Functions of the AP-1 transcription factor Fra-2 in epidermal development and disease

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 15-05-2015Altered epidermal differentiation and the production of inflammatory cytokines and chemokines by epidermal cells are hallmarks of numerous skin diseases affecting over 25% of the human population. Here I have identified Fra-2, an AP-1 transcription factor, as a key regulator of terminal epidermal differentiation and cytokine expression in keratinocytes. Mechanistically, Fra-2 binds and transcriptionally regulates gene promoters of epidermal differentiation genes, located within the Epidermal Differentiation Complex (EDC). EDC gene promoters are co-occupied by the transcriptional repressor Ezh2. Fra-2 remains transcriptionally inactive in non-differentiated keratinocytes, where it was found mono- and dimethylated on lysine 104, and interacted with Ezh2. Upon keratinocyte differentiation, Fra-2 is C-terminally phosphorylated on serine 320 and threonine 322 by ERK1/2, leading to transcriptional activation. Thus, the induction of epidermal differentiation by Fra-2 is controlled by a dual mechanism involving Ezh2-dependent methylation and activation by ERK1/2-dependent phosphorylation. During skin morphogenesis in mice, epithelial-restricted, ectopic expression of Fra-2 induced EDC gene expression. Moreover, in a papilloma-prone background, reduced tumor burden was observed due to precocious keratinocyte differentiation by Fra-2 expression. Importantly, loss of Fra-2 in suprabasal keratinocytes is sufficient to cause skin barrier defects due to reduced expression of differentiation genes. Besides impairing keratinocyte differentiation, loss of epidermal Fra-2 (Fra-2Δep) results in inflammation characterized by epidermal hyperplasia, infiltration of inflammatory cells into the dermis and epidermis and high serum cytokine levels. Additionally, Fra-2-deficient keratinocytes display increased p65/NF-κB activity and the concurrent removal of epithelial p65 partially attenuates the skin and systemic phenotype of Fra-2Δep mutants. These findings identify an important function of epidermal p65 in initiating inflammatory processes. Additionally, a cell autonomous but indirect regulation of Fra-2 on the expression of the cytokine TSLP by keratinocytes was uncovered. Interestingly, transplanting Fra-2Δep skin onto immune-compromised SCID mice gave rise to the formation of skin papillomas, suggesting a tumor protective role of the acute inflammatory response observed in Fra-2Δep skin. My data demonstrate a cell-autonomous function of Fra-2 in epithelial homeostasis by regulating keratinocyte differentiation, p65 activity and epithelial cytokine expression, which are causally involved in the development of inflammatory skin diseases and skin tumors.Las alteraciones en la diferenciación epidérmica y la producción de citoquinas inflamatorias y quimiocinas por las células epidérmicas son señas de identidad de numerosas enfermedades de la piel que afectan a más del 25% de la población humana. Aquí he identificado Fra-2, un factor de transcripción AP-1, como un regulador clave de la diferenciación epidérmica y la expresión de citoquinas en los queratinocitos. Mecanísticamente, Fra-2 se une y regula transcripcionalmente promotores de genes de diferenciación epidérmica ubicados dentro del Complejo de Diferenciación Epidérmica (EDC del inglés Epidermal Differentiation Complex). Los promotores de los genes EDC son co-ocupados por el represor transcripcional Ezh2. Fra-2 permanece transcripcionalmente inactiva en los queratinocitos no diferenciados, donde se encontró mono y dimetilado en la lisina 104, e interactuando con Ezh2. Tras la diferenciación de los queratinocitos, Fra-2 es fosforilado en el extremo C-terminal, en la serina 320 y la treonina 322, por ERK1/2, lo cual conduce a su activación transcripcional. Por lo tanto, la inducción de la diferenciación epidérmica por Fra-2 se controla mediante un doble mecanismo de metilación dependiente de Ezh2 y la activación por fosforilación dependiente de ERK1/2. Durante la morfogénesis de la piel en ratones, la expresión ectópica de Fra-2, restringida a células epiteliales, indujo la expresión de los genes EDC. Además, en ratones susceptibles a papilomas se observó una reducción de los tumores debido a una diferenciación precoz de queratinocitos por la sobreexpresión de Fra-2. Es importante destacar que la pérdida de Fra-2 en los queratinocitos suprabasales es suficiente para causar defectos de barrera de la piel debido a la reducción de la expresión de genes de diferenciación. Además de alterar la diferenciación de los queratinocitos, la pérdida de Fra-2 (Fra-2Δep) en la epidermis resulta en una inflamación que se caracteriza por hiperplasia epidérmica, infiltración de células inflamatorias en la dermis y la epidermis y niveles de citoquinas séricas elevadas. Además, los queratinocitos deficientes en Fra-2 muestran una mayor actividad de p65/NF-kB y la eliminación simultánea de p65 epitelial atenúa parcialmente el fenotipo de la piel y sistémico de los mutantes Fra-2Δep. Estos hallazgos identifican una función importante de p65 epidérmico en la iniciación de los procesos inflamatorios. Además, se pudo constatar una regulación autónoma pero indirecta de Fra-2 sobre la expresión de la citoquina TSLP por los queratinocitos. Sorprendentemente, el trasplante de piel Fra-2Δep en ratones inmunocomprometidos (SCID) dio lugar a la formación de papilomas en la piel, lo que sugiere un papel protector frente a tumores de la respuesta inflamatoria aguda observada en la piel Fra-2Δep. Mis datos demuestran una función autónoma de Fra-2 en la homeostasis epitelial mediante la regulación de la diferenciación de queratinocitos, la actividad de p65 y la expresión de citoquinas epiteliales que están implicadas causalmente en el desarrollo de enfermedades inflamatorias de la piel y tumores de pielThis work was supported by the following grants and fellowships: FPU pre-doctoral fellowship by the Ministry of Economy and Competitiveness, 2012 Call; BIF travel grant (May 2013 - August 2013) - Stefanie Wur

SchussenAktivplus: reduction of micropollutants and of potentially pathogenic bacteria for further water quality improvement of the river Schussen, a tributary of Lake Constance, Germany

The project focuses on the efficiency of combined technologies to reduce the release of micropollutants and bacteria into surface waters via sewage treatment plants of different size and via stormwater overflow basins of different types. As a model river in a highly populated catchment area, the river Schussen and, as a control, the river Argen, two tributaries of Lake Constance, Southern Germany, are under investigation in this project. The efficiency of the different cleaning technologies is monitored by a wide range of exposure and effect analyses including chemical and microbiological techniques as well as effect studies ranging from molecules to communities

Reducing the environmental impact of surgery on a global scale: systematic review and co-prioritization with healthcare workers in 132 countries

Abstract Background Healthcare cannot achieve net-zero carbon without addressing operating theatres. The aim of this study was to prioritize feasible interventions to reduce the environmental impact of operating theatres. Methods This study adopted a four-phase Delphi consensus co-prioritization methodology. In phase 1, a systematic review of published interventions and global consultation of perioperative healthcare professionals were used to longlist interventions. In phase 2, iterative thematic analysis consolidated comparable interventions into a shortlist. In phase 3, the shortlist was co-prioritized based on patient and clinician views on acceptability, feasibility, and safety. In phase 4, ranked lists of interventions were presented by their relevance to high-income countries and low–middle-income countries. Results In phase 1, 43 interventions were identified, which had low uptake in practice according to 3042 professionals globally. In phase 2, a shortlist of 15 intervention domains was generated. In phase 3, interventions were deemed acceptable for more than 90 per cent of patients except for reducing general anaesthesia (84 per cent) and re-sterilization of ‘single-use’ consumables (86 per cent). In phase 4, the top three shortlisted interventions for high-income countries were: introducing recycling; reducing use of anaesthetic gases; and appropriate clinical waste processing. In phase 4, the top three shortlisted interventions for low–middle-income countries were: introducing reusable surgical devices; reducing use of consumables; and reducing the use of general anaesthesia. Conclusion This is a step toward environmentally sustainable operating environments with actionable interventions applicable to both high– and low–middle–income countries

Terminal epidermal differentiation is regulated by the interaction of Fra-2/AP-1 with Ezh2 and ERK1/2.

Altered epidermal differentiation characterizes numerous skin diseases affecting >25% of the human population. Here we identified Fra-2/AP-1 as a key regulator of terminal epidermal differentiation. Epithelial-restricted, ectopic expression of Fra-2 induced expression of epidermal differentiation genes located within the epidermal differentiation complex (EDC). Moreover, in a papilloma-prone background, a reduced tumor burden was observed due to precocious keratinocyte differentiation by Fra-2 expression. Importantly, loss of Fra-2 in suprabasal keratinocytes is sufficient to cause skin barrier defects due to reduced expression of differentiation genes. Mechanistically, Fra-2 binds and transcriptionally regulates EDC gene promoters, which are co-occupied by the transcriptional repressor Ezh2. Fra-2 remains transcriptionally inactive in nondifferentiated keratinocytes, where it was found monomethylated and dimethylated on Lys104 and interacted with Ezh2. Upon keratinocyte differentiation, Fra-2 is C-terminally phosphorylated on Ser320 and Thr322 by ERK1/2, leading to transcriptional activation. Thus, the induction of epidermal differentiation by Fra-2 is controlled by a dual mechanism involving Ezh2-dependent methylation and activation by ERK1/2-dependent phosphorylation.We thank Dr. Falk Weih for providing FoxN1 Cre transgenic mice, and Dr. David Santamaria for providing lentiviral expression plasmids. We are very grateful to the members of the Wagner and Ezhkova laboratory for constructive input and criticism. S.W. is funded by a FPU predoctoral fellowship from the Spanish Ministry of Education and a BIF travel fellowship. E.F.W is funded by the Banco Bilbao Vizcaya Argentaria (BBVA) Foundation and a European Research Council Advanced Grant (ERC FCK/2008/37). J.Z. is supported by the Shandong Provincial Natural Science Foundation, China. J.G.-V. is supported by the Spanish Ministry of Education (SAF2012_39670). J.M. is supported by Ramon y Cajal Programme (MINECO, RYC-2012-10651). The CNIO Proteomics Unit belongs to ProteoRed, PRB2-ISCIII, supported by grant PT13/0001. E.E. is supported by the National Institute of Arthritis and Musculoskeletal and Skin Diseases of the National Institutes of Health (R01 AR063724).S

Assessment of fish health status in the Upper Danube River by investigation of ultrastructural alterations in the liver of barbel Barbus barbus

Despite intensive efforts and tightened guidelines for improvement of water quality over the last 2 decades, declines of fish populations have been reported for several rivers around the world. The present study forms part of a comprehensive weight-of-evidence approach, which aims to identify potential causes for the decline in fish catches observed in the Upper Danube River. The major focus of the present study is the investigation of the health status of wild barbel Barbus barbus L. collected from 3 locations along the Danube River, which experienced different levels of contamination. Whereas the comparison of the condition factor (CF) of field fish with that of control fish revealed no differences, ultrastructural investigations indicated severe disturbance of hepatic cell metabolism in field fish from the more contaminated sites Rottenacker and Ehingen, compared to both control fish and field fish from the less contaminated site Riedlingen. The ultrastructural analysis provided information about reactions of e.g. the rough endoplasmic reticulum, peroxisomes, andmitochondria, indicating an impaired health status of barbel at the sampling sites Rottenacker and Ehingen. Even though a straightforward cause-effect relationship between sediment contamination and ultrastructural alterations could not be established, based on a meta-analysis and toxicity assays it may be suggested that sediment-bound xenobiotics at least partly account for the hepatocellular changes. A relationship between impaired fish health status and the decline of fish catches along the Upper Danube River cannot be excluded

Elevated Plasma Vitamin B12 in Patients with Hepatic Glycogen Storage Diseases

Background: Hepatic glycogen storage diseases (GSDs) are inborn errors of metabolism affecting the synthesis or breakdown of glycogen in the liver. This study, for the first time, systematically assessed vitamin B(12)status in a large cohort of hepatic GSD patients.Methods: Plasma vitamin B-12, total plasma homocysteine (tHcy) and methylmalonic acid concentrations were measured in 44 patients with hepatic GSDs and compared to 42 healthy age- and gender-matched controls. Correlations of vitamin B(12)status with different disease markers of GSDs (including liver transaminase activities and triglycerides) as well as the vitamin B(12)intake were studied.Results: GSD patients had significantly higher plasma vitamin B(12)concentrations than healthy controls (p= 0.0002). Plasma vitamin B(12)concentration remained elevated in GSD patients irrespective of vitamin B(12)intake. Plasma vitamin B(12)concentrations correlated negatively with triglyceride levels, whereas no correlations were detected with liver transaminase activities (GOT and GPT) in GSD patients. Merging biomarker data of healthy controls and GSD patients showed a positive correlation between vitamin B(12)status and liver function, which suggests complex biomarker associations. A combined analysis of biomarkers permitted a reliable clustering of healthy controls versus GSD patients.Conclusions: Elevated plasma concentration of vitamin B-12(irrespective of B(12)intake) is a common finding in patients with hepatic GSD. The negative correlation of plasma vitamin B(12)with triglyceride levels suggests an influence of metabolic control on the vitamin B(12)status of GSD patients. Elevated vitamin B(12)was not correlated with GOT and GPT in our cohort of GSD patients. Merging of data from healthy controls and GSD patients yielded positive correlations between these biomarkers. This apparent dichotomy highlights the intrinsic complexity of biomarker associations and argues against generalizations of liver disease and elevated vitamin B(12)in blood. Further studies are needed to determine whether the identified associations are causal or coincidental, and the possible impact of chronically elevated vitamin B(12)on GSD

Interleukin‑2 Functionalized Nanocapsules for T Cell-Based Immunotherapy

A major demand on immunotherapy is the direct interference with specific immune cells <i>in vivo</i>. In contrast to antibody-engineered nanoparticles to control dendritic cells function, targeting of T cells for biomedical applications still remains an obstacle as they disclose reduced endocytic activities. Here, by coupling the cytokine interleukin-2 (IL-2) to the surface of hydroxyethyl starch nanocapsules, we demonstrated a direct and specifc T cell targeting <i>in vitro</i> and <i>in vivo</i> by IL-2 receptor-mediated internalization. For this purpose, defined amounts of azide-functionalized IL-2 were linked to alkyne-functionalized hydroxyethyl starch nanocapsules <i>via</i> copper-free click reactions. In combination with validated quantification of the surface-linked IL-2 with anthracen azide, this method allowed for engineering IL-2-functionalized nanocapsules for an efficient targeting of human and murine T cell populations with various IL-2 receptor affinities. This nanocapsule-mediated technique is a promising strategy for T cell-based immunotherapies and may be translated to other cytokine-related targeting systems

Impact of ITS-Based Sequencing on Antifungal Treatment of Patients with Suspected Invasive Fungal Infections

Molecular techniques including the sequencing of fungal-specific DNA targets are increasingly used in the diagnosis of suspected invasive fungal infections. In contrast to established biomarkers like galactomannan or 1-3-&beta;-d-glucan, the clinical impact of these methods remains unknown. We retrospectively investigated the impact of ITS1-sequencing on antifungal treatment strategies in 71 patients (81 samples) with suspected invasive fungal infections. ITS-sequencing either confirmed already ongoing antifungal therapy (19/71 patients, 27%), led to a change in antifungal therapy (11/71, 15%) or supported the decision to withhold antifungal treatment (34/71, 48%) (in seven of 71 patients, ITS-sequencing results were obtained postmortem). ITS-sequencing results led to a change in antifungal therapy in a relevant proportion of patients, while it confirmed therapeutic strategies in the majority. Therefore, ITS-sequencing was a useful adjunct to other fungal diagnostic measures in our cohort

SchussenAktiv-Eine Modellstudie zur Effizienz der Reduktion der Gehalte an anthropogenen Spurenstoffen durch Aktivkohle in Kläranlagen: Expositions-und Effektmonitoring vor Inbetriebnahme der Adsorptionsstufe auf der Kläranlage Langwiese des AZV Mariatal, Ravensburg

SchussenAktiv-a Model Study on the Efficacy in Reducing Anthropogenic Micropollutants by Activated Carbon Filtration in Wastewater Treatment Plants-Exposure and Effect Monitoring Prior to the Startup of the Charcoal Adsorption System at the Wastewater Treatment Plant Langwiese, Association for Wastewater Treatment Mariatal, City of Ravensburg, GermanyInternational audienceZusammenfassung Durch den kombinierten Einsatz verschiedener Methoden ist nachweisbar, dass sich Spurenstoffe auf den Gesundheitszustand wasserlebender Organismen und die Integrität aquatischer Le-bensgemeinschaft negativ auswirken. Im Projekt SchussenAktiv konnte die Präsenz von Spurenstoffen mit toxischen (z.B. gento-xischen) und hormonellen (z.B. östrogenartigen) Potentialen so-wie tatsächlichen Wirkungen in Verbindung gebracht werden. Die große Variabilität im Nachweis östrogenartig wirkender Chemi-kalien spiegelt sich auch in der Variabilität der nachgewiesenen östrogenen Wirkpotenziale und Wirkungen bei Fischen und Fisch-nährtieren wider. Die reduzierte Anzahl sensitiver Taxa unterhalb der untersuchten Kläranlage Langwiese (AZV Mariatal, Ravens-burg) an der Schussen spricht dafür, dass sich negative Effekte be-reits auf biozönotischer Ebene manifestiert haben. Ein Zusam-menspiel toxischer und hormoneller Einflüsse auf die Organismen in der Schussen ist hierbei aufgrund der erzielten Resultate wahr-scheinlich. Für die als Referenzgewässer ausgewählte Argen konn-te gezeigt werden, dass die untersuchte Probenahmestelle zwar insgesamt als deutlich weniger belastet gelten kann als die Probe-nahmestellen an der Schussen, dass a ber auch hier Bedarf be-steht, bestimmte Expositionen (z. B.-Sitosterol, Cadmium, Ar-sen, Quecksilber, Zink) und Effekte (z. B. Acetylcholinesterase-hemmung bei Fischen, fehlende Abundanz von Gammariden) ge-nauer zu betrachten um gegebenenfalls ihre Ursachen zu eruieren. Using a combination of different chemical and biological methods the project SchussenAktiv provided evidence that micropollutants negatively influence the health status of aquatic organisms and the integrity of aquatic ecosystems. It was possible to establish plausible connections between the presence of distinct micropol-lutants in the environment, toxic (e.g. genotoxic) or hormonal (e.g. estrogen-like) potentials in effluent or surface water samples , and the respective effects in feral fish. Large variability in the abundance of estrogen-like substances was reflected in the variation of both estrogenic potentials and estrogenic effects in fish and invertebrates. The decreased number of sensitive taxa in the river Schussen downstream the wastewater treatment plant Langwiese revealed evidence for detrimental effects also on the community structure. In general, the results of SchussenAktiv led to the suggestion that both toxic and endocrine-active chemicals interact in influencing organisms abundant in the Schussen river downstream sewage treatment plant. In contrast, the sampling site at the Argen river, chosen as a reference, was found to be clearly less polluted than the investigated sites along the Schussen. Nevertheless , detected pollutants (-sitosterol, cadmium, arsenic, mercury , zinc) and effects (AChE inhibition in fish, absence of gam-marids) call for a causal analysis also in the river Argen