Contribution of activin signals to progression of malignant pleural mesothelioma and evaluation of therapeutic implications

Hintergrund: Das maligne Pleuramesotheliom (MPM) ist ein Asbest-bedingter bösartiger Tumor mit häufig auftretender Resistenz gegen Chemo- und Strahlentherapie. Wachstumsfaktoren aus der Aktivin Superfamilie spielen beim malignen Wachstum von verschiedenen Tumorerkrankungen, einschließlich dem Nicht-Kleinzelligen Bronchialkarzinom und Ösophaguskarzinom eine wichtige Rolle. Das Ziel dieser Studie war es zu untersuchten, welche Rolle Activin Signale in MPM Zellmodellen spielen und inwieweit diese zu Wachstum und Migration beitragen. Methoden: Die Expression von Activin βA, βB, βC und βE (kodiert durch die Gene INHBA, INHBB, INHBC and INHBE), Aktivin-Rezeptoren (ACVR2, ACVR2B, ALK4 und ALK7) und Aktivin-Antagonisten wurde in 9 MPM Zelllinien und in nicht-malignen Mesothelzellen mittels RT- und qRT-PCR untersucht. Die Aktivin A Expression wurde weiters mittels Immunhistochemie in Paraffin-eingebetteten MPM-Gewebeproben untersucht. Zur funktionellen Analyse von Aktivin Signalen wurden MPM Zelllinien exogen mit Aktivin A, Aktivin-Rezeptor-Inhibitoren und INHBA-regulierenden siRNAs behandelt. Die Ermittlung der Zellproliferation erfolgte durch MTT und Clonogenic Assays und Zellmigration wurde durch Scratch und Transwell Assays ermittelt. Die Phosphorylierung von SMAD2 wurde mittels Western Blot untersucht als Nachweis für die Aktivierung des kanonischen Aktivin/TGF-β Signalweges. Ergebnisse: Die mit PCR durchgeführten Expressionsanalysen zeigten eine hohe Expression von Aktivin A und Aktivin-Rezeptoren in den meisten Zelllinien im Vergleich zu nicht malignen Mesothelzellen. Immunhistochemisch konnte in Gewebeproben von MPM Patienten eine intensive zytoplasmatische Färbung der Tumorzellen gezeigt werden. Die Behandlung mit exogenem Aktivin A führte zu einer Phosphorylierung von SMAD2 in allen Zelllinien, was auf die Funktionalität der Aktivin-Signalachse in MPM Zelllinien schließen lässt. Im Gegensatz zur humanen Leberkrebszelllinie HepG2, die durch eine Behandlung mit Aktivin A inhibiert wird, konnte die Proliferation von Mesotheliom-Zelllinien durch Behandlung mit Aktivin A stimuliert werden, wie MTT Assays und Clonogenic Assays zeigten. Eine Behandlung mit zwei verschiedenen Kinase-Inhibitoren für Aktivin-Rezeptoren (SB-431542, A8301) führte hingegen zu einer klaren Inhibierung der Proliferation, Klonogenität und Migration von Mesotheliom-Zellen. Da diese Kinase-Inhibitoren jedoch nicht spezifisch für Aktivin-Rezeptoren sind und zusätzlich auch auf TGF-β Rezeptoren wirken, wurde die Expression von INHBA zusätzlich durch siRNA-Oligonukleotide reduziert. Wie erwartet wurde durch die Transfektion mit siRNA spezifisch für Aktivin A, jedoch nicht durch Scrambled Kontroll-siRNA die Aktivin A Expression reduziert und die Zellproliferation geschwächt. Abschließend wurde der Einfluß von Trichostatin A und 5-Azacytidine als Einzelsubstanzen und als Kombination auf die Genexpression von INHBA getestet, allerdings konnte kein klarer Trend bezüglich einer Erhöhung oder Reduktion der Expression festgestellt werden. Schlussfolgerung: Diese Daten zeigen, dass deregulierte Aktivin A Expression zum malignen Phänotyp von Mesotheliom-Zellen beiträgt und dass Aktivin-Signale als neues therapeutisches Target weiter untersucht werden sollten.Background: Malignant pleural mesothelioma (MPM) is an asbestos-related malignancy characterized by frequent resistance to chemo- and radiotherapy. Growth factors of the activin family are involved in malignant growth in several tumor types including non-small cell lung cancer (NSCLC) and esophageal cancer. The aim of the present study was to investigate activin signals in MPM cell models and their contribution to malignant growth and spreading. Methods: The expression of the activin βA, βB, βC and βE subunits (encoded by the genes INHBA, INHBB, INHBC, INHBE), activin receptors (ACVR2, ACVR2B, ALK4 and ALK7) and activin antagonizing factors was analyzed in a panel of 9 MPM cell lines and in non-malignant mesothelial cells by RT- and qRT-PCR. Activin A expression was also analyzed by immunohistochemistry in paraffin embedded tissues. For functional analysis of activin signals, MPM cell lines were treated with exogenous activin A, activin receptor inhibitors or INHBA-targeting siRNA. Cell growth was assessed by MTT and clonogenic growth assays and cell migration by scratch and transwell assays. Phosphorylation of SMAD2 was analyzed by Western blotting as readout for activation of the canonical activin/TGF-β signaling pathway. Results: Expression data in mesothelioma cell models obtained by real-time PCR revealed high expression of activin-βA and activin receptors in most cell lines compared to non-malignant mesothelial cells. Likewise, immunohistochemistry in paraffin embedded tissue sections of MPM patients showed intense cytoplasmic staining for activin A in the tumor cells of a subset of the cases analyzed. Treatment with exogenous activin A induced SMAD2 phosphorylation in all cell lines tested, thus demonstrating functionality of the activin signaling axis in mesothelioma cell models. In contrast to the human hepatocarcinoma cell line HepG2, which in agreement with previous reports was inhibited, proliferation of mesothelioma cell models was stimulated by activin A as observed by MTT assays and clonogenic assays. Treatment with two different kinase inhibitors of activin receptors (SB-431542, A8301) in contrast, clearly inhibited mesothelioma cell proliferation and clonogenicity as well as migration. As kinase inhibitors are not absolutely specific for activin receptors and also co-target TGF-β receptors, INHBA was also silenced with siRNA oligonucleotides. As expected, transfection with INHBA-targeting siRNA but not scrambled control siRNA decreased activin-βA expression level and impaired cell proliferation. Finally, the influence of Trichostatin A and 5-azacytidine on the expression of INHBA was tested as single agents, as well as in combination, but no clear trend towards up- or down-regulation could be observed. Conclusions: The data generated during this investigation clearly suggest that deregulated activin A signaling contributes to the malignant phenotype of mesothelioma cells and might therefore represent a valuable candidate for therapeutic interference

Impact of Short-Term Isoflavone Intervention in Polycystic Ovary Syndrome (PCOS) Patients on Microbiota Composition and Metagenomics

Background: Polycystic ovary syndrome (PCOS) affects 5–20% of women of reproductive age worldwide and is associated with disorders of glucose metabolism. Hormone and metabolic signaling may be influenced by phytoestrogens, such as isoflavones. Their endocrine effects may modify symptom penetrance in PCOS. Equol is one of the most active isoflavone metabolites, produced by intestinal bacteria, and acts as a selective estrogen receptor modulator. Method: In this interventional study of clinical and biochemical characterization, urine isoflavone levels were measured in PCOS and control women before and three days after a defined isoflavone intervention via soy milk. In this interventional study, bacterial equol production was evaluated using the log(equol: daidzein ratio) and microbiome, metabolic, and predicted metagenome analyses were performed. Results: After isoflavone intervention, predicted stool metagenomic pathways, microbial alpha diversity, and glucose homeostasis in PCOS improved resembling the profile of the control group at baseline. In the whole cohort, larger equol production was associated with lower androgen as well as fertility markers. Conclusion: The dynamics in our metabolic, microbiome, and predicted metagenomic profiles underline the importance of external phytohormones on PCOS characteristics and a potential therapeutic approach or prebiotic in the future

Gene expression profiling in whole blood samples from Polycystic Ovary Syndrome (PCOS) patiens compared to healthy women

Das polyzystische Ovarien-Syndrom (PCOS) stellt die häufigste endokrine Erkrankung bei Frauen im reproduktionsfähigen Alter dar. Die genauen pathophysiologischen und geneti-schen Hintergründe dieser Erkrankung sind bis heute ungeklärt. Mononukleäre Blutzellen stellen eine leicht zugängliche Quelle für humanes Zellmaterial dar. Bisher wurden diese Zel-len jedoch kaum genutzt um Informationen über eine mögliche deregulierte Genexpression bei PCOS-Patientinnen zu erhalten. Ziel der Studie: Ziel dieser Studie war es, eine umfassende Genexpressionsanalyse in RNA aus Vollblut-Proben von Patientinnen mit PCOS und gesunden Frauen durchzuführen. Material und Methoden: Insgesamt wurden 12 Patientinnen mit diagnostiziertem PCOS und 11 gesunde Probandin-nen in die Studie eingeschlossen. Das PAXgene RNA Blood System (BD, PreAnalytix) wurde zur Gewinnung von qualitativ hochwertiger RNA aus Vollblut verwendet. Zahlreiche Gene, die in die Regulation der Reproduktionsfähigkeit, Glukose-Metabolismus und Steroidhor-mon-Synthese (FSH-Rezeptor, LH-Rezeptor, Kisspeptin, Kisspeptin-Rezeptor, Androgen-Rezeptor, Östrogen-Rezeptor alpha, AMH-Rezeptor, CYP11A1, CYP17A1, CYP19A1, FGF21, StAR, SRD5A1, SRD5A2, Follistatin, IGF-1, Insulin-Rezeptor, Leptin, Leptin-Rezeptor, Cheme-rin, RBP4, Visfatin, Inhibin alpha) involviert sind, wurden mittels quantitativer real-time PCR untersucht. Ergebnisse: Patientinnen mit PCOS zeigten eine signifikant höhere Expression an CYP19A1 (p=0,029), StAR (p=0,002), visfatin (p=0,0281) und KISS1R (p=0,029) in mononukleären Blutzellen im Vergleich zu gesunden Frauen. Es wurde keine Korrelation mit metabolischen und biochemi-schen Parametern gefunden. Schlussfolgerung: In PCOS Patientinnen wurde ein stark verändertes Genexpressionsprofil in mononukleären Blutzellen gefunden, was auf eine Involvierung dieser Zellen in die pathophysiologischen Hintergründe des PCOS hindeuten könnte. Weitere Studien sind jedoch von Nöten.The polycystic ovary syndrome (PCOS) represents the most common endocrine disorder in women of reproductive age. However, the pathophysiologic and genetic background of this disorder still remain to be elucidated. Peripheral mononuclear blood cells are an easy-accessible source for gene expression analysis in human cells. Nevertheless, these cells have hardly been used for assessing information about deregulations in gene expression levels in PCOS patients. Aim of this Study: The aim of this study was to perform a comprehensive gene expression analysis in RNA iso-lated from whole blood samples of PCOS patients and healthy women. Materials and Methods: Altogether, 12 patients diagnosed with PCOS and 11 healthy study participants were includ-ed in this study. The PAXgene RNA Blood System (BD, PreAnalytix) was used for the isolation of high-quality RNA from whole blood samples. Several genes known to be involved in the regulation of fertility and reproduction, glucose metabolism and steroid hormone synthesis (FSH receptor, LH receptor, kisspeptin, kisspeptin receptor, androgen receptor, estrogen receptor alpha, AMH receptor, CYP11A1, CYP17A1, CYP19A1, FGF21, StAR, SRD5A1, SRD5A2, follistatin, IGF-1, insulin receptor, leptin, leptin receptor, chemerin, RBP4, visfatin, inhibin alpha) were analysed via quantitative real-time PCR. Gene expression levels were correlated with biochemical and metabolic parameters. Results: Patients with PCOS showed a significant higher expression of CYP19A1 (p=0.029), StAR (p=0.002), visfatin (p=0.0281) und KISS1R (p=0.029) in peripheral mononuclear blood cells compared to healthy women. No correlations with metabolic or biochemical parameters were found. Conclusion: In PCOS patients, a strongly deregulated gene expression profile in peripheral blood mono-nuclear cells was found, suggesting an involvement of these cells in the pathophysiologic background of this disorder. Further studies in this area are needed.Julia MünzkerZsfassung in dt. und engl. SpracheGraz, Univ., Masterarb., 2015(VLID)489534

Performance of Marmoset Monkeys as Embryo Donors Is Reflected by Different Stress-Related Parameters

Non-human primates (NHPs) serve as embryo donors for embryo collection in order to mimic genetic diseases in humans by genetic modification. Reproductive health of the embryo donors is crucial, and chronic distress needs to be avoided. Embryo retrieval rates (ERR), anti-Müllerian hormone (AMH) concentrations, cortisol levels, and body weight fluctuations were assessed as markers for fertility and distress. With regard to successful embryo retrievals (total n = 667), the animals were either used for extended periods (long-term group; LTG) or only for short periods (short-term group; STG). Retrospective evaluation expectedly showed that animals in the LTG had a higher ERR than animals in the STG (p p = 0.0002). High ERR were associated with high AMH and low cortisol levels, and minimal body weight fluctuations following anesthesia, indicating a superior ability of the LTG animals to handle distress. We conclude that the long-term experimental use of marmosets does not impair their fertility or health status per se, supporting the view that animal reuse can be in accordance with the 3R-principle, implying reduction, replacement, and refinement in animal experimentation

Impact of Short-Term Isoflavone Intervention in Polycystic Ovary Syndrome (PCOS) Patients on Microbiota Composition and Metagenomics

Background: Polycystic ovary syndrome (PCOS) affects 5–20% of women of reproductive age worldwide and is associated with disorders of glucose metabolism. Hormone and metabolic signaling may be influenced by phytoestrogens, such as isoflavones. Their endocrine effects may modify symptom penetrance in PCOS. Equol is one of the most active isoflavone metabolites, produced by intestinal bacteria, and acts as a selective estrogen receptor modulator. Method: In this interventional study of clinical and biochemical characterization, urine isoflavone levels were measured in PCOS and control women before and three days after a defined isoflavone intervention via soy milk. In this interventional study, bacterial equol production was evaluated using the log(equol: daidzein ratio) and microbiome, metabolic, and predicted metagenome analyses were performed. Results: After isoflavone intervention, predicted stool metagenomic pathways, microbial alpha diversity, and glucose homeostasis in PCOS improved resembling the profile of the control group at baseline. In the whole cohort, larger equol production was associated with lower androgen as well as fertility markers. Conclusion: The dynamics in our metabolic, microbiome, and predicted metagenomic profiles underline the importance of external phytohormones on PCOS characteristics and a potential therapeutic approach or prebiotic in the future

Impact of Short-Term Isoflavone Intervention in Polycystic Ovary Syndrome (PCOS) Patients on Microbiota Composition and Metagenomics

Background: Polycystic ovary syndrome (PCOS) affects 5–20% of women of reproductive age worldwide and is associated with disorders of glucose metabolism. Hormone and metabolic signaling may be influenced by phytoestrogens, such as isoflavones. Their endocrine effects may modify symptom penetrance in PCOS. Equol is one of the most active isoflavone metabolites, produced by intestinal bacteria, and acts as a selective estrogen receptor modulator. Method: In this interventional study of clinical and biochemical characterization, urine isoflavone levels were measured in PCOS and control women before and three days after a defined isoflavone intervention via soy milk. In this interventional study, bacterial equol production was evaluated using the log(equol: daidzein ratio) and microbiome, metabolic, and predicted metagenome analyses were performed. Results: After isoflavone intervention, predicted stool metagenomic pathways, microbial alpha diversity, and glucose homeostasis in PCOS improved resembling the profile of the control group at baseline. In the whole cohort, larger equol production was associated with lower androgen as well as fertility markers. Conclusion: The dynamics in our metabolic, microbiome, and predicted metagenomic profiles underline the importance of external phytohormones on PCOS characteristics and a potential therapeutic approach or prebiotic in the future

Systematic in vivo evaluation of the time-dependent inflammatory response to steel and Teflon insulin infusion catheters

Abstract Continuous subcutaneous insulin infusion (CSII) catheters are considered the weak link of insulin pump therapy. Wear-time considerably varies between patients and the choice of catheter material is based on personal preferences rather than scientific facts. Therefore, we systematically assessed and quantified the inflammatory tissue response to steel versus Teflon CSII catheters over a maximum wear-time of 7 days in swine. Tissue surrounding catheters was analysed using histopathology and quantitative real-time PCR. The area of inflammation increased significantly over time independent of material which was confirmed by an increase in CD68 expression and an increase in mononuclear and neutrophil cell infiltrate around the catheters. We observed substantially higher fibrin deposition (p < 0.05) around steel on day 4 of wear-time. IL-6 gene expression increased within 24 hours after insertion, returned to normal levels around Teflon (p < 0.05) but remained high around steel (p < 0.05). IL-10 and TGF-β levels did not resolve over time, indicating impaired wound healing. In conclusion, there was a major temporal effect in the acute inflammatory response to CSII catheters but we found little difference between materials. This study setup presents a robust tool for the systematic analysis of the tissue response to CSII catheters

Autoimmunity to the Follicle-Stimulating Hormone Receptor (FSHR) and Luteinizing Hormone Receptor (LHR) in Polycystic Ovarian Syndrome

Hyperandrogenemia and ovulatory dysfunction are hallmarks of polycystic ovary syndrome (PCOS), pointing to a deranged hypothalamus-pituitary-ovarian (HPO) axis. An autoimmune etiology of PCOS is suspected in a subset of patients due to the relatively high concordance of PCOS with common autoimmune diseases. For this reason, we tested the hypothesis that natural autoantibodies (aAb) to the follicle-stimulating hormone receptor (FSHR) or luteinizing hormone receptor (LHR) are prevalent in PCOS. To this end, new luminometric assays for quantifying aAb to the FSHR (FSHR-aAb) or LHR (LHR-aAb) were developed using full-length recombinant human receptors as fusion proteins with luciferase as reporter. Prevalence of FSHR-aAb and LHR-aAb was determined in serum samples from healthy controls and PCOS patients. Steroid hormone profiles were compared between patients with and without FSHR-aAb or LHR-aAb. Signal linearity and detection ranges were characterized and both methods passed basic performance quality checks. The analysis revealed a relatively low prevalence, with 4 out of 430 samples positive for FSHR-aAb in the control versus 11 out of 550 samples in the PCOS group, i.e., 0.9% versus 2.0%, respectively. Similarly, there were only 5 samples positive for LHR-aAb in the control versus 2 samples in the PCOS group, i.e., 1.2% versus 0.4%, respectively. Samples positive for FSHR-aAb displayed steroid hormones in the typical range of PCOS patients, whereas the two samples positive for LHR-aAb showed relatively elevated free testosterone in relation to total testosterone concentrations with unclear significance. We conclude that the FSHR and LHR constitute potential autoantigens in human subjects. However, the prevalence of specific autoantibodies to these receptors is relatively low, both in control subjects and in women with PCOS. It is therefore unlikely that autoimmunity to the LHR or FSHR constitutes a frequent cause of hyperandrogenemia or ovulatory dysfunction in PCOS

Direct Determination of Ni²⁺-Capacity of IMAC Materials Using Near-Infrared Spectroscopy

The present paper reports a new method for the quantification of the Ni2+-capacity of an immobilized metal affinity chromatography (IMAC) material using near-infrared spectroscopy (NIRS). Conventional analyses using UV absorption spectroscopy or atomic absorption spectrometry (AAS) need to dissolve the silica-based metal chelate sorbent as sample pretreatment. In the first step, those methods were validated on the basis of an ideal homogenous NiSO4-solution and unveiled that UV with an intermediate precision of 2.6% relative standard deviation (RSD) had an advantage over AAS with an intermediate precision of 6.5% RSD. Therefore, UV analysis was chosen as reference method for the newly established NIRS model which has the advantage of being able to measure the material directly in diffuse reflection mode. Partial least squares regression (PLSR) analysis was used as multivariate data analysis tool for quantification. The best PLSR result obtained was: coefficient of determination (R2) = 0.88, factor = 2, root mean square error of prediction (RMSEP) = 22 µmol/g (test-set validation) or 7.5% RSDPLSR. Validation of the Ni2+-capacity using UV absorption spectroscopy resulted in an intermediate precision of ±18 µmol/g or 5.0% RSD. Therefore, NIRS provides a fast alternative analysis method without the need of sample preparation