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Cellular and nuclear morphology…and calcium signaling: revealing the interplay between structure and function (2012)

Markus Breit Peter von der Bengtson Anna Hagenston Hilmar Bading Gillian Queisser

Poster presentation: Calcium plays a pivotal role in relaying electrical signals of the cell to subcellular compartments, such as the nucleus. Since this one ion type is used by the cell for many processes a neuron needs to establish finely tuned calcium pathways in order to be able to differentiate multiple tasks, [1-3]. While it is known that neurons can actively change their shape upon neuronal activity, [4-7], we here present novel findings of activity-regulated nuclear morphology, [8,9]. With the help of an experimental and computational modeling approach, we show that hippocampal neurons can change the previously spherical shape of their nuclei to complex and infolded morphologies. This morphology regulation is demonstrated to be regulated by NMDA-receptor gated calcium, while synaptic and extra-synaptic NMDA-receptors elicit opposing effects on nuclear morphology, [8]. The structural alterations of the cell nucleus have significant effects on nuclear calcium dynamics. Compartmentalization of the nucleus, due to membrane infoldings, changes calcium frequencies, amplitudes and spatial distributions, [8,10]. Since these parameters have been shown to control downstream events towards gene transcription, [11,12], the results elucidate the cellular control of nuclear function with the help of morphology modulation. With respect to processes downstream of calcium, we show that histone H3 phosphorylation is closely linked to nuclear morphology. Investigating the nuclear morphologies of hippocampal neurons, two major classes were identified [9,10]. One class contains non-infolded nuclei that have the function of calcium signal integrators, while the other class contains highly infolded nuclei, which function as frequency detectors of nuclear calcium, [10]. Extending this interdisciplinary approach of investigating structure/function relationships in neurons, the effects of cellular morphology – as well as the morphology of the endoplasmic reticulum and other organelles – on neuronal calcium signals is currently being investigated. This endeavor makes use of highly detailed, three-dimensional models of neuronal calcium dynamics, including the three-dimensional morphology of the cell and its organelles.

Tagungsbericht : "Gegenbilder - literarisch/filmisch/fotografisch" ; Internationales und interdisziplinäres Forschungskolloquium der Bamberger Graduiertenschule für Literatur, Kultur und Medien, 8./9. Juni 2012 in Bamberg (2012)

Kathrin Wimmer

Wir leben in einer Welt voller Bilder. Unser Denken funktioniert in Bildern, die menschliche Kommunikation bedient sich verschiedener Sprachbilder und das mediale Zeitalter überrollt uns mit einer wahren Flut aus bewegten und unbewegten Bildern. Die Moderne greift dabei nicht nur auf den reichen Bilderschatz vergangener Jahrhunderte zurück, sondern es werden auch traditionelle Motive variiert, verändert und verworfen. Das ursprüngliche Bild wird ergänzt durch Gegenbilder und Alternativkonzepte. Letztlich ringen sie alle, Bilder und ihre Gegenbilder, um die Deutungshoheit. Die Veranstalter Corina Erk, M.A., und Christoph Naumann, M.A., eröffneten als Promovierendenvertreter der Bamberger Graduiertenschule für Literatur, Kultur und Medien den Workshop „Gegenbilder – literarisch/filmisch/fotografisch“ und stellten das anspruchsvolle Tagungsprogramm vor, das sich in fünf Sektionen gliederte. Jedes Panel näherte sich aus einer anderen Perspektive dem Themenbündel „Gegenbild“ an.

Priorisierung in der Medizin – Diskussion einer Realität (2012)

Ulrich Finke Gerd Hoffmann

Welche gesellschaftlichen Zwänge wirken auf die Medizin und ihre Anwender ein? Wie ist das Verhältnis von Ökonomie und medizinisch Gebotenem? Wie steht es mit der Finanzierung der nicht evidenzbasierten Behandlung? Stellen Rationierung und Rationalisierung die möglichen Prinzipien der Priorisierung dar? Führt die Priorisierung zur Qualitätsminderung oder gar Sorgfaltsverletzung? Diese Fragen behandelte der 4. Ärztetag am Dom in Frankfurt am Main. Feine Unterschiede – Über Vorliebe und Abneigung im ärztlichen Alltag (Prof. Dr. med. Ralph Bickeböller, niedergelassener Arzt, Frankfurt am Main) Die Ärzte gehen ihrem Handwerk im Zeichen der Regelleistungsvolumen, der diagnosebezogenen Entgeltpauschalen, der Leitlinien, der Evidence-based-medicine, der Regelung des Sozialgesetzbuches V, der Arznei- und Heilmittelrichtlinie und anderer vielfältiger Bestimmungen nach, so dass allein durch die Vielfalt der Regelungen und durch die Vielzahl der Kontroll- und Regressmechanismen ein System etabliert ist, das einen mehr oder weniger starken Druck auf die Ärzte entfaltet, das Notwendige, Zweckmäßige und Wirtschaftliche am Patienten durchzuführen. Wie steht es mit dem Symbolwert von operativen Eingriffen, wenn man als Operateur für eine Inkontinenzoperation, die vielleicht eine halbe Stunde dauert und zwei postoperative Visiten benötigt, deutlich mehr erlösen kann als für eine organerhaltende Nierentumorresektion von drei Stunden mit sieben postoperativen Visiten? Nicht nur Ärzte haben ihre Prioritäten, u. a. tritt der Patient als Kunde auf, der durch seine Entscheidung für eine Dienstleistung ein Entgelt für den Dienstleistenden generiert. Warum bedürfen wir überhaupt einer Priorisierung in der Medizin? Weil die gesundheitlichen Bedürfnisse der Bürger unbegrenzt sind, weil es den medizinischen Fortschritt gibt und weil die Ressourcen begrenzt sind. Es geht unter anderem deshalb um die Frage, ob es für das Gesamtsystem Medizin gelingen kann, Präferenzen mit einer der festgelegten Rangordnung folgenden Ressourcenverteilung zu entwickeln, die nachvollziehbar, transparent, demokratisch und so weit wie möglich gerecht ist. Aufgrund der notwendigen Rationierung innerhalb des Gesundheitswesens ist deshalb für eine offene Priorisierung das Wort zu reden, die unterschiedliche Präferenzen, unterschiedliche Partizipationsmöglichkeiten, vor allem konkrete Ungerechtigkeiten für ein Leben in Wohlergehen für uns alle wahrnehmen und demokratisch diskutierten lässt. Die Allokation der stets zu knappen Ressourcen im Gesundheitswesen aus volkswirtschaftlicher Sicht (Prof. Dr. Klaus-Dirk Henke, Fachgebiet Finanzwissenschaft und Gesundheitsökonomie, Technische Universität Berlin) Die Allokation der stets zu knappen Ressourcen wird anhand einer Fünf-Ebenen-Betrachtung erläutert. Beim top-down und beim bottom-up Ansatz wird deutlich, dass und in welcher Form die unterschiedlichen Verwendungszwecke miteinander konkurrieren. Zu einer wünschenswerten Allokation der Ressourcen gibt es Hinweise aus der Wohlfahrtstheorie, aber keine empirische Basis für eine optimale Zuordnung der knappen Mittel. Anreizkompatible Finanzierungs- und Vergütungssysteme sowie die Ergebnisse der Krankheitskostenrechnungen helfen bei der Verwirklichung einer besseren Allokation. Von zentraler Bedeutung sind jedoch die vier ausgewählten Wettbewerbsfelder im Gesundheitswesen. Außerdem wird für die zukünftige Entwicklung die wachsende Bedeutung des Humankapitals für Wachstum und Produktivität in den Vordergrund gestellt und das Gesundheitswesen innerhalb der vielen Wirtschaftszweige angesichts der Wirtschafts- und Finanzkrise in 2008/2009 als besonders stabil eingeschätzt. Priorisierung und die deutsche Vergangenheit – Warum die Priorisierungsdebatte so heikel ist (Prof. Dr. Weyma Lübbe, Lehrstuhl für Praktische Philosophie, Universität Regensburg) Die Debatte über eine Priorisierung medizinischer Maßnahmen ist in Deutschland nach wie vor überwiegend eine akademische Debatte. Von politischer Seite wird das Thema als „unethisch“ bezeichnet und die Diskussion wird durch Negierung der Knappheit vermieden. Diese im internationalen Vergleich auffällige Zurückhaltung der Politik ist auch durch die Angst bedingt, offizielle Stellen könnten durch eine solche Debatte gezwungen werden, vergleichende Urteile über den „Wert“ des (Über-)Lebens verschiedener Patienten oder Patientengruppen zu fällen. Lebenswerturteile von staatlicher Seite waren wesentlicher Bestandteil der Kampagnen, mit denen im Nationalsozialismus das Euthanasie-Programm vorbereitet wurde. Damit möchte niemand auch nur entfernt in Verbindung gebracht werden. Der vorliegende Beitrag argumentiert, dass interpersonell vergleichende Lebenswerturteile kein unvermeidbarer Bestandteil einer Priorisierungsdebatte sind und dass sie in der Tat vermieden werden sollten. Im Rahmen politiknaher Fachkontroversen, namentlich in der Kontroverse zwischen deutschen Gesundheitsökonomen und dem Institut für Qualität und Wirtschaftlichkeit im Gesundheitswesen (IQWiG) über die Methoden der Kosten-Nutzen-Bewertung für Arzneimittel nach § 35 b SGB V, werden sie freilich nicht konsequent vermieden. Eine kontinuierliche kritische Beobachtung und Beurteilung der Implementation ökonomischer Evaluationsmethoden im Blick auf rechtliche und ethische Maßstäbe ist daher unbedingt zu empfehlen.

[Tagungsbericht zu:] Das Atrium von San Marco in Venedig. Die Genese der Genesismosaiken : Bad Homburg, Forschungskolleg Humanwissenschaften, 22. - 23.06.2012 (2012)

Rebecca Müller Herbert L. Kessler Martin Büchsel

Bericht der Organisatoren: Die Tagung wollte ein Zeichen setzen: ein Zeichen, dass es geboten sei, sich erneut der Cotton Genesis und ihrem ausdrücklichsten mittelalterlichen Nachfahren, den Schöpfungsmosaiken der Vorhalle von San Marco in Venedig, zuzuwenden. Die Diskussion dieser Verbindung von frühchristlichen Illuminationen, die nur noch in wenigen verkohlten Fragmenten überliefert sind, mit den mittelalterlichen Mosaiken ist seit der Entdeckung durch Johan Jakob Tikkanen 1889 geführt worden. Sie kam 1986 mit der Edition der Cotton Genesis durch Kurt Weitzmann und Herbert Kessler zu einem vorläufigen Abschluss. Die Mosaiken erschienen als weitgehend getreue Kopie der Buchmalereien der Handschrift, die dabei allein redaktionelle, aber keine konzeptionelle Veränderungen durch die Mosaizisten erfahren hätten...

Sepsis 2012 : Paris, France, 7 - 9 November 2012 ; meeting abstracts (2012)

5-Lipoxygenase contributes to PPAR [gamma] activation in macrophages in response to apoptotic cells (2012)

Andreas von Knethen Lisa Eifler Laura Kuchler Annika Heeg Heinrich Heide Ilka Wittig Thorsten Jürgen Maier Dieter Steinhilber Bernhard Brüne

Background: One hallmark contributing to immune suppression during the late phase of sepsis is macrophage polarization to an anti-inflammatory phenotype upon contact with apoptotic cells (AC). Taking the important role of the nuclear receptor PPARγ for this phenotype switch into consideration, it remains elusive how AC activate PPARγ in macrophages. Therefore, we were interested to characterize the underlying principle. Methods: Apoptosis was induced by treatment of Jurkat T cells for 3 hours with 0.5 μg/ml staurosporine. Necrotic cells (NC) were prepared by heating cells for 20 minutes to 65°C. PPARγ activation was followed by stably transducing RAW264.7 macrophages with a vector encoding the red fluorescent protein mRuby after PPARγ binding to 4 × PPRE sites downstream of the reporter gene sequence. This readout was established by treatment with the PPARγ agonist rosiglitazone (1 μM) and AC (5:1). Twenty-four hours after stimulation, mRuby expression was analysed by fluorescence microscopy. Lipid rafts of AC, NC, as well as living cells (LC) were enriched by sucrose gradient centrifugation. Fractions were analysed for lipid raft-associated marker proteins. Lipid rafts were incubated with transduced RAW264.7 macrophages as described above. 5-Lipoxygenase (5-LO) involvement was verified by pharmacological inhibition (MK-866, 1 μM) and overexpression. Results: Assuming that the molecule responsible for PPARγ activation in macrophages is localized in the cell membrane of AC, most probably associated to lipid rafts, we isolated lipid rafts from AC, NC and LC. Mass spectrometric analysis of lipid rafts of AC showed the expression of 5-LO, whereas lipid rafts of LC did not. Moreover, incubating macrophages with lipid rafts of AC induced mRuby expression. In contrast, lipid rafts of NC and LC did not. To verify the involvement of 5-LO in activating PPARγ in macrophages, Jurkat T cells were incubated for 30 minutes with the 5-LO inhibitor MK-866 (1 μM) before apoptosis induction. In line with our hypothesis, these AC did not induce mRuby expression. Finally, although living Jurkat T cells overexpressing 5-LO did not activate PPARγ in macrophages, mRuby expression was significantly increased when AC were generated from 5-LO overexpressing compared with wild-type Jurkat cells. Conclusion: Our results suggest that induction of apoptosis activates 5-LO, localizing to lipid rafts, necessary for PPARγ activation in macrophages. Therefore, it will be challenging to determine whether 5-LO activity in AC, generated from other cell types, correlates with PPARγ activation, contributing to an immune-suppressed phenotype in macrophages.

LPS-induced Pellino3 degradation is mediated by p62-dependent autophagy (2012)

Annika Heeg Laura Kuchler Lisa Eifler Tilo Knape Hermann Heide Bernhard Brüne Andreas von Knethen

Background: In macrophages Toll-like receptor 4 (TLR4) is activated in response to lipopolysaccharide (LPS) and induces proinflammatory cytokine expression [1]. Therefore, mechanisms terminating proinflammatory gene expression are important. Autophagy plays a central role in controlling innate immune responses by lysosomal degradation of signaling proteins, thus contributing to the resolution of inflammation [2]. Autophagic proteins like p62 directly interact with molecules involved in the TLR4-signaling pathway, but a correlation with the IRAK E3 ligase and scaffold protein Pellino3 remains obscure [3,4]. Hence, we are interested in elucidating the function of Pellino3 to prove our hypothesis that it is a key regulator in the TLR4-signaling cascade [5]. Methods: We used the cecal ligation and puncture (CLP) mouse model causing polymicrobial sepsis to analyze Pellino3 protein and mRNA expression. Furthermore, we induced endotoxemia in RAW264.7 mouse macrophages by LPS treatment to verify in vivo experiments. Lentiviral Pellino3 knockdown in RAW264.7 macrophages was used for cytokine measurements at mRNA level. To analyze potential Pellino3 binding partners in TLR4-signaling by mass spectrometry (MS), we overexpressed FLAG-tagged Pellino3 in RAW264.7 macrophages, treated cells for 3, 6 and 24 hours with LPS and immunoprecipitated Pellino3 via its FLAG-tag. To consider Pellino3 degradation as a result of p62-mediated autophagy, we transiently knocked down p62 by siRNA in RAW264.7 macrophages and also pharmacologically blocked LPS-induced autophagy by Bafilomycin A1. Results: We demonstrated Pellino3 protein degradation in primary CD11b+ splenocytes after 24 hours following CLP operation and confirmed this in RAW264.7 macrophages after 24-hour LPS stimulation. Knockdown of Pellino3 attenuates proinflammatory cytokines, for example IL-6 mRNA, after 6 hours of LPS. Furthermore, we found by MS and verifying immunoprecipitation experiments that p62 is a Pellino3 binding partner, thus targeting Pellino3 for degradation. In line, both p62 knockdown and Bafilomycin A1 treatment prevent Pellino3 degradation, supporting an autophagic mechanism. Conclusion: Our observations highlight a regulatory role of Pellino3 on TLR4 signaling. Thus, antagonism of Pellino3 in the hyperinflammatory phase of sepsis may counteract the cytokine storm. Furthermore, stabilization of Pellino3 by inhibition of autophagy in the hypoinflammatory phase of sepsis may improve immunity. In consideration of these two conflictive sepsis phases, modulation of Pellino3 may provide a new strategy for the development of a therapy approach in sepsis.

Attenuated NOX2 expression impairs ROS production during the hypoinflammatory phase of sepsis (2012)

Laura Kuchler V. Morbitzer Annika Heeg Lisa Eifler Tilo Knape Bernhard Brüne Andreas von Knethen

Background: The multicomponent phagocytic NADPH oxidase produces reactive oxygen species (ROS) after activation by microorganisms or inflammatory mediators [1]. In the hypoinflammatory phase of sepsis, macrophages are alternatively activated by contact with apoptotic cells or their secretion products. This inhibits NADPH oxidase and leads to attenuated ROS production [2] and furthermore contributes among others to a hyporeactive host defense. Due to this immune paralysis, sepsis patients suffer from recurrent and secondary infections [3]. We focused on the catalytic subunit of NADPH oxidase, the transmembrane protein NOX2 [4]. We assume that after induction of sepsis the expression of NOX2 is reduced and hence ROS production is decreased. Methods: We induced polymicrobial sepsis in mice by cecal ligation and puncture. The ability of peritoneal macrophages (PMs) to produce ROS was determined by FACS via hydroethidine assay. NOX2 expression of PMs was determined by western blot and qPCR. To elucidate the mechanism causing mRNA destabilization, we performed in vitro experiments using J774 macrophages. To obtain an alternatively activated phenotype, macrophages were stimulated with conditioned medium from apoptotic T cells (CM). By luciferase assays we figured out a 3'UTR-dependent regulation of NOX2 mRNA stability. Assuming that a protein is involved in the mRNA degradation, we performed a RNA pulldown with biotinylated NOX2-3'UTR constructs followed by mass spectrometry. We verified the role of SYNCRIP by siRNA approach. Additionally, we overexpressed NOX2 in J774 cells and analyzed the ROS production (w/wo CM treatment) by FACS. Results: We found an impaired expression of NOX2 at RNA and protein level along with decreased ROS production after induction of sepsis in mice as well as stimulating J774 macrophages with CM of apoptotic T cells. This is due to a time-dependent NOX2 mRNA degradation depending on SYNCRIP, a RNA-binding protein, which stabilizes NOX2 mRNA through binding to its 3'UTR under normal conditions. In line, knockdown of SYNCRIP also decreases NOX2 mRNA expression. We assume that a CM-dependent modification or degradation of SYNCRIP prevents its stabilizing function. As the overexpression of NOX2 restores ROS production of CM-treated J774 cells, we assume that NOX2 expression is crucial for maintaining NADPH activity during the hypoinflammatory phase of sepsis. Conclusion: Our data imply a regulatory impact of SYNCRIP on NOX2 stability during the late phase of sepsis. Therefore, further understanding of the regulation of NADPH oxidase could lead to the design of a therapy to reconstitute NADPH oxidase function, finally improving immune function in sepsis patients.

Kinetic characterization of selective peroxisome-proliferator-activated receptor gamma modulators in vitro (2012)

Tilo Knape Lisa Eifler Annika Heeg Laura Kuchler Bernhard Brüne Michael J. Parnham Andreas von Knethen

Background: The ligand-activated transcription factor, peroxisome-proliferator-activated receptor gamma (PPARγ), has been shown to play an essential role in immunosuppression during sepsis. PPARγ is upregulated in T cells of septic patients, sensitizing these cells to PPARγ-dependent apoptosis and thus contributing to T-cell depletion [1,2]. In the polymicrobial cecum ligation and puncture (CLP) sepsis model in mice, both T-cell-specific gene knockout (Lck-Cre PPARγfl/fl) and systemic pharmacological PPARγ antagonism by GW9662 improved survival [3]. Because GW9662 was only effective when applied 3 hours after CLP, we were interested to extend this time frame. For this reason we characterized the kinetics of SPPARγMs when administered before or in combination with the agonist thiazolidinedione, rosiglitazone. Methods: A PPARγ-dependent transactivation assay was used in HEK293T cells. It is based on the vector pFA-PPARγ-LBD-GAL4-DBD encoding the hybrid protein PPARγ-LBD-GAL4-DBD and the reporter vector pFR-Luc, carrying a GAL4-responsive element in front of the Firefly luciferase gene. These two vectors were co-transfected, in combination with a control vector encoding Renilla luciferase (pRL-CMV) to normalize Firefly luciferase activity for transfection efficiency. Following transfection, cells were incubated with the SPPARγMs F-MOC and MCC-555 and the PPARγ antagonist GW9662 for different times (2 to 48 hours) and at increasing doses (0.01 to 10 μM), with or without rosiglitazone (0.01 to 10 μM). Transactivation was analyzed using a 96-well plate format. Results: Rosiglitazone transactivated PPARγ in a time-dependent and dose-dependent manner, the response gradually increasing to a maximum at 48 hours with 10 μM. Low concentrations (0.01 to 0.1 μM) of SPPARγMs F-MOC and MCC-555 and the PPARγ antagonist GW9662 all exerted dose-independent antagonistic effects at an early incubation time point (2 hours). From 10 hours onwards, MCC-555 and GW9662, given alone, both exerted PPARγ agonistic effects, MCC-555 in parallel to responses to rosiglitazone, but GW9662 with characteristics of partial antagonism. F-MOC showed no dose-dependent effect at any concentration at later time points. Only GW9662 (1 to 10 μM) was able to inhibit rosiglitazone (0.1 to 1 μM)-induced PPARγ transactivation after 10 hours. Conclusion: Our kinetic analysis reveals clear differences in the modulatory characteristics of PPARγ inhibitors, with previously unreported early inhibitory effects and late agonistic or partial agonistic activity. New SPPARγMs with extended inhibitory activity may prove useful in the therapy of sepsis.

Ninjurin 1 contributes to TLR-induced inflammation in endothelial cells (2012)

Carla Jennewein Kai Zacharowski

Background: Nerve injury induced protein 1 (Ninjurin 1 (Ninj1)) was first identified in Schwann cells and neurons contributing to cell adhesion and nerve regeneration. Recently, the role of Ninj1 has been linked to inflammatory processes in the central nervous system where functional repression reduced leukocyte infiltration and clinical disease activity during experimental autoimmune encephalomyelitis in mice [1]. But Ninj1 is also expressed outside the nervous system in various organs such as the liver and kidney as well as on leukocytes [2,3]. Therefore, we hypothesized that Ninj1 contributes to inflammation in general; that is, also outside the nervous system, with special interest in the pathogenesis of sepsis. Methods: Ninj1 was repressed by transfecting HMEC-1 cells, a human dermal microvascular endothelial cell line with siRNA targeting Ninj1 (siNinj1) or a negative control (siC). Subsequently, cells were stimulated with 100 ng/ml LPS (TLR4 agonist), 3 μg/ml LTA (TLR2 agonist) or 100 n/ml poly(I:C) (TLR3 agonist) for 3 hours. The inflammatory response was analyzed by real-time PCR. In addition, transmigration of neutrophils across a HMEC-1 monolayer was measured using transwell plates (pore size 3 μm). Results: Repression of Ninj1 by siRNA reduced Ninj1 mRNA expression in HMEC about 90% (Figure 1A). Reduced Ninj1 expression decreased neutrophil migration to 62.5% (Figure 1B) and TLR signaling. In detail, knockdown of Ninj1 significantly reduced TLR-2 and TLR-4 triggered expression of ICAM-1 and IL-6 (Figure 1C,D) while poly(I:C)-induced expression was only slightly reduced. To analyze a more specific TLR-3 target, we measured IP-10 mRNA expression, which was also significantly reduced in siNinj1-transfected cells (Figure 1E). Conclusion: Our in vitro data strongly indicated that Ninj1 is involved in regulation of TLR signaling and therewith contributes to inflammation. In vivo experiments will clarify its impact on systemic inflammation.

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