Discovery and validation of essential modules of T cell migration into the central nervous system in a genome-wide CRISPR/Cas9 screening in experimental autoimmune encephalomyelitis

Multiple sclerosis (MS) is an autoimmune, neuroinflammatory disease where peripheral immune cells infiltrate the central nervous system (CNS), causing inflammation and subsequent demyelination that ultimately leads to neuronal degeneration. The pathogenesis of MS is thought to be primarily driven by autoreactive T cells, which are the first immune cells to migrate into the CNS. Although efforts to restrict T cell migration into the CNS have yielded some success in treating MS, the need for additional treatment options remains. With this in mind, this study aimed to identify crucial genes necessary for T cell migration in the CNS in experimental autoimmune encephalomyelitis (EAE), an animal model for MS. In this work we used an adoptive T-cell transfer EAE model in Lewis rats to conduct an in vivo genome-wide loss-of-function CRISPR/Cas9 screening in encephalitogenic T cells. Through a subsequent validation screening, a diverse set of molecules essential for T cell migration during the initial phase of EAE pathogenesis was identified. To gain comprehensive insights into the functional role of these identified candidates in regulating T cell migration, we performed validation experiments involving the generation of single knockout TMBP cells and elucidated their underlying mechanisms. This systematic approach facilitated the clustering of these genes into functionally coherent modules with significant implications for understanding T cell migration regulation. The adhesion module comprises of the α4-integrin, its binding partner β1-integrin and the integrin chaperone Hsp90b1. Loss of those genes leads to impaired attachment of T cells to vascular endothelial cells, consequently hindering the process of transmigration into the CNS. The second chemotaxis module involves the Chemokine receptor Cxcr3, its intracellular binding partner Gnai2, and its transcription factor Tbx21. Perturbation of this module alters the overall chemoattractive response of T cells. The third module, centered around the kinase Grk2, influences T cell egress. In vivo microscopy experiments revealed that Grk2-deficient T cells are capable of adhering to vascular endothelial cells but fail to complete the diapedesis process. Mechanistically, this impairment arises from defective S1PR1 internalization mediated by Grk2 phosphorylation. Moreover, we identified two interacting proteins, Arih1 and Ube2l3, both involved in the ubiquitination process to affect T cell migration. Lastly, we discovered that Ets1 acts as an inhibitor of T cell migration, as evidenced by the accumulation of Ets1-deficient T cells within the CNS. Collectively, our study offers valuable insights into the regulatory mechanisms underlying T cell migration in the context of EAE, thereby holding promising implications for the development of innovative therapeutic strategies.Multiple Sklerose (MS) ist eine autoimmune, neuroinflammatorische Erkrankung, die durch die Infiltration von Immunzellen in das zentrale Nervensystem (ZNS) gekennzeichnet ist und zu Entzündungen und anschließender Entmarkung führt, was letztendlich in der Degeneration von Neuronen endet. Die Pathogenese von MS wird hauptsächlich durch autoreaktive T-Zellen getrieben, die als erste Immunzellen in das ZNS migrieren. Obwohl Bemühungen, die Migration von T-Zellen in das ZNS zu begrenzen, teilweise erfolgreich waren, besteht weiterhin Bedarf an zusätzlichen Behandlungsoptionen. Vor diesem Hintergrund zielte diese Arbeit darauf ab, essentielle Gene zu identifizieren, die für die T-Zell-Migration in das ZNS während der experimentellen autoimmunen Enzephalomyelitis (EAE), einem Tiermodel für MS, erforderlich sind. In dieser Arbeit verwendeten wir ein EAE Modell des adoptiven T-Zell-Transfers in Lewis Ratten, um ein in vivo genomweites CRISPR/Cas9-Screening in enzephalitogenen T-Zellen durchzuführen. Durch ein anschließendes Validierungsscreening konnten wir eine Gruppe von Molekülen identifizieren, die während der initialen Phase der EAE-Pathogenese für die Migration von T-Zellen essentiell sind. Um weitere Erkenntnisse über die funktionelle Rolle dieser identifizierten Kandidaten bei der Regulation der T-Zell-Migration zu gewinnen, führten wir Validierungsexperimente mit individuellem Gen-Knockout in T-Zellen durch. Dieser Ansatz ermöglichte die Gruppierung dieser Gene in funktional kohärente Module die entscheidend zur Regulation der T-Zell-Migration beitragen. Das Adhäsionsmodul besteht aus α4-Integrin, seinem Bindungspartner β1-Integrin und dem Integrin-Chaperon Hsp90b1. Der Verlust dieser Gene führt zu einer beeinträchtigten Anheftung von T-Zellen an die endothelialen Zellen der Blutgefäße, was die Transmigration in das ZNS behindert. Das zweite Chemotaxis-Modul umfasst den Chemokinrezeptor Cxcr3, seinen intrazellulären Bindungspartner Gnai2 und seinen Transkriptionsfaktor Tbx21. Die Beeinträchtigung dieses Moduls hat Auswirkungen auf die Reaktion von T-Zellen auf chemo-taktische Signale. Das dritte Modul, das sich um die Kinase Grk2 zentriert, beeinflusst den Austritt von T-Zellen. In vivo Mikroskopie-Experimente zeigten, dass Grk2-defiziente T-Zellen in der Lage sind, an endotheliale Zellen zu binden, jedoch den Prozess der Diapedese nicht vollständig abschließen können. Mechanistisch gesehen entsteht diese Beeinträchtigung durch eine fehlerhafte Internalisierung von S1PR1, welcher von Grk2 phosphoryliert wird. Darüber hinaus identifizierten wir zwei interagierende Proteine, Arih1 und Ube2l3, die beide am Prozess der Ubiquitinierung beteiligt sind und die T-Zell-Migration beeinflussen. Schließlich entdeckten wir, dass Ets1 eine inhibitorische Rolle bei der T-Zell-Migration spielt, was sich durch die Ansammlung von Ets1-defizienten T-Zellen im ZNS zeigte. Zusammenfassend liefert unsere Studie wertvolle Erkenntnisse über die regulatorischen Mechanismen, die der T-Zell-Migration im Kontext von EAE zugrunde liegen, und hat somit vielversprechende Implikationen für die Entwicklung innovativer therapeutischer Strategie

Einige Pendenzen. Weben und Text in der antiken Literatur

The metaphorical relation between the craft of weaving and singing or writing has often been noted and explained, not least with view to the etymological relation of text and textile. It is unsurprising, then, that it has become a commonplace assumption among literary critics to interpret the description of woven garments or scenes of weaving in literature as unequivocal representations of the literary texts themselves. The present contribution proposes to defamiliarise these overly familiar relations and make a fresh attempt at explaining the success of the metaphor of weaving for textual production: Engaging with the various stages and procedures of textile production at the upright loom common in Antiquity, the paper identifies two fundamental qualities of weaving that lie at the heart of its metaphorical appropriation: the linearity and coherence of the working process in weaving and its instantaneous materialisation in the woven garment. Drawing on a wide range of texts from ancient literature (Plautus, Cicero, Optatian, ps.Virgil’s Ciris, and ps.Tibullus’ Pagyricus Messallae), it is shown that the metaphor of weaving owes its success to the fact that it allows for reflection on the dialectics of ideality and materiality in texts. Against this backdrop, a reading of the Metamorphoses is put forward that interprets the Ovidian weaving scenes as a meditation on the metaphor iself

On Cicero’s De domo. A survey of recent work

The contribution offers a brief survey of research on De domo and locates it in the changing landscape of Ciceronian studies. After a consideration of the old debate on the speech’s authenticity, it identifies four dominant lines of inquiry in recent scholarship on De domo. Il contributo offre una breve prospettiva di ricerca sul De domo di Cicerone, collocando l’orazione nel panorama in continua trasformazione degli studi ciceroniani. Dopo una riflessione sullo storico dibattito riguardante l’autenticità del dialogo, si identificano quattro campi di ricerca dominanti nella critica recente al De domo

Protein C Replacement in Severe Meningococcemia: Rationale and Clinical Experience

Severe meningococcemia, which is associated with hemodynamic instability, purpura fulminans and disseminated intravascular coagulation, still has a high mortality rate, and patients who survive are often left invalids because of amputations and organ failure. Clinical studies have shown that levels of protein C are markedly decreased in patients with severe meningococcemia and that the extent of the decrease correlates with a negative clinical outcome. There is a growing body of data demonstrating that activated protein C, in addition to being an anticoagulant, is also a physiologically relevant modulator of the inflammatory response. The dual function of protein C may be relevant to the treatment of individuals with severe meningococcal sepsis. In the present review we give a basic overview of the protein C pathway and its anticoagulant activity, and we summarize experimental data showing that activated protein C replacement therapy clearly reduces the mortality rate for fulminant meningococcemi

You Are What You Eat and So Is Our Planet: Identifying Dietary Groups Based on Personality and Environmentalism

Behavioral change interventions promoting the reduction of animal product consumption are valuable tools to improve ecological sustainability as well as public health and help the mitigation of climate change. Recent findings revealed improved efficacy of interventions targeted at barriers (e.g., self-efficacy) of three different types of meat consumers over non-targeted interventions (e.g., completion of unrelated surveys). However, such interventions have yet to factor in the role of individual differences in personality. Therefore, in a first step, we performed segmentation analysis on barriers and benefits of reducing animal product consumption (e.g., meat attachment, environmentalism) with the inclusion of personality. In an online sample of N=1135 participants, latent profile analysis revealed five distinct dietary groups: “plant-based eaters”, “meat-reducers”, “medium-hindrance meat eaters”, “medium strong-hindrance meat eaters, and “strong-hindrance meat eaters”, based on inhibitors and facilitators of meat reduction. Groups differed in terms of consumption of different animal products (η2=0.08 to η2=0.80) as well as the Big Five (η2=0.08 to η2=0.80) and Dark Triad (η2=0.08 to η2=0.80). Strong-hindrance meat eaters were characterized by low Conscientiousness, Agreeableness, and Openness as well as high dark trait expression, implying new targets for future intervention design.Medical School HamburgPeer Reviewe

Dotierung von Cu(In,Ga)Se2-Schichten mit Natrium und Kalium zur Steigerung des Wirkungsgrads

Dünnschichtsolarzellen mit dem höchsten Laborwirkungsgrad von 21,7% basieren auf dem Verbindungshalbleiter Cu(In,Ga)Se2. Um hocheffiziente Cu(In,Ga)Se2-Solarzellen herzustellen ist die Dotierung der Cu(In,Ga)Se2-Schicht mit Natrium und Kalium mit einem optimierten Verfahren notwendig. Gegenstand dieser Arbeit ist daher die Untersuchung der Natrium- und Kaliumdiffusion in Cu(In,Ga)Se2-Schichten und die Entwicklung eines Dotierverfahrens zur gezielten Dotierung der Cu(In,Ga)Se2-Schicht

Bringing Light Into the Dark: Associations of Fire Interest and Fire Setting With the Dark Tetrad

Fire setting is a significant problem for society, costing many human lives and causing great property damage. One important risk factor of fire setting observed in forensic samples is fire interest. However, less is known about the relationship of fire interest and fire setting to other variables such as personality traits in subclinical samples. In this study, we observed the relationship of potentially important personality traits with fire interest and fire setting in a sample of N = 222 students. In addition to zero-order correlations, we calculated path models and a logistic regression including all predictor variables. From the Dark Tetrad, consisting of psychopathy, narcissism, Machiavellianism, and three facets of sadism, psychopathy, and physical sadism were found to be associated with fire interest and fire setting. Furthermore, vicarious sadism was associated with fire interest. The other Dark Tetrad traits and four sensation seeking facets did not substantially add to the predictions. This confirms the results of previous studies with clinical and forensic samples with psychopathy and sadism as relevant predictors for fire interest and fire setting. Our results also provide evidence for viewing sadism as the multidimensional construct discriminating between vicarious and other forms of sadism, for the distinction of psychopathy and Machiavellianism, and for the Dark Tetrad being linked to object related violence.Peer Reviewe