Amplifikationen des ß-Cateningens bei juvenilen Angiofibromen

Hintergrund: Das juvenile Angiofibrom ist eine gutartige Neoplasie mit lokal destruktivem Wachstum, das fast ausschließlich bei männlichen Jugendlichen im Alter zwischen neun und 19 Jahren auftritt. Der Tumor ist histologisch durch seine irregulären Gefäßstrukturen, die in ein bindegewebig imponierendes Stroma eingebettet sind, gekennzeichnet. Eine eindeutige Klärung der Pathogenese ist bis jetzt nicht gelungen. In molekularpathologischen Untersuchungen konnten sowohl Mutationen im ß-Catenin-Gen als auch eine Überexpression der ß-Catenin m-RNA nachgewiesen werden. Dies führte zu der Feststellung, dass der WNT Pathway und mit ihm ß-Catenin eine wichtige Rolle in der Pathogenese des Angiofibroms spielt. In der vorliegenden Arbeit sollte geklärt werden, ob neben den bekannten Mutationen auch eine Amplifikation des ß-Cateningens vorliegt, die einen erhöhten Proteinlevel ohne Mutation der GSK3ß erklären können. Methoden: Um Veränderungen von ß-Catenin auf genomischer Ebene nachzuweisen, wurden im Rahmen der vorliegenden Arbeit spezifische Sonden mittels PCR als auch mittels kommerziell erworbenen bacterial artificial chromosomes (BAC) generiert, die FISH an unterschiedlichen Ausgangsmaterialien (4 Zellkulturpräparate, Paraffinschnitte von 6 Tumoren) etabliert und angewandt werden konnten. Ergebnisse: Während mit den PCR-Sonden keine visuelle Auswertung der Signale möglich war, konnte im Rahmen der vorliegenden Arbeit die FISH mit BAC-Sonden erfolgreich an den zu untersuchenden Ausgangsgeweben etabliert werden. In vier von sechs untersuchten Angiofibromen wurden sowohl Gewinne als auch Amplifikationen von ß-Catenin in Paraffingewebeschnitten nachgewiesen. Zwei Tumore zeigten weder Gewinne noch Amplifikationen. In der untersuchten Zellkultur zeigten zwei der vier untersuchten Tumoren Amplifikationen, einer Gewinne und einer sowohl Gewinne als auch Amplifikationen. Schlussfolgerungen: Im Rahmen dieser Arbeit konnte erstmals gezeigt werden, dass Amplifikationen des ß-Cateningens im juvenilen Angiofibrom vorliegen und neben den bereits bekannten Mutationen einen weiteren Mechanismus zur Erhöhung des intrazellulären ß-Catenins darstellen. Mit dem Nachweis von Gewinnen und Amplifikationen des ß-Catenin-Gens haben wir einen weiteren Mechanismus der Beteiligung des WNT-Pathways in der Tumorbiologie gefunden.Background: Juvenile angiofibromas are benign neoplasms presenting with local destructive behavior and growth patterns. They are diagnosed predominantly in young males between 9 and 19 years old. The tumor histology shows irregular vessels embedded in a fibrous stroma. Its pathogenesis is still unclear. However, CTNNB1 (gene encoding for β-catenin) mutations as well as the overexpression of the ßcatenin m-RNA have been demonstrated in these tumors leading to the assumption of WNT pathway importance in JA pathogenesis. The presented work intends to answer if beside reported mutations gains as well as amplifications of the CTNNB1 gene contribute further to elevated protein levels in JAs. Methods:To detect changes at the genomic level, FISH probes were generated with PCR as well as using bacterial artificial chromosomes (BAC). These probes were applied to paraffin sections of 6 tumors as well as on 4 cell cultures of juvenile angiofibromas. Results: While the PCR-based probes didn`t allow a visual interpretation the BACprobes worked well. In 4 out of the 6 analyzed JAs CTNNB1 gains and amplifications were proved. In two specimens whether gains nor amplifications were found. In cell culture two times amplifications, in one case gains and in the other probe gains as well amplifictaions were noted. Conclusion: The presented study proves for the first time gains and amplifications of the CTNNB1 gene in JAs as a further contribution in the tumor pathphysiology stressing on the importance of the WNT pathway in JAs

Differential nasal swab cytology represents a valuable tool for therapy monitoring but not prediction of therapy response in chronic rhinosinusitis with nasal polyps treated with Dupilumab

Introduction: Chronic Rhinosinusitis with nasal polyps (CRSwNP) is a common chronic disease with a high impact on patients’ quality of life. If conservative and surgical guideline treatment cannot sufficiently control disease burden, biologicals can be considered as a comparably new treatment option that has revolutionized CRSwNP therapy since the first approval of Dupilumab in 2019. With the aim to select patients who benefit from this new treatment and to find a marker for therapy monitoring, we investigated the cellular composition of nasal mucous membranes and inflammatory cells of patients suffering from CRSwNP and undergoing Dupilumab therapy using non-invasive nasal swab cytology. Methods: Twenty CRSwNP patients with the indication for Dupilumab therapy have been included in this prospective clinical study. In total, five study visits were conducted with ambulatory nasal differential cytology using nasal swabs starting with the beginning of therapy and followed by visits every 3 months for 12 months. First, these cytology samples were stained with the May-GrunwaldGiemsa method (MGG) and the percentage of ciliated cells, mucinous cells, eosinophil cells, neutrophil cells, and lymphocytes was analyzed. Secondly, an immunocytochemical (ICC) ECP-staining was performed to detect eosinophil granulocytes. Additionally, during each study visit the nasal polyp score, SNOT20 questionnaire, olfactometry, the total IgE concentration in peripheral blood as well as the eosinophil cell count in peripheral blood were recorded. The change of parameters was evaluated over one year and the correlation between clinical effectiveness and nasal differential cytology was analyzed. Results: In both MGG (p<0.0001) and ICC analysis (p<0.001) a significant decrease of eosinophils was seen under Dupilumab treatment. When patients were divided into a Eo-low- (<21%) and Eo-high- (≥21%) group according to the percentage eosinophils in nasal swab catology in the first study visit, the Eo-highgroup showed a greater change of eosinophils over time (D17.82) compared to the Eo-low-group (D10.67) but, however, no better response to therapy. The polyp score, SNOT20 questionnaire, and total IgE concentration in peripheral blood showed a significant decrease during the observation period (p<0.0001). Discussion: Nasal swab cytology as an easy-to-apply diagnostic method allows detection and quantification of the different cell populations within the nasal mucosa at a given time. The nasal differential cytology showed a significant decrease of eosinophils during Dupilumab therapy and can therefore be used as non-invasvive method for monitoring therapy success of this cost intensive therapy and potentially can allow an optimized individual therapy planning and management for CRSwNP patients. Since the validity of initial nasal swab eosinophil cell count as a predictive biomarker for therapy response was limited in our study, additional studies including larger number of participants will be necessary to further evaluate the potential benefits for clinical practice of this new diagnostic method

New genetic loci link adipose and insulin biology to body fat distribution.

Body fat distribution is a heritable trait and a well-established predictor of adverse metabolic outcomes, independent of overall adiposity. To increase our understanding of the genetic basis of body fat distribution and its molecular links to cardiometabolic traits, here we conduct genome-wide association meta-analyses of traits related to waist and hip circumferences in up to 224,459 individuals. We identify 49 loci (33 new) associated with waist-to-hip ratio adjusted for body mass index (BMI), and an additional 19 loci newly associated with related waist and hip circumference measures (P < 5 × 10(-8)). In total, 20 of the 49 waist-to-hip ratio adjusted for BMI loci show significant sexual dimorphism, 19 of which display a stronger effect in women. The identified loci were enriched for genes expressed in adipose tissue and for putative regulatory elements in adipocytes. Pathway analyses implicated adipogenesis, angiogenesis, transcriptional regulation and insulin resistance as processes affecting fat distribution, providing insight into potential pathophysiological mechanisms

Sex-stratified Genome-wide Association Studies Including 270,000 Individuals Show Sexual Dimorphism in Genetic Loci for Anthropometric Traits

Peer reviewe

Crystal Structure and Interactions of the PAS Repeat Region of the Drosophila Clock Protein PERIOD

International audiencePERIOD proteins are central components of the Drosophila and mammalian circadian clock. Their function is controlled by daily changes in synthesis, cellular localization, phosphorylation, degradation, as well as specific interactions with other clock components. Here we present the crystal structure of a Drosophila PERIOD (dPER) fragment comprising two tandemly organized PAS (PER-ARNT-SIM) domains (PAS-A and PAS-B) and two additional C-terminal alpha helices (alphaE and alphaF). Our analysis reveals a noncrystallographic dPER dimer mediated by intermolecular interactions of PAS-A with PAS-B and helix alphaF. We show that alphaF is essential for dPER homodimerization and that the PAS-A-alphaF interaction plays a crucial role in dPER clock function, as it is affected by the 29 hr long-period perL mutation

Crystal structure and interactions of the PAS repeat region of the Drosophila clock protein PERIOD

PERIOD proteins are central components of the Drosophila and mammalian circadian clock. Their function is controlled by daily changes in synthesis, cellular localization, phosphorylation, degradation, as well as specific interactions with other clock components. Here we present the crystal structure of a Drosophila PERIOD (dPER) fragment comprising two tandemly organized PAS (PER-ARNT-SIM) domains (PAS-A and PAS-B) and two additional C-terminal alpha helices (alphaE and alphaF). Our analysis reveals a noncrystallographic dPER dimer mediated by intermolecular interactions of PAS-A with PAS-B and helix alphaF. We show that alphaF is essential for dPER homodimerization and that the PAS-A-alphaF interaction plays a crucial role in dPER clock function, as it is affected by the 29 hr long-period perL mutation

Table_1_Differential nasal swab cytology represents a valuable tool for therapy monitoring but not prediction of therapy response in chronic rhinosinusitis with nasal polyps treated with Dupilumab.docx

IntroductionChronic Rhinosinusitis with nasal polyps (CRSwNP) is a common chronic disease with a high impact on patients’ quality of life. If conservative and surgical guideline treatment cannot sufficiently control disease burden, biologicals can be considered as a comparably new treatment option that has revolutionized CRSwNP therapy since the first approval of Dupilumab in 2019. With the aim to select patients who benefit from this new treatment and to find a marker for therapy monitoring, we investigated the cellular composition of nasal mucous membranes and inflammatory cells of patients suffering from CRSwNP and undergoing Dupilumab therapy using non-invasive nasal swab cytology.MethodsTwenty CRSwNP patients with the indication for Dupilumab therapy have been included in this prospective clinical study. In total, five study visits were conducted with ambulatory nasal differential cytology using nasal swabs starting with the beginning of therapy and followed by visits every 3 months for 12 months. First, these cytology samples were stained with the May-Grunwald-Giemsa method (MGG) and the percentage of ciliated cells, mucinous cells, eosinophil cells, neutrophil cells, and lymphocytes was analyzed. Secondly, an immunocytochemical (ICC) ECP-staining was performed to detect eosinophil granulocytes. Additionally, during each study visit the nasal polyp score, SNOT20 questionnaire, olfactometry, the total IgE concentration in peripheral blood as well as the eosinophil cell count in peripheral blood were recorded. The change of parameters was evaluated over one year and the correlation between clinical effectiveness and nasal differential cytology was analyzed.ResultsIn both MGG (pDiscussionNasal swab cytology as an easy-to-apply diagnostic method allows detection and quantification of the different cell populations within the nasal mucosa at a given time. The nasal differential cytology showed a significant decrease of eosinophils during Dupilumab therapy and can therefore be used as non-invasvive method for monitoring therapy success of this cost intensive therapy and potentially can allow an optimized individual therapy planning and management for CRSwNP patients. Since the validity of initial nasal swab eosinophil cell count as a predictive biomarker for therapy response was limited in our study, additional studies including larger number of participants will be necessary to further evaluate the potential benefits for clinical practice of this new diagnostic method.</p

DataSheet_1_Differential nasal swab cytology represents a valuable tool for therapy monitoring but not prediction of therapy response in chronic rhinosinusitis with nasal polyps treated with Dupilumab.pdf

IntroductionChronic Rhinosinusitis with nasal polyps (CRSwNP) is a common chronic disease with a high impact on patients’ quality of life. If conservative and surgical guideline treatment cannot sufficiently control disease burden, biologicals can be considered as a comparably new treatment option that has revolutionized CRSwNP therapy since the first approval of Dupilumab in 2019. With the aim to select patients who benefit from this new treatment and to find a marker for therapy monitoring, we investigated the cellular composition of nasal mucous membranes and inflammatory cells of patients suffering from CRSwNP and undergoing Dupilumab therapy using non-invasive nasal swab cytology.MethodsTwenty CRSwNP patients with the indication for Dupilumab therapy have been included in this prospective clinical study. In total, five study visits were conducted with ambulatory nasal differential cytology using nasal swabs starting with the beginning of therapy and followed by visits every 3 months for 12 months. First, these cytology samples were stained with the May-Grunwald-Giemsa method (MGG) and the percentage of ciliated cells, mucinous cells, eosinophil cells, neutrophil cells, and lymphocytes was analyzed. Secondly, an immunocytochemical (ICC) ECP-staining was performed to detect eosinophil granulocytes. Additionally, during each study visit the nasal polyp score, SNOT20 questionnaire, olfactometry, the total IgE concentration in peripheral blood as well as the eosinophil cell count in peripheral blood were recorded. The change of parameters was evaluated over one year and the correlation between clinical effectiveness and nasal differential cytology was analyzed.ResultsIn both MGG (pDiscussionNasal swab cytology as an easy-to-apply diagnostic method allows detection and quantification of the different cell populations within the nasal mucosa at a given time. The nasal differential cytology showed a significant decrease of eosinophils during Dupilumab therapy and can therefore be used as non-invasvive method for monitoring therapy success of this cost intensive therapy and potentially can allow an optimized individual therapy planning and management for CRSwNP patients. Since the validity of initial nasal swab eosinophil cell count as a predictive biomarker for therapy response was limited in our study, additional studies including larger number of participants will be necessary to further evaluate the potential benefits for clinical practice of this new diagnostic method.</p

DataSheet_2_Differential nasal swab cytology represents a valuable tool for therapy monitoring but not prediction of therapy response in chronic rhinosinusitis with nasal polyps treated with Dupilumab.docx

IntroductionChronic Rhinosinusitis with nasal polyps (CRSwNP) is a common chronic disease with a high impact on patients’ quality of life. If conservative and surgical guideline treatment cannot sufficiently control disease burden, biologicals can be considered as a comparably new treatment option that has revolutionized CRSwNP therapy since the first approval of Dupilumab in 2019. With the aim to select patients who benefit from this new treatment and to find a marker for therapy monitoring, we investigated the cellular composition of nasal mucous membranes and inflammatory cells of patients suffering from CRSwNP and undergoing Dupilumab therapy using non-invasive nasal swab cytology.MethodsTwenty CRSwNP patients with the indication for Dupilumab therapy have been included in this prospective clinical study. In total, five study visits were conducted with ambulatory nasal differential cytology using nasal swabs starting with the beginning of therapy and followed by visits every 3 months for 12 months. First, these cytology samples were stained with the May-Grunwald-Giemsa method (MGG) and the percentage of ciliated cells, mucinous cells, eosinophil cells, neutrophil cells, and lymphocytes was analyzed. Secondly, an immunocytochemical (ICC) ECP-staining was performed to detect eosinophil granulocytes. Additionally, during each study visit the nasal polyp score, SNOT20 questionnaire, olfactometry, the total IgE concentration in peripheral blood as well as the eosinophil cell count in peripheral blood were recorded. The change of parameters was evaluated over one year and the correlation between clinical effectiveness and nasal differential cytology was analyzed.ResultsIn both MGG (pDiscussionNasal swab cytology as an easy-to-apply diagnostic method allows detection and quantification of the different cell populations within the nasal mucosa at a given time. The nasal differential cytology showed a significant decrease of eosinophils during Dupilumab therapy and can therefore be used as non-invasvive method for monitoring therapy success of this cost intensive therapy and potentially can allow an optimized individual therapy planning and management for CRSwNP patients. Since the validity of initial nasal swab eosinophil cell count as a predictive biomarker for therapy response was limited in our study, additional studies including larger number of participants will be necessary to further evaluate the potential benefits for clinical practice of this new diagnostic method.</p