Caffeine Inhibits EGF-Stimulated Trophoblast Cell Motility through the Inhibition of mTORC2 and Akt.

Impaired trophoblast invasion is associated with pregnancy disorders such as early pregnancy loss and preeclampsia. There is evidence to suggest that the consumption of caffeine during pregnancy may increase the risk of pregnancy loss; however, little is known about the direct effect of caffeine on normal trophoblast biology. Our objectives were to examine the effect of caffeine on trophoblast migration and motility after stimulation with epidermal growth factor (EGF) and to investigate the intracellular signaling pathways involved in this process. Primary first-trimester extravillous trophoblasts (EVT) and the EVT-derived cell line SGHPL-4 were used to study the effect of caffeine on EGF-stimulated cellular motility using time-lapse microscopy. SGHPL-4 cells were further used to study the effect of caffeine and cAMP on EGF-stimulated invasion of fibrin gels. The influence of caffeine and cAMP on EGF-stimulated intracellular signaling pathways leading to the activation of Akt were investigated by Western blot analysis. Caffeine inhibits both EGF-stimulated primary EVT and SGHPL-4 cell motility. EGF stimulation activates phosphatidylinositol 3-kinase, and Akt and caffeine inhibit this activation. Although cAMP inhibits both motility and invasion, it does not inhibit the activation of Akt, indicating that the effects of caffeine seen in this study are independent of cAMP. Further investigation indicated a role for mammalian target of rapamycin complex 2 (mTORC2) as a target for the inhibitory effect of caffeine. In conclusion, we demonstrate that caffeine inhibits EGF-stimulated trophoblast invasion and motility in vitro and so could adversely influence trophoblast biology in vivo

Endothelin-1 down-regulates matrix metalloproteinase 14 and 15 expression in human first trimester trophoblasts via endothelin receptor type B

$\textbf{STUDY QUESTION}$: Does endothelin-1 (ET-1) regulate matrix metalloproteinase (MMP) 14 and 15 production and invasion of human first trimester trophoblasts? $\textbf{SUMMARY ANSWER}$: ET-1 in pathophysiological concentrations down-regulates MMP14 and MMP15 expression via endothelin receptor (ETR) type B and decreases trophoblast migration and invasion. $\textbf{WHAT IS KNOWN ALREADY}$: MMP14 and MMP15 are involved in trophoblast invasion. Impairment of invasion has been linked to pregnancy complications such as pre-eclampsia (PE). ET-1 is up-regulated in PE. $\textbf{STUDY DESIGN, SIZE, DURATION}$: $\textit{In vitro}$ study using primary human trophoblasts from 50 first trimester placentas (gestational week 7-12). $\textbf{PARTICIPANTS/MATERIALS, SETTING, METHODS}$: Trophoblasts were cultured in the absence or presence of 10-100 nM ET-1. MMP14 and MMP15 mRNA and protein were quantified by RT-qPCR and Western blotting, respectively. Selective antagonists for ETRA (BQ-123) or ETRB (BQ-788) were used to identify ETR subtypes involved. Functional ET-1 effects were tested in first trimester chorionic villous explants and transwell invasion assays. The roles of tumor necrosis factor (TNF)-α (25 ng/ml) and oxygen (1%) in ET-1 regulation of MMP14 and 15 expression were assessed by Western blotting. $\textbf{MAIN RESULTS AND THE ROLE OF CHANCE}$: ET-1 down-regulated MMP14 and MMP15 mRNA (-21% and -26%, respectively, $\textit{P}$ < 0.05) and protein levels (-18% and -22%, respectively, $\textit{P}$ < 0.05). This effect was mediated via ETRB. ET-1 decreased trophoblast outgrowth in placental explants (-24%, $\textit{P}$ < 0.05) and trophoblast invasion (-26%, $\textit{P}$ ≤ 0.01). TNF-α enhanced ET-1 mediated MMP15 down-regulation (by 10%, $\textit{P}$ < 0.05), whereas hypoxia abolished the effect of ET-1 on both MMPs. $\textbf{LARGE SCALE DATA}$: N/A. $\textbf{LIMITATIONS, REASONS FOR CAUTION}$: Only primary trophoblasts were used in this study. Since trophoblast yield from first trimester placental material is limited, further aspects of MMP14 and 15 regulation could not be characterized. Other anti-invasive factors may be altered by ET-1 in trophoblasts and, thus, contribute to the reduced invasion, but have not been investigated. Oxygen levels similar to those found in the decidua (5-8% O2) were not analyzed in this study. $\textbf{WIDER IMPLICATIONS OF THE FINDINGS}$: ET-1 modifies placental function already during the first trimester of pregnancy, the time-window when the placental changes implicated in PE occur. Thus, our results improve the understanding of the placental mechanisms underlying trophoblast invasion and PE.The study was funded by the Oesterreichische Nationalbank (Anniversary Fund, project number: 14796) and the Herzfelder'sche Familienstiftung (to J.P.; number: 00685). AMM received funding from the Austrian Science Fund FWF (W1241) and the Medical University Graz through the PhD Program Molecular Fundamentals of Inflammation (DK-MOLIN)

Tumour invasiveness, the local and systemic environment and the basis of staging systems in colorectal cancer

background: The present study aimed to examine the relationship between tumour invasiveness (T stage), the local and systemic environment and cancer-specific survival (CSS) of patients with primary operable colorectal cancer. methods: The tumour microenvironment was examined using measures of the inflammatory infiltrate (Klintrup-Makinen (KM) grade and Immunoscore), tumour stroma percentage (TSP) and tumour budding. The systemic inflammatory environment was examined using modified Glasgow Prognostic Score (mGPS) and neutrophil:lymphocyte ratio (NLR). A 5-year CSS was examined. results: A total of 331 patients were included. Increasing T stage was associated with colonic primary, N stage, poor differentiation, margin involvement and venous invasion (P&lt;0.05). T stage was significantly associated with KM grade (P=0.001), Immunoscore (P=0.016), TSP (P=0.006), tumour budding (P&lt;0.001), and elevated mGPS and NLR (both P&lt;0.05). In patients with T3 cancer, N stage stratified survival from 88 to 64%, whereas Immunoscore and budding stratified survival from 100 to 70% and from 91 to 56%, respectively. The Glasgow Microenvironment Score, a score based on KM grade and TSP, stratified survival from 93 to 58%. conclusions: Although associated with increasing T stage, local and systemic tumour environment characteristics, and in particular Immunoscore, budding, TSP and mGPS, are stage-independent determinants of survival and may be utilised in the staging of patients with primary operable colorectal cancer

Increased angiogenic factor secretion by decidual natural killer cells from pregnancies with high uterine artery resistance alters trophoblast function.

STUDY QUESTION Are the concentrations of factors secreted by decidual natural killer (dNK) cells from pregnancies at high risk of poor spiral artery remodelling different to those secreted from pregnancies at low risk? SUMMARY ANSWER Expression levels of PLGF, sIL-2R, endostatin and angiogenin were significantly increased by dNK cells from high-risk pregnancies, and angiogenin and endostatin were found to alter trophoblast function. WHAT IS KNOWN ALREADY During early pregnancy, maternal uterine spiral arteries are remodelled from small diameter, low-flow, high-resistance vessels into larger diameter, higher flow vessels, with low-resistance. This change is essential for the developing fetus to obtain sufficient oxygen and nutrients. dNK cells have been implicated in this process. STUDY DESIGN, SIZE, DURATION dNK cells were isolated from first trimester terminations of pregnancies (obtained with local ethical approval) screened for normal- or high-resistance index, indicative of cases least (21%) likely to have developed pre-eclampsia had the pregnancy not been terminated (n = 18 each group). Secreted factors and the effects of these on the trophoblast cell line, SGHPL-4, were assessed in vitro. PARTICIPANTS/MATERIALS, SETTING, METHODS A multiplex assay was used to assess dNK cell-secreted factors. SGHPL-4 cell functions were assessed using time-lapse microscopy, 3D invasion assays, endothelial-like tube formation ability and western blot analysis. MAIN RESULTS AND THE ROLE OF CHANCE The expression levels of PLGF (P < 0.01), sIL-2R (P < 0.01), endostatin (P < 0.05) and angiogenin (P < 0.05) were significantly increased by dNK cells from high-risk pregnancies. Endostatin significantly decreased SGHPL-4 invasion (P < 0.05), SGHPL-4 tube formation (P < 0.05) and SGHPL-4 Aktser473 phosphorylation (P < 0.05). Angiogenin significantly decreased SGHPL-4 invasion (P < 0.05), but increased SGHPL-4 tube formation (P < 0.01) and decreased SGHPL-4 Aktser473 phosphorylation (P < 0.05). LIMITATIONS, REASONS FOR CAUTION The culture of dNK cells and protein concentrations in vitro may not fully represent the in vivo situation. Although SGHPL-4 cells are extravillous trophoblast derived, further studies would be needed to confirm the roles of angiogenin and endostatin in vivo. WIDER IMPLICATIONS OF THE FINDINGS The altered expression of secreted factors of dNK cells may contribute to pregnancy disorders associated with poor spiral artery remodelling. STUDY FUNDING/COMPETING INTEREST(S) This study was supported by the Wellcome Trust (project reference 091550). R.F. was a recipient of a PhD studentship from the Division of Biomedical Sciences, St. George's, University of London. The authors have no conflict of interests

Anspruch und Wirklichkeit in der universitären Lehrerinnen- und Lehrerbildung am Beispiel des Universitätsstandortes Wien

Anspruch und Wirklichkeit in der universitären Lehrerinnen- und Lehrerbildung am Beispiel des Universitätsstandortes Wien. Evaluation der allgemeinen pädagogischen Ausbildung und des Schul-praktikums. Schwerpunkt der Arbeit ist die Evaluation der allgemeinen pädagogischen Ausbildung und des Schulpraktikums im Rahmen des universitären Lehramtsstudiums im Wintersemester 200/01. Mittels Fragebögen wurden rund 40.000 Einzeldaten erhoben, ergänzt durch verbale Stellungnahmen. Das Sample umfasst rund 140 Studierende und ca. 80 BetreuungslehrerInnen aus dem Raum Ostösterreich. Die Ergebnisse dieser Befragung werden mit der Auswertung einer ähnlich konzipierten Studie aus dem Jahr 1991 verglichen. Es zeigt sich, dass durch die Weiterentwicklung und Neuorientierung des pädago-gischen Begleitstudiums wesentliche qualitative Verbesserungen erreicht werden konnten und dass der Stellenwert dieser Bereiche (allgemeine pädagogische Aus-bildung, Schulpraktikum) innerhalb des gesamten Lehramtsstudiums für die Studierenden deutlich gestiegen ist. Gleichzeitig werden konkrete Kritikpunkte und Problemzonen in der prinzipiellen Struktur, in der Durchführung der Lehrver-anstaltungen und im Bereich der Hochschuldidaktik herausgearbeitet. Deutlich wird auf die Notwendigkeit der Ausbildung einer theoriegestützten Refle-xionskompetenz, einer frühzeitigen Praxisbegegnung und einer reflektierten Aus-einandersetzung mit der Schulrealität während des Studiums deutlich. Diese notwendige Qualitätssteigerung kann und soll durch ein konkret formuliertes konsekutives Curriculum, durch die kontinuierliche Einbindung von Praxisphasen, durch begleitende Supervision und durch die Forcierung von Lehrveranstaltungen in seminaristischer Form erreicht werden. Der abschließende Gedanke der Arbeit lautet, dass LehrerInnen-Ausbildung und LehrerInnenbildung im umfassenden Sinne innerhalb des universitären Lehramts-studiums ermöglicht werden müssen. Eine Darstellung der Geschichte der universitären LehrerInnenbildung mit dem Schwerpunkt an der Wiener Universität, die Auseinandersetzung mit dem Begriff bzw. die versuchsweise Klärung des Begriffes Evaluation, die Diskussion des Theorie-Praxis-Verhältnisses und die Darlegung notwendiger Schritte in der universitären LehrerInnenbildung („Zukunftsperspektiven“) ergänzen die Arbeit bzw. runden die Arbeit ab. Die Erhebungsgrundlagen (Fragebögen, Textergänzungen Auswertungen) sind auf einer CD-Rom abgespeichert und der schriftlichen Arbeit beigelegt, um weitere detaillierte Forschungen zu ermöglichen.Demands, Claims and Bottom-Line Reality in University-Level Teacher Education at the University of Vienna Evaluation of general teacher education and practical teacher training The focus of this study is the evaluation of general teacher education as well as practical teacher training within the framework of university-level teacher training courses of study. The study took place in the first semester of the 2000/2001 university year. 40,000 pieces of data were collected by means of a questionnaire, complemented partially by extensive personal interviews and statements made by the participants in verbal form. The sample encompasses approximately 140 students together with about 80 qualified teachers from the eastern part of Austria who serve as supervisors. The results of this survey will be compared to the analysis of a similarly-conceived study which was implemented in 1991. The accompanying study demonstrates that the ongoing development and reorientation of general teacher training courses and practical teacher training have resulted in a significant qualitative improvement. Furthermore, their added value and status within the context of the overall teacher training courses of study leading to a university degree, as perceived by the students themselves, have clearly increased. At the same time, specific weaknesses, criticisms and problem areas have been defined - whether in the approach to teaching training, how courses and seminars are actually conducted or in relation to the field of teaching methodologies. This study points out that it is essential to provide students with practical training at an early stage, to ensure that they confront the day-to-day realities of working in schools throughout their university studies, and that they are given broad-based practical competence supported by theoretical know-how. This necessary boost in quality can be achieved by a variety of instruments: through a clearly-defined, evolving curriculum; the ongoing integration of practical training periods; hands-on supervision by qualified teachers and by encouraging the university to hold courses in the form of seminars. Finally, the study concludes that a complete and comprehensive training and education of teachers must be made possible within the framework of university-level courses of study. The evaluation is complemented by a comprehensive summary of the history of university-level teacher education, particularly at Vienna University, a discussion of the issue of evaluation in general, an analysis of the relationship between theory and practice as well as an attempt to define necessary steps to be taken ("future perspectives"). The fundamental basis for this study – questionnaire, text appendices, and evaluation – has been stored on a CD Rom which accompanies the written version. This is designed to allow for further detailed research to take place

Cholangiocytes derived from human induced pluripotent stem cells for disease modeling and drug validation.

The study of biliary disease has been constrained by a lack of primary human cholangiocytes. Here we present an efficient, serum-free protocol for directed differentiation of human induced pluripotent stem cells into cholangiocyte-like cells (CLCs). CLCs show functional characteristics of cholangiocytes, including bile acids transfer, alkaline phosphatase activity, γ-glutamyl-transpeptidase activity and physiological responses to secretin, somatostatin and vascular endothelial growth factor. We use CLCs to model in vitro key features of Alagille syndrome, polycystic liver disease and cystic fibrosis (CF)-associated cholangiopathy. Furthermore, we use CLCs generated from healthy individuals and patients with polycystic liver disease to reproduce the effects of the drugs verapamil and octreotide, and we show that the experimental CF drug VX809 rescues the disease phenotype of CF cholangiopathy in vitro. Our differentiation protocol will facilitate the study of biological mechanisms controlling biliary development, as well as disease modeling and drug screening.This work was funded by ERC starting grant Relieve IMDs (L.V., N.H.), the Cambridge Hospitals National Institute for Health Research Biomedical Research Center (L.V., N.H., F.S.), the Evelyn trust (N.H.) and the EU Fp7 grant TissuGEN (M.CDB.). FS has been supported by an Addenbrooke’s Charitable Trust Clinical Research Training Fellowship and a joint MRC-Sparks Clinical Research Training Fellowship.This is the author accepted manuscript. The final version is available from Nature Publishing Group via http://dx.doi.org/10.1038/nbt.327

Evaluation of human first trimester decidual and telomerase-transformed endometrial stromal cells as model systems of in vitro decidualization

<p>Abstract</p> <p>Background</p> <p>Decidualization, the differentiation process of maternal uterine stromal cells into secretory decidual cells, is a prerequisite for successful implantation and progression of pregnancy. For in vitro differentiation mostly primary human endometrial stromal cells (HESC) isolated from uterine samples after hysterectomy for benign gynaecological diseases are utilised. However, a continuous supply of endometrial tissue is often lacking. Hence, we analysed whether cultivated human decidual stromal cells (HDSC) prepared from first trimester pregnancy terminations may represent an alternative model system for in vitro decidualization. Moreover, based on the expression of critical marker genes these cells were compared to a previously established endometrial stromal cell line during in vitro differentiation.</p> <p>Methods</p> <p>HDSC isolated from decidual tissue attached to first trimester placentae, and telomerase-transformed human endometrial stromal cells (THESC) were characterised by immunofluorescence and differentiated in vitro using either cyclic adenosine monophosphate (cAMP) and/or estrogen (E2)/progesterone (P4). Proliferation was measured by analyzing cumulative cell numbers. Expression of mRNAs encoding progesterone receptor (PR), prolactin (PRL), insulin-like growth factor binding protein-1 (IGFBP1), and Dickkopf-1 (DKK1) was evaluated using quantitative PCR after 3, 6, 9 and 12 days of in vitro differentiation. PRL and IGFBP-1 protein expression was investigated by enzyme-linked immunosorbent assay (ELISA) and Western blotting, respectively. Furthermore, forkhead box O1A (FOXO1A), a critical transcription factor in decidualization, was analysed by immunofluorescence and Western blotting at two different time points of differentiation.</p> <p>Results</p> <p>Treatment with cAMP provoked morphological changes and growth arrest of THESC and HDSC, the latter showing loss of cells after 6 days of treatment. E2P4 stimulation did neither affect cell morphology nor proliferation of THESC and HDSC. Upon cAMP stimulation PR mRNA was suppressed in HDSC but not in THESC, whereas E2P4 did not alter transcript levels in both cell types. Protein expression of PR-A and PR-B was detectable in HDSC and diminished under cAMP, whereas THESC failed to produce the nuclear receptors. Supplementation of cAMP induced mRNA and protein expression of PRL and IGFBP-1 in both cell types at day 3, 6, 9, and 12 of treatment. In HDSC stimulation with E2P4 increased PRL and IGFBP-1 mRNA and protein production, whereas hormone treatment did not induce the two factors in THESC. E2P4 increased DKK1 mRNA at all time points in HDSC and cAMP provoked induction at day 9 and 12 of differentiation. In contrast, cAMP suppressed DKK1 mRNA in THESC, whereas E2P4 was ineffective. In both cell types combined treatments with cAMP and E2P4 provoked higher expression levels of PRL and IGFBP1 mRNA and protein as compared to cAMP stimulation alone. FOXO1A protein and its nuclear abundance were increased by cAMP in both cell types. However, reduction of its nuclear localisation upon E2P4 treatment could only be observed in HDSC.</p> <p>Conclusion</p> <p>Both HDSC and THESC may represent suitable model systems for cAMP-dependent in vitro decidualization. Since cAMP decreases cell viability of HDSC after 6 days of incubation, this substance should be preferentially used in short-term experiments. Progesterone treatment of THESC might not be applicable since these cells lack progesterone response and PR protein. In contrast, stimulation of PR-expressing HDSC with E2P4 or cAMP/E2P4 may represent an appropriate protocol for human in vitro decidualization inducing and maintaining expression of critical marker genes in a time-dependent manner.</p

Human trophoblast invasion: new and unexpected routes and functions

Stem cells & developmental biolog

Psychophysiological stress response after a 6-week Mindful Self-Compassion training in psychiatric rehabilitation inpatients: a randomized post-test only study

Objectives: Mindfulness-based interventions (including self-compassion interventions) are effective in improving stress management at psychological and physical levels. Mindful Self-Compassion (MSC) is a newly developed program particularly aimed at increasing self-compassion. The main objective of this study was to determine whether the psychophysiological stress response during a social-evaluative speaking task differs in inpatients participating in the MSC or the Progressive Muscle Relaxation (PMR) program at the end of their 6-week psychiatric rehabilitation stay (i.e., post-test only design). Method: Data from 50 inpatients (25 MSC, 25 PMR, 35 female) aged 19 to 76 years (M = 47.22, SD = 12.44) were analyzed in terms of psychophysiological stress response. For this purpose, heart rate variability, heart rate, and blood pressure were assessed together with several psychometric variables: positive and negative affect (PANAS), subjective stress perception (Visual Analog Scale), self-compassion (Self-Compassion Scale), cognitive reappraisal and suppression (Emotion Regulation Questionnaire), psychological distress (Brief Symptom Inventory-18), and appraisal and rumination (selected items). Results: After correction for alpha inflation no differences in the psychophysiological stress response and psychometric parameters between the MSC and PMR group were found. Discussion: In general, our results indicate that MSC is not superior to PMR training. However, more research with clinical randomized controlled trials investigating larger samples are needed to further affirm these initial findings

Vitamin D modulates biliary fibrosis in ABCB4-deficient mice

PURPOSE: Impaired vitamin D receptor signaling represents an aggravating factor during liver injury, and recent studies suggest that vitamin D might exert a protective role in chronic hepatobiliary diseases. We hypothesized that vitamin D supplementation would ameliorate liver fibrosis in ATP-binding cassette transporter B4 knockout (Abcb4(−/−)) mice as a preclinical model of sclerosing cholangitis. METHODS: Abcb4(−/−) and wild-type mice were fed a regular chow diet (600 IU vitamin D/kg food) or diets with lower (100 IU/kg) and higher (2,400 IU/kg) vitamin D concentrations for 12 weeks. Serum 25-hydroxyvitamin D concentrations were measured by chemiluminescence immunoassays. Liver injury and biliary fibrosis were assessed by liver enzyme activities, histopathology and hepatic collagen contents. Hepatic mRNA expression of markers for fibrosis, vitamin D and bile acid metabolism were analyzed by quantitative PCR. RESULTS: Different vitamin D concentrations were observed depending on genotype and diet group, with Abcb4(−/−) mice on the control diet showing lower vitamin D concentrations compared to wild-type mice. Abcb4(−/−) animals on the low vitamin D diet demonstrated the most advanced liver fibrosis and highest hepatic collagen contents. Feeding Abcb4(−/−) mice a high vitamin D diet enriched serum vitamin D levels, lowered liver enzyme activities, altered expression levels of profibrogenic genes and ameliorated, in part, liver injury. CONCLUSIONS: This is the first report to demonstrate that fibrogenesis in the established Abcb4(−/−) model is influenced by vitamin D supplementation. Since vitamin D modulates sclerosing cholangitis in vivo, we speculate that sufficient vitamin D intake might improve liver damage and induce antifibrotic effects in chronic cholestasis in humans. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s12072-014-9548-2) contains supplementary material, which is available to authorized users