, such as inflammatory bowel disease (IBD), HIV infection, and ankylosing spondylitis. Objectives In a cohort of 85 patients with inflammatory bowel disease-associated spondyloarthritis (SpA/IBD), we assessed gut bacterial infiltration and intestinal damage. In systemic circulation, GI epithelial damage, microbial translocation and immune system activation were assessed with intestinal-fatty acid binding protein (I-FABP), lipopolysaccharide (LPS), soluble CD14 (sCD14), respectively. Moreover, the in vitro activity of the latter two was evaluated on osteoblast cells. Methods I-FABP, LPS, sCD14, sclerostin (SOST) and anti-SOST antibodies (anti-SOST-IgG) were assayed with ELISAs. LPS and sCD14 were used in vitro to stimulate the MG63 human osteoblast-like cell line. Occludin, claudin-1, claudin-4, and the presence of bacteria were assessed, respectively, by real-time-PCR analysis and immunohystochemical staining of the ileum. Results Bacteria were detectable in the ileal epithelium of IBD patients, but only in SpA/IBD they were associated with epithelial damage and downregulation of occludin, claudin-1 and claudin-4 (figure 1A-B). The serum levels of I-FABP, LPS and sCD14 resulted significantly higher in axial (187.9, 14.03, and 26.97, respectively) and peripheral SpA/IBD (130.3, 11.55, and 18, respectively) than in IBD patients (IFABP 43.65, p<0.0001 for both patients’ groups; LPS 9.625, p<0.0001 vs Ax-SpA/IBD and=0.007 vs Per-SpA/IBD; sCD14 12.34, p<0.0001 for both patients’ groups) (figure 1C). In the SpA/IBD cohort, SOST was weakly correlated with I-FABP (r=-0.2683), LPS (r=-0.3063) and sCD14 (r=-0.3075), and anti-SOST-IgG with LPS (r=- 0.3959) and sCD14 (r=-0.3414). Moreover, sCD14 showed significant correlation with I-FABP (r=0.3316) and LPS (r=0.5649). In vitro, LPS, but not sCD14, significantly induced SOST expression through the upregulation of both Wnt3a and Wnt5a and the downregulation of the b-catenin proteins (figure 1D). On the opposite, the combination of LPS and sCD14 downregulated SOST expression through the upregulation of ERK1/2 and b-catenin protein (figure 1D).Abstract OP0271 – Figure 1 A. immunoistochemical staining of ileal bacteria. Ten ileal samples of patients affected by SpA/IBD or IBD were stained for bacteria infiltration (upper panel). Lower (from left to right): Gram and LPS staining of the same samples. B. Analysis of ¡leal tight-junctions proteins expression. From the left to the right: Ten ileal samples of patients affected by SpA/IBD or IBD were stained for occludin (and claudin-1 and -4, data not shown); count of occludin positive cells and quantitative real-time-PCR of occludin (and claudin-1 and -4, data not shown) expression in the same ¡leal samples. C. analysls of I-FABP, LPS and sCD14 serum levels in SpA/IBD and IBD patients. ELISAs assays were carried out in 45 patients with axial and 40 patients with peripheral SpA/IBD, and compared with IBD or HC. D. Western-blot analysis of MG-63 osteoblast-cells. The MG-63 osteoblast-like cell line was stimulated with LPS ± sCD14 in vitro and then cells were harvested for western blot analisys. Semi-quantitative densitometric analysis of the protein bands was carried out on the blot (data not shown). Statistical analysis: Kruskal-Wallis analysis. ••p<0.01; ••••p<0.0001; if not reported: p non significant. Abbreviations. SpA/IBD: inflammatory bowel disease-associated spondyloarthritis; ax-SpA/IBD: axial SpA/IBD: peripheral SpA/IBD; IBD: Inflammatory bowel disease; HC: healthy controls; I-FABP: intestinal fatty acid binding protein; LPS: bacterial lypopolysaccaride; sCD14: solubie CD14; ERK 1/2: extracellular Signal-regulated Kinase-1/2. Wnt: wingless protein family: SOST: sclerostin. Conclusions The role of gut inflammation and microbial translocation in the onset of arthritis in IBD patients are still under investigation. We have demonstrated that in SpA/IBD there is a significant bacterial infiltration of the ileal tract, associated with the downregulation of tight-junctions’ proteins (occludin, claudin-1 and claudin-4) and epithelial damage, that cause microbial translocation and higher plasma levels of I-FABP, LPS, and sCD14. LPS and sCD14, thus, could trigger a complex systemic inflammatory response acting on several biochemical pathways, linking the immune system (anti-SOST-IgG) and the bone (SOST). Disclosure of Interest None declared

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.

Analysis of cell-cell junctions in human amnion and chorionic plate affected by chorioamnionitis

Chorioamnionitis is an acute inflammatory reaction associated with the premature rupture of the fetal membranes. It is caused mainly by invasion of bacteria from the vaginal tract that can penetrate the intact membranes and invade the amnion cavity and the decidua. Tight junctions (TJs) and adherent junctions (AJs) are intercellular junctions crucial for epithelia adhesion and permeability regulation in a wide variety of tissues and organs. Our aim is to investigate if TJ and AJ molecules are involved in human chorioamnionitis. We studied the protein expression (by immunohistochemistry and western blotting) and the mRNA levels (by RT-PCR) of some junction proteins such as Zonula Occludens-1 (ZO-1), occludin, VE-cadherin and β-catenin in fetal membranes from women with chorioamnionitis compared to those membranes derived from idiopathic pregnancies. Western blotting and immunohistochemical data established that occludin expression was decreased in amnion with chorioamnionitis compared to amnion from idiopathic pregnancies. Samples tested for ZO-1, VE-cadherin and β-catenin (proteins and mRNAs) showed no differences between idiopathic and pathological membranes. One of the most relevant results is the decrease of occludin in membranes with chorioamnionitis. Since we have previously demonstrated that some cytokines, particularly elevated in the chorioamnionitis, cause the disruption of TJs in placental villi, we suggest that the decrease of occludin in amnion may be the first change that leads to the rupture of the amniotic membrane in this pathology.

Expression of Trop2 in bladder cancer is modulated by miR125b: in vivo and in vitro analyses

Human trophoblastic cell surface antigen 2 (Trop-2) is a 40-kDa transmembrane glycoprotein, first identified as a cell surface marker for human trophoblast cells (1). Elevated expression of Trop-2 has been shown in several types of epithelial cancers and correlated with tumour aggressive and poor prognosis (2-3). The first aim of this study was to evaluate the variation of the Trop-2 expression in normal urothelium and urothelial bladder cancer. The immunohistochemical results showed an increase of Trop-2 levels in bladder cancer tissues with the increase of the severity of the pathology. Recent data identified Trop-2 as a target for miR-125b suggesting a pos sible role of miR-125b in the modulation of Trop-2 protein expression (4). The second aim was to verify if Trop-2 could be a target for miR-125b in bladder cells and to evaluate the possible role of miR-125b in the modulation of Trop-2 protein expression in normal bladder as well as in urothelial bladder cancer. In vitro we showed a contribution of miR-125b in deregulation of Trop-2 protein expression in a bladder cell line and we found that the expression of miR-125b was inversely correlated with the expression of Trop-2 protein on a cohort of bladder cancer tissues. We concluded to investigate in a larger population the use of Trop-2 and/or miR-125b as potential diagnostic markers in urothelial bladder cancer.

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.