Zebrafish as a Model Organism to Study the <em>In Vivo</em> Role of G-Protein Coupled Receptor 17 in Myelination

The sheathing of axons with myelin by oligodendrocytes constitutes a necessity for sufficient and rapid nerve conduction within the vertebrate central nervous system (CNS). However to date, the complex molecular mechanisms controlling distinct signalling cascades of myelination are still not decrypted. Recent studies have proposed G-protein coupled receptors (GPCRs), such as Gpr17, as crucial regulators of oligodendrocyte development and myelination. Herein, this study investigates the implication of Gpr17 in myelination in its native signalling environment by using zebrafish (Danio rerio), an ideal suited vertebrate model system for studying myelination in vivo. Additionally, further functionality studies evaluate responsiveness of zebrafish and chimeric human/zebrafish Gpr17 receptors in extrinsic mammalian cellular backgrounds. While zebrafish Gpr17 transiently transfected in HEK293 cells remained unresponsive towards the small molecule agonist MDL29,951, stimulation of chimeric human/zebrafish receptors containing the human binding domain led to concentration-dependent response curves in Dynamic mass redistribution assays. These results, further confirmed by in vivo assays revealing unaffected oligodendrocyte development and myelination after bath treatment with MDL29,951, imply an impaired zebrafish receptor-ligand interaction both in vivo and in vitro. Investigations with zebrafish demonstrated sustained Gpr17 expression in oligodendrocyte lineage cells during CNS development from 1 to 5 dpf. Moreover, morpholino-induced receptor knockdown provoked a significant reduction of pre-mature and mature oligodendrocyte numbers and disturbed myelination. The reduced cell numbers are assumed to result from impaired migration of pre- mature pre-oligodendrocytes (pre-OLs) accumulating in the ventral spinal cord region. On the contrary, overexpression of Gpr17 in early stages did not influence oligodendrocyte development. Together, the data confirm a crucial role of zebrafish Gpr17 in vivo. Clearly, future studies with knockout animals are strictly necessary to corroborate these findings and conclusively delineate the impact of Gpr17 on oligodendrocyte development and myelination

The retreat of the state from entrepreneurial activities: an extended survey

There is a huge body of literature in the social sciences that deals with the privatization of public enterprises in developed OECD countries after World War II. In the first part of this article, we present and critically examine empirical findings of comparative inquiries on the factors influencing cross-national differences in privatization efforts across the OECD world. Although these empirical studies provide valuable and interesting evidence, it turns out that these studies are incomplete insofar, as they mostly focus just on one dimension of privatization: the monetary or ‘material’ aspect typically measured with privatization proceeds raised by governments. Against this background, in the second part of this paper we point out that there are also formal privatization (i.e., ‘privatizing’ the legal form of a public enterprise) and functional privatization (i.e., contracting out public service production). Moreover it is emphasized that privatization does not automatically imply a retreat of the state from entrepreneurial activities. From there we plead for taking into account the development of regulatory policies and public subsidies to private enterprises over time as well, in order to get a more comprehensive picture of the development of the entrepreneurial state in the OECD world. Therefore, for each of these dimensions of entrepreneurial activity the current ‘state of the art’ regarding theory and empirics is surveyed and enriched by own empirical data. -- Dieser Aufsatz zieht eine Zwischenbilanz des Rückzugs des Staates aus unternehmerischen Tätigkeiten in der OECD-Welt seit 1980. Der Beitrag präsentiert einen kritischen Überblick über empirische Studien zu den Bestimmungsfaktoren der nationalen Unterschiede in den Bereichen Privatisierung, Subventionszahlungen und Regulierung. Es wird gezeigt, dass erhebliche Forschungslücken bestehen; abschließend werden Wege für die künftige Forschung skizziert, um zu einem umfassenderen Bild der Entwicklung des unternehmerisch tätigen Staates zu gelangen.

Combined Cyclosporin A and Hypothermia Treatment Inhibits Activation of BV-2 Microglia but Induces an Inflammatory Response in an Ischemia/Reperfusion Hippocampal Slice Culture Model

Introduction: Hypothermia attenuates cerebral ischemia-induced neuronal cell death associated with neuroinflammation. The calcineurin inhibitor cyclosporin A (CsA) has been shown to be neuroprotective by minimizing activation of inflammatory pathways. Therefore, we investigated whether the combination of hypothermia and treatment with CsA has neuroprotective effects in an oxygen-glucose deprivation/reperfusion (OGD/R) injury model in neuronal and BV-2 microglia monocultures, as well as in an organotypic hippocampal slice culture (OHSC). Methods: Murine primary neurons, BV-2 microglia, and OHSC were pretreated with CsA and exposed to 1 h OGD (0.2% O2) followed by reperfusion at normothermia (37°C) or hypothermia (33.5°C). Cytotoxicity was measured by lactate dehydrogenase and glutamate releases. Damage-associated molecular patterns (DAMPs) high mobility group box 1 (HMGB1), heat shock protein 70 (Hsp70), and cold-inducible RNA-binding protein (CIRBP) were detected in cultured supernatant by western blot analysis. Interleukin-6 (IL-6), Interleukin-1α and -1β (IL-1α/IL1-β), tumor necrosis factor-α (TNF-α), monocyte chemotactic protein 1 (MCP1), inducible nitric oxide synthase (iNOS), glia activation factors ionized calcium-binding adapter molecule 1 (Iba1), and transforming growth factor β1 (TGF-β1) gene expressions were analyzed by RT-qPCR. Results: Exposure to OGD plus 10 μM CsA was sufficient to induce necrotic cell death and subsequent release of DAMPs in neurons but not BV-2 microglia. Moreover, OGD/R-induced secondary injury was also observed only in the neurons, which was not attenuated by cooling and no increased toxicity by CsA was observed. BV-2 microglia were not sensitive to OGD/R-induced injury but were susceptible to CsA-induced toxicity in a dose dependent manner, which was minimized by hypothermia. CsA attenuated IL-1β and Iba1 expressions in BV-2 microglia exposed to OGD/R. Hypothermia reduced IL-1β and iNOS expressions but induced TNF-α and Iba1 expressions in the microglia. However, these observations did not translate to the ex vivo OHCS model, as general high expressions of most cytokines investigated were observed. Conclusion: Treatment with CsA has neurotoxic effects on primary neurons exposed to OGD but could inhibit BV-2 microglia activation. However, CsA and hypothermia treatment after ischemia/reperfusion injury results in cytotoxic neuroinflammation in the complex ex vivo OHSC

Optimierung des CD4-spezifischen Gentransfers in primäre humane T-Zellen mittels [MLV(HIV-1)]-Vektoren im Hinblick auf die Gentherapie des Immunschwächesyndroms AIDS

Für verschiedene gentherapeutische Ansätze zur Behandlung der HIV-Infektion oder anderer erworbener oder angeborener Krankheiten wie z. B. der angeborenen Immunschwäche SCID ist ein hocheffizienter Gentransfer in CD4-positive T- Lymphozyten erforderlich. In der vorliegenden Arbeit wurden daher geeignete retrovirale Pseudotypvektoren weiterentwickelt. Unter Einsatz von Markergenen wurden Methoden der Transduktion primärer humaner Lymphozyten optimiert. Schließlich wurden verschiedene potentielle therapeutische anti-HIV-Gene durch retroviralen Gentransfer in humane T-Zelllinien übertragen und hinsichtlich der Hemmung der in-vitro Replikation verschiedener HIV-Stämme verglichen. Zunächst wurden stabile Verpackungszelllinien zur Herstellung von [MLV(HIV-1)]- und [MLV(GaLV)]-Pseudotypvektoren entwickelt, die ein für die Analyse der Transduktionseffizienz geeignetes Markergen übertragen. [MLV(HIV-1)]-Vektoren konnten mit Titern bis zu 2 x 10 hoch 5 i.E. / ml hergestellt werden. Die Optimierung der Kultivierung primärer humaner T-Lymphozyten vor dem ex- vivo Gentransfer ergab, dass eine 24-stündige PHA/IL-2 Stimulation mit anschließender 48-stündiger Kultivierung in IL-2 Medium optimal für die Transduktion primärer CD4-positiver T-Lymphozyten unter weitgehender Erhaltung des Expressionsmusters der Chemokinrezeptoren CXCR4 und CCR5 ist. Bei längerer Stimulation mit PHA und IL-2 verändert sich sowohl das CD4/CD8-Verhältnis als auch die CCR5-Expression gegenüber nativem Blut signifikant. Die Analyse der Expression des übertragenen Markergens und anderen Oberflächenmarkern der Zellen nach der Transduktion zeigte eine strikte Abhängigkeit der Transduktion der [MLV(HIV-1)]-Vektoren vom HIV-Rezeptor CD4, während herkömmliche [MLV(GaLV)]-Vektoren sowohl CD4-positive als auch CD4-negative Zellen transduzierten. Die Effizienz von [MLV(HIV-1CXCR4)]- Vektoren für CD4-positive Zellen war signifikant höher als die der [MLV(GaLV)]-Vektoren, während die Transduktionseffizienz der [MLV(HIV-1CCR5)]-Vektoren aufgrund der geringen Anzahl CCR5-positiver CD4-T-Zellen am niedrigsten war. Zwei Tage nach der Transduktion wurde eine reduzierte Korezeptorexpression in den Zellen nachgewiesen. Gründe hierfür könnten die Internalisierung der Korezeptoren nach der Transduktion oder eine durch die Kultivierung der Zellen bedingte Änderung der Expression sein. Nach weiterer Optimierung des retroviralen Gentransferprotokolls, u.a. durch Verwendung autologen Plasmas, konnten schließlich bei einmaliger Transduktion mit einer m.o.i. von 5 mit den [MLV(HIV-1CXCR4)]-Vektoren Transduktionsraten von bis zu 80 % erreicht werden. Zum Vergleich der Wirkung potentieller anti-HIV-Gene, die mit den neuen Vektoren in der Gentherapie des Immunschwächesyndroms AIDS eingesetzt werden könnten, wurden fünf verschiedene HIV-Inhibitoren (zwei intrazellulär exprimierte Antikörperfragmente (scFv) gegen HIV-1 Integrase und Reverse Transkriptase, zwei Ribozyme, die die HIV-1 RNA in der 5´-LTR oder im Pol- Leserahmen spezifisch spalten, sowie Interleukin-16) in den gleichen Transfervektor kloniert und durch retroviralen Gentransfer in die T-Zelllinie SupT1 übertragen. In Infektionsversuchen mit zwei unterschiedlichen HIV-1 Stämmen vermittelte jedoch keiner der potentiellen Inhibitoren eine signifikante Resistenz gegenüber HIV-1. Erst nach Sortierung der Kulturen auf starke Expression der übertragenen Gene konnte in den sortierten Zellen eine geringe Hemmwirkung des 5´-LTR-spezifischen Ribozyms auf die in-vitro Replikation des Stammes HIV-1IIIB, nicht jedoch auf die des Stammes HIV-1NL4-3 gezeigt werden. Die Signifikanz dieser Beobachtung muß über den Vergleich der Hemmwirkung weiterer Inhibitorgene geklärt werden

Acute necrotizing encephalopathy (ANE1): rare autosomal-dominant disorder presenting as acute transverse myelitis

The term "acute transverse myelitis (ATM)” comprises various non-traumatic disorders that eventually can be associated with a focal myelopathy. Patients characteristically present with an acutely occurring paraparesis/plegia and require a comprehensive and timely diagnostic work up for the initiation of an appropriate treatment. We present a case of a 36-year-old female patient with a rare genetic disorder (ANE1: Acute Necrotizing Encephalopathy due to a RANBP2 mutation) who presented with an acute quadriplegia. Following an acute pulmonal infection, she rapidly (< 24h) developed a severe quadriplegia (total motor score 38) with some facial sensory symptoms (perioral hypoesthesia). Magnetic resonance imaging (MRI) revealed a combination of longitudinal extensive transverse myelitis and symmetrical thalamic lesions. A work-up for infectious and systemic diseases was negative; specifically, no findings related to multiple sclerosis, neuromyelitis optica or vascular disorders. After empirical high dose steroid treatment and rehabilitation therapy, the patient gained almost normal gait and upper limb function. She was found to carry an autosomal-dominant missense mutation in the RANBP2 gene predisposing for ANE. Gene segregation was confirmed in other family members that had been affected by other episodes of acute steroid-responsive encephalopathies. We propose that a redefined diagnostic workup of ATM might include ANE1, as the frequency of this rare disorder might be underestimate

Adjektivgebrauch in Bildbeschreibungen – Eine korpuslinguistische Untersuchung zum Gebrauch von Adjektiven in Texten von Schülerinnen und Schülern

In this article, written descriptions by students in the third to sixth grade (N = 288) are examined with regard to the use of syntactically differently integrated and differently complex adjectives. Descriptively, attributive adjectives were used most frequently and participially attributive adjectives were used least frequently. The use of different adjectives correlated positively with each other almost universally across the sample. The use of attributive adjectives increased significantly with increasing age. The use of participial attributive, adverbial, compositional, and derivative adjectives was significantly associated with increasing grade level. The use of compositional and derivative adjectives was significantly associated with increasing grade level. Predicative adjective use did not differ significantly between grades. The results provide important insights into the language acquisition process of adjectives and give indications for didactic starting points for teaching text-sort-specific word types

Post-TTM Rebound Pyrexia after Ischemia-Reperfusion Injury Results in Sterile Inflammation and Apoptosis in Cardiomyocytes

Introduction. Fever is frequently observed after acute ischemic events and is associated with poor outcome and higher mortality. Targeted temperature management (TTM) is recommended for neuroprotection in comatose cardiac arrest survivors, but pyrexia after rewarming is proven to be detrimental in clinical trials. However, the cellular mechanisms and kinetics of post- TTM rebound pyrexia remain to be elucidated. Therefore, we investigated the effects of cooling and post-TTM pyrexia on the inflammatory response and apoptosis in a cardiomyocyte ischemia-reperfusion (IR) injury model. Methods. HL-1 cardiomyocytes were divided into the following groups to investigate the effect of oxygen-glucose deprivation/reperfusion (OGD/R), hypothermia (33.5°C), and pyrexia (40°C): normoxia controls maintained at 37°C and warmed to 40°C, OGD/R groups maintained at 37°C and cooled to 33.5°C for 24 h with rewarming to 37°C, and OGD/R pyrexia groups further warmed from 37 to 40°C. Caspase-3 and RBM3 were assessed by Western blot and TNF-α, IL-6, IL-1β, SOCS3, iNOS, and RBM3 transcriptions by RT-qPCR. Results. OGD-induced oxidative stress (iNOS) in cardiomyocytes was attenuated post-TTM by cooling. Cytokine transcriptions were suppressed by OGD, while reperfusion induced significant TNF-α transcription that was exacerbated by cooling. Significant inductions of TNF-α, IL-6, IL-1β, and SOCS3 were observed in noncooled, but not in cooled and rewarmed, OGD/R-injured cardiomyocytes. Further warming to pyrexia induced a sterile inflammatory response in OGD/R-injured groups that was attenuated by previous cooling, but no inflammation was observed in pyrexic normoxia groups. Moreover, cytoprotective RBM3 expression was induced by cooling but suppressed by pyrexia, correlating with apoptotic caspase-3 activation. Conclusion. Our findings show that maintaining a period of post-TTM “therapeutic normothermia” is effective in preventing secondary apoptosis-driven myocardial cell death, thus minimizing the infarct area and further release of mediators of the innate sterile inflammatory response after acute IR injury

ADJEKTIVE IN BILDBESCHREIBUNGEN. Eine Korpuslinguistische Untersuchung zum Adjektivgebrauch von bilingual und monolingual aufwachsenden Schülerinnen und Schülern

Im vorliegenden Beitrag werden schriftliche Bildbeschreibungen von monolingual und bilingual aufwachsenden Schüler*innen der dritten bis sechsten Klasse (N = 288) hinsichtlich ihres Adjektivgebrauchs untersucht. Deskriptiv zeigt sich, dass monolinguale Schüler*innen mehr Adjektive in der Freitextproduktion verwenden als bilinguale Schüler*innen. Mit Ausnahme der prädikativen und wertenden Adjektive, die prädikativ und attributiv vorkommen, zeigt sich ein vermehrter Adjektivgebrauch bei monolingual Lernenden im Vergleich zu bilingual Lernenden. Für die prädikativen Adjektive zeigen sich keine signifikanten Unterschiede; für die wertenden Adjektive zeigt sich ein Mehrgebrauch der bilingualen Schüler*innen. Die gefundenen Effekte können als klein bis mittel klassifiziert werden. Es zeigt sich keine Evidenz für das Vorliegen einer Interaktion zwischen Spracherwerb und Klassenstufe in der Verwendung attributiver und wertender Adjektive. Die Auswertung liefert wichtige Erkenntnisse zum Sprachaneignungsprozess von Adjektiven und zeigt schulischen Handlungsbedarf auf