Executive summary. Expert consensus statement on the diagnosis and treatment of paediatric pulmonary hypertension. The European Paediatric Pulmonary Vascular Disease Network, endorsed by ISHLT and DGPK

The European Paediatric Pulmonary Vascular Disease (PVD) Network is a registered, non-profit organisation that strives to define and develop effective, innovative diagnostic methods and treatment options in all forms of paediatric pulmonary hypertensive vascular disease, including specific forms such as pulmonary arterial hypertension (PAH)-congenital heart disease, pulmonary hypertension (PH) associated with bronchopulmonary dysplasia, persistent PH of the newborn, and related cardiac dysfunction. Methods The writing group members conducted searches of the PubMed/MEDLINE bibliographic database (1990-2015) and held five face-to-face meetings with votings. Clinical trials, guidelines, and reviews limited to paediatric data were searched using the terms 'pulmonary hypertension' and 5-10 other keywords, as outlined in the other nine articles of this special issue. Class of recommendation (COR) and level of evidence (LOE) were assigned based on European Society of Cardiology/American Heart Association definitions and on paediatric data only, or on adult studies that included >10% children. Results A total of 9 original consensus articles with graded recommendations (COR/LOE) were developed, and are summarised here. The topics included diagnosis/monitoring, genetics/biomarker, cardiac catheterisation, echocardiography, cardiac magnetic resonance/chest CT, associated forms of PH, intensive care unit/ventricular assist device/lung transplantation, and treatment of paediatric PAH. Conclusions The multipaper expert consensus statement of the European Paediatric PVD Network provides a specific, comprehensive, detailed but practical framework for the optimal clinical care of children with PH

Development of innovative ecotoxicological evaluation methods as fertility tests and alternative procedures to animal testing

Titelblatt und Inhalt 1\. Einleitung 6 2\. Material und Methoden 19 Teil I: Spezielle Versuche zur Motilität, Kryokonservierung und Befruchtung 27 Teil II: Ökotoxikologische Versuche 36 3\. Ergebnisse Teil I: Motilität, Kryokonservierung und Befruchtung 56 Teil II: Ökotoxikologische Versuche 79 4\. Diskussion Teil I: Motilität, Kryokonservierung und Befruchtung 103 Teil II: Ökotoxikologische Versuche 123 5\. Zusammenfassung / Summary 147 6\. Literaturverzeichnis 154 AnhangZiel der vorliegenden Dissertation war die Entwicklung eines Biotestverfahrens mit Fischspermien zur Bewertung der Reproduktionstoxizität umweltrelevanter Chemikalien und Abwasserproben. Fischspermien wurden ausgewählt, da sie ökologisch relevant und im Vergleich zu Keimzellen anderer Wasserorganismen gut verfügbar sind. Die uneingeschränkte Beweglichkeit (Motilität) der Spermien ist die Hauptvoraussetzung für eine erfolgreiche Befruchtung und damit ein Messparameter für ihre Fertilität. Die Motilität ist mit Hilfe von computergestützter Videomikrographie (CASA = computer assisted sperm analysis) gut und objektiv messbar. Außer der Motilität wurden der ATP-Gehalt und die Membranintegrität durch DNA-Färbung als alternative Endpunkte für den Test untersucht. Nach einer Reihe von Voruntersuchungen an diversen limnischen und marinen Fischarten blieben zwei Arten übrig, deren Sperma den Qualitätsanforderungen gerecht werden konnten: Karpfen (Cyprinus carpio L.) und Sterlet (Acipenser ruthenus L.). Neben der Analyse der grundsätzlichen Motilitätseigenschaften, wurde das Sperma kryokonserviert, um die ganzjährige Verfügbarkeit von qualitativ einheitlichem Zellmaterial sicherzustellen. Dabei wurden für beide Arten die Kryomittel und Einfriermethoden so optimiert, um nach dem Auftauen möglichst hohe Motilitätsraten zu erhalten. In Befruchtungsversuchen konnte gezeigt werden, dass das so konservierte Sperma in der Lage ist, frische Eier zu befruchten und einen hohen Prozentsatz an normal entwickelten Embryos zu produzieren. Sperma beider Arten konnte durch Verdünnung in geeigneten Immobilisierungslösungen über mehrere Stunden im Eisbad aufbewahrt und anschließend ohne nennenswerten Qualitätsverlust aktiviert werden. Diese Lösungen wurden vor allem als Inkubationsmedium für die Spermazellen erarbeitet. Testsubstanzen und Abwasserproben wurden darin gelöst bzw. verdünnt und anschließend mit der Spermaprobe vermischt. Die Exposition wurde für 4 h im Eisbad vorgenommen. In den ökotoxikologischen Versuchen wurden hauptsächlich vier Substanzen auf Karpfenspermien getestet: Cadmium, 4-Nonylphenol, Crotonaldehyd und Rotenon. Je Testsubstanz wurden mehrere voneinander unabhängige Testreihen durchgeführt, um die Konzentrations-Wirkungs-Beziehungen und EC50-Werte zu ermitteln. Zusätzlich wurde eine Abwasserprobe aus der chemischen Industrie eingesetzt. Die Testmethoden für frische und kryokonservierte Proben wurden so aneinander angeglichen, dass die Ergebnisse vergleichbar sind. Der Endpunkt Motilität konnte aufgrund der Instabilität der Proben nach dem Auftauen jedoch nur mit frischen Spermien durchgeführt werden. Dieser Endpunkt reagierte im Vergleich zu den anderen Endpunkten auf alle vier Schadstoffe sowie auf die Abwasserprobe am empfindlichsten, gefolgt vom ATP-Gehalt. Mit der DNA- Färbemethode zum Endpunkt Membranintegrität konnten lediglich mit zwei der vier Testsubstanzen sinnvolle Konzentrations-Wirkungs-Beziehungen ermittelt werden. Die kryokonservierten Zellen reagierten in fast allen Fällen empfindlicher als die nativen Spermien. Beim Vergleich der Ergebnisse mit entsprechenden Literaturangaben zu EC50- und G-Werten aus etablierten DIN- Verfahren wie dem Fisch-, dem Daphnien- und dem Algentest zeigte sich, dass der Endpunkt Motilitätsrate ähnlich empfindlich reagiert bzw. sogar deutlich niedrigere EC50-Werte produziert. Dieses Ergebnis wird besonders durch den mit 90 ng/l enorm niedrigen EC50-Wert für Rotenon und durch die sehr empfindliche Reaktion auf das Abwasser unterstrichen.The aim of the following dissertation was the development of an ecotoxicological test method using fish sperm for evaluating the reproduction toxicity of environmentally relevant chemicals and sewage samples. Fish sperm were selected because they are ecologically relevant and are, compared to the gametes of other water organisms, easily attainable. The unrestricted motility of the sperm is a major precondition for successful fertilization. Motility can be well and objectively measured by means of computer assisted sperm analysis (CASA). Besides motility, ATP content and membrane integrity through DNA-staining were measured as alternative endpoints for the biotest. Following a series of preliminary tests on fresh- and saltwater fish species, two remained whose sperm meet the established quality criteria: carp (Cyprinus carpio L.) and sterlet (Acipenser ruthenus L.). In addition to the analysis of fundamental motility characteristics sperm were cryopreserved in order to insure the year-round availability of qualitatively uniform cell material. For both species the preservation media and freezing methods were optimized to maintain the highest possible motility rates after thawing. In fertilization trials it was shown that such cryopreserved sperm was capable of fertilizing fresh eggs and producing a high percentage of normally developed embryos. The use of appropriate immobilizing solutions allowed sperm samples of both species to be immobilized for several hours and eventually re-activated without any noteworthy loss in quality. These solutions were prepared primarily as incubation media for the sperm cells. Test substances and sewage samples were diluted/thinned in the solutions and then mixed with the respective sperm sample. The exposition was set at four hours on crushed ice. In the ecotoxicological experiments mainly four substances were tested on carp sperm: cadmium, 4-nonylphenol, crotonaldehyde, and rotenone. For each substance several independent test series were conducted in order to determine the resulting concentration-effect relationships and EC50-values. Additionally, a sewage sample derived from the chemical industry was examined. The test methods for native and cryopreserved sperm were attuned to each other, such that the results could be compared. The endpoint motility rate could be performed only with native sperm, due to the storage sensitivity of the thawed sperm samples. Compared to the other endpoints, motility rate reacted most sensitively to all four toxins as well as to the sewage sample, followed by ATP content. The DNA-staining method used for the endpoint membrane integrity indicated in only two out of four test substances credible concentration-effect relationships. The cryopreserved cells employed in both tests for ATP content and membrane integrity reacted in nearly all cases more sensitively than the native sperm. A comparison of these results with related reports in the literature on EC50- and G-values from established DIN procedures such as the fish, daphnia, and algae tests shows that the endpoint motility rate reacts in a similarly sensitive way, or produces even remarkably lower EC50-values. This conclusion is especially emphasized by the very low EC50-values for rotenone of 90 ng/l and the very sensitive reaction to the sewage sample

Upregulation of miR-17-92 cluster is associated with progression and lymph node metastasis in oesophageal adenocarcinoma

The occurrence of lymph node metastasis (LNM) and depth of tumour infiltration are significant prognostic factors in oesophageal adenocarcinoma (OAC), however no reliable prognostic biomarkers have been established so far. Aim of this study was to characterize microRNAs (miRs) of OAC patients, who primarily underwent oesophagectomy, in order to identify specific alterations during tumour progression and LNM. MicroRNA array-based quantification analysis of 754 miRs, including tumour specimens of 12 patients with pT2 OAC from three different centres (detection group), was performed. We identified miR-17, miR-19a/b, miR-20a, and miR-106a, showing the best predictive power for LNM. These miRs were validated by quantitative real time-PCR (qRT-PCR) in 43 patients with different tumour stages (pT1: n = 21; pT2: n = 12 and pT3: n = 10) (training group) (p < 0.05), demonstrating that increasing levels of identified miRs were associated with advanced depth of tumour infiltration. These findings were verified in another independent group of 46 pT2 OAC patients (validation group). Quantitative RT-PCR analysis of the miR-panel confirmed these results except for miR-19a (p < 0.05 each). Logistic regression analysis identified miR-17 and miR-20a (p = 0.025 and p = 0.022, respectively) to be independent variables for prediction of LNM. The mathematical prediction model was used in the validation group, and the estimated prognosis was compared to the actual postsurgical follow-up. This comprehensive data demonstrated the importance of miR-17-92 cluster and miR-106a for progression as well as LNM in OAC indicating that those might be feasible prognostic biomarkers

2019 updated consensus statement on the diagnosis and treatment of pediatric pulmonary hypertension: The European Pediatric Pulmonary Vascular Disease Network (EPPVDN), endorsed by AEPC, ESPR and ISHLT

The European Pediatric Pulmonary Vascular Disease Network is a registered, non-profit organization that strives to define and develop effective, innovative diagnostic methods and treatment options in all forms of pediatric pulmonary hypertensive vascular disease, including pulmonary hypertension (PH) associated with bronchopulmonary dysplasia, PH associated with congenital heart disease (CHD), persistent PH of the newborn, and related cardiac dysfunction. The executive writing group members conducted searches of the PubMed/MEDLINE bibliographic database (1990-2018) and held face-to-face and web-based meetings. Ten section task forces voted on the updated recommendations, based on the 2016 executive summary. Clinical trials, meta-analyses, guidelines, and other articles that include pediatric data were searched using the term "pulmonary hypertension" and other keywords. Class of recommendation (COR) and level of evidence (LOE) were assigned based on European Society of Cardiology/American Heart Association definitions and on pediatric data only, or on adult studies that included >10% children or studies that enrolled adults with CHD. New definitions by the World Symposium on Pulmonary Hypertension 2018 were included. We generated 10 tables with graded recommendations (COR/LOE). The topics include diagnosis/monitoring, genetics/biomarkers, cardiac catheterization, echocardiography, cardiac magnetic resonance/chest computed tomography, associated forms of PH, intensive care unit/lung transplantation, and treatment of pediatric PH. For the first time, a set of specific recommendations on the management of PH in middle- and low-income regions was developed. Taken together, these executive, up-to-date guidelines provide a specific, comprehensive, detailed but practical framework for the optimal clinical care of children and young adults with PH.status: publishe

2019 updated consensus statement on the diagnosis and treatment of pediatric pulmonary hypertension : The European Pediatric Pulmonary Vascular Disease Network (EPPVDN), endorsed by AEPC, ESPR and ISHLT

The European Pediatric Pulmonary Vascular Disease Network is a registered, non-profit organization that strives to define and develop effective, innovative diagnostic methods and treatment options in all forms of pediatric pulmonary hypertensive vascular disease, including pulmonary hypertension (PH) associated with bronchopulmonary dysplasia, PH associated with congenital heart disease (CHD), persistent PH of the newborn, and related cardiac dysfunction. The executive writing group members conducted searches of the PubMed/MEDLINE bibliographic database (1990–2018) and held face-to-face and web-based meetings. Ten section task forces voted on the updated recommendations, based on the 2016 executive summary. Clinical trials, meta-analyses, guidelines, and other articles that include pediatric data were searched using the term “pulmonary hypertension” and other keywords. Class of recommendation (COR) and level of evidence (LOE) were assigned based on European Society of Cardiology/American Heart Association definitions and on pediatric data only, or on adult studies that included >10% children or studies that enrolled adults with CHD. New definitions by the World Symposium on Pulmonary Hypertension 2018 were included. We generated 10 tables with graded recommendations (COR/LOE). The topics include diagnosis/monitoring, genetics/biomarkers, cardiac catheterization, echocardiography, cardiac magnetic resonance/chest computed tomography, associated forms of PH, intensive care unit/lung transplantation, and treatment of pediatric PH. For the first time, a set of specific recommendations on the management of PH in middle- and low-income regions was developed. Taken together, these executive, up-to-date guidelines provide a specific, comprehensive, detailed but practical framework for the optimal clinical care of children and young adults with PH