Enhanced Immunomodulation in Inflammatory Environments Favors Human Cardiac Mesenchymal Stromal-Like Cells for Allogeneic Cell Therapies

Rising numbers of patients with cardiovascular diseases and limited availability of donor hearts require new and improved therapy strategies. Human atrial appendage-derived cells (hAACs) are promising candidates for an allogeneic cell-based treatment. In this study, we evaluated their inductive and modulatory capacity regarding immune responses and underlying key mechanisms in vitro. For this, cryopreserved hAACs were either cultured in the presence of interferon-gamma (IFNγ) or left unstimulated. The expression of characteristic mesenchymal stromal cell markers (CD29, CD44, CD73, CD105, CD166) was revealed by flow cytometry that also highlighted a predominant negativity for CD90. A low immunogeneic phenotype in an inflammatory milieu was shown by lacking expression of co-stimulatory molecules and upregulation of the inhibitory ligands PD-L1 and PD-L2, despite de novo expression of HLA-DR. Co-cultures of hAACs with allogeneic peripheral blood mononuclear cells, proved their low immunogeneic state by absence of induced T cell proliferation and activation. Additionally, elevated levels of IL-1β, IL-33, and IL-10 were detectable in those cell culture supernatants. Furthermore, the immunomodulatory potential of hAACs was assessed in co-cultures with αCD3/αCD28-activated peripheral blood mononuclear cells. Here, a strong inhibition of T cell proliferation and reduction of pro-inflammatory cytokines (IFNγ, TNFα, TNFβ, IL-17A, IL-2) were observable after pre-stimulation of hAACs with IFNγ. Transwell experiments confirmed that mostly soluble factors are responsible for these suppressive effects. We were able to identify indolamin-2,3-dioxygenase (IDO) as a potential key player through a genome-wide gene expression analysis and could demonstrate its involvement in the observed immunological responses. While the application of blocking antibodies against both PD-1 ligands did not affect the immunomodulation by hAACs, 1-methyl-L-tryptophan as specific inhibitor of IDO was able to restore proliferation and to lower apoptosis of T cells. In conclusion, hAACs represent a cardiac-derived mesenchymal stromal-like cell type with a high potential for the application in an allogeneic setting, since they do not trigger T cell responses and even increase their immunomodulatory potential in inflammatory environments

Stille Jugendliche in der Berufsberatung : wie gelingt Beratung mit ihnen?

Introversion ist eines von vielen Persönlichkeitsmerkmalen. Rund 30 % der Menschen sind introvertiert. Introvertierte Menschen zeichnen sich durch ihr Stillsein und ihre Zurückhaltung aus. Sie ziehen ihre Energie aus der Ruhe. Sie machen vieles mit sich selber aus. Dies macht es für eine Beratungsperson schwierig herauszufinden, was diese Menschen bewegt, beschäftigt, interessiert und blockiert. Diese Arbeit geht deshalb der Frage nach, unter welchen Voraussetzungen Beratung mit introvertierten Jugendlichen gelingen. kann Dabei werden vier Bereiche beleuchtet: 1. Methoden und Arbeitsmittel, 2. Beziehungsaufbau, 3. Herausforderungen und 4. der Einfluss von Introversion auf die Berufswahl. Zur Beantwortung dieser Fragen wurden verschiedene Theorien beigezogen und Experteninterviews mit vier Berufsberatenden geführt. Wichtige Erkenntnisse aus der Befragung und der Theorie sind folgende: • Methoden, die nicht nur das Gespräch zwischen Beratungsperson und Klientin oder Klient voraussetzen, haben sich in der Beratung von introvertierten Jugendlichen bewährt. • Die Kommunikation mit Introvertierten ist langsamer und es entstehen mehr Gesprächspausen. Es ist wichtig, dass Beratungspersonen, diese Pausen zulassen und sie nicht mit weiteren Fragen füllen. • Extravertierte Personen können direkter mit ihren Widersprüchlichkeiten und blinden Flecken konfrontiert werden. Bei Introvertierten ist es ratsam dies weniger direkt zu tun, da sie konfliktscheuer sind. • Extraversion gilt als Ideal in unserer Gesellschaft. Von Introvertierten wird erwartet, dass sie sich nach diesem Ideal strecken und ihre Stärken werden oft nicht wahrgenommen. Als Beratungsperson ist es wichtig, zu wissen, was Introversion ist, um den Klientinnen und Klienten erklären zu können, was es bedeutet, introvertiert zu sein. • Die Selbstwirksamkeitserwartungen von introvertierten Jugendlichen werden als tiefer wahrgenommen als diejenigen von extravertierten. Wertschätzung ist deshalb besonders wichtig. • Vorbereitung ist eine der Stärken von Introvertierten und kann beim Thema Bewerbung von Vorteil sein

Cardiac Extracellular Vesicles (EVs) Released in the Presence or Absence of Inflammatory Cues Support Angiogenesis in Different Manners

Cells release extracellular vesicles (EVs) to communicate in a paracrine manner with other cells, and thereby influence processes, such as angiogenesis. The conditioned medium of human cardiac-derived adherent proliferating (CardAP) cells was recently shown to enhance angiogenesis. To elucidate whether their released EVs are involved, we isolated them by differential centrifugation from the conditioned medium derived either in the presence or absence of a pro-inflammatory cytokine cocktail. Murine recipient cells internalized CardAP-EVs as determined by an intracellular detection of human proteins, such as CD63, by a novel flow cytometry method for studying EV-cell interaction. Moreover, endothelial cells treated for 24 h with either unstimulated or cytokine stimulated CardAP-EVs exhibited a higher tube formation capability on Matrigel. Interestingly, unstimulated CardAP-EVs caused endothelial cells to release significantly more vascular endothelial growth factor and interleukin (IL)-6, while cytokine stimulated CardAP-EVs significantly enhanced the release of IL-6 and IL-8. By nCounter® miRNA expression assay (NanoString Technologies) we identified microRNA 302d-3p to be enhanced in unstimulated CardAP-EVs compared to their cytokine stimulated counterparts, which was verified by quantitative polymerase chain reaction. This study demonstrates that both CardAP-EVs are pro-angiogenic by inducing different factors from endothelial cells. This would allow to select potent targets for a safe and efficient therapeutic application

Philosophie de la logique et philosophie du langage : la prédication

Marion Carel et Pascale Haag, maîtres de conférencesOswald Ducrot, directeur d’études S’inscrivant dans la lignée de la Grammaire de Port Royal, Du Marsais et Beauzée admettent que nos énoncés ont la fonction de dépeindre nos pensées. Ils fournissent pour cela deux ingrédients : d’une part leurs termes pleins signifient les Idées à combiner pour découvrir la pensée reflétée ; d’autre part, la construction syntaxique fournit les opérations que l’interlocuteur doit faire pour retrouver la pensé..

Philosophie de la logique et philosophie du langage

Marion Carel, Pascale Haag, maîtres de conférencesOswald Ducrot, directeur d’études Les descriptions définies On s’est Interrogé sur l’importance linguistique de trois conceptions logico-philosophiques des descriptions définies : celles de Frege, de Russell et de Donnellan. On a d’abord rappelé les spécificités de ces théories, qui se distinguent par le rôle qu’elles donnent, dans la valeur sémantique de l’énoncé, à la qualification des objets contenue dans les descriptions définies. En les c..

Extracellular vesicles from regenerative human cardiac cells act as potent immune modulators by priming monocytes

Background: Nano-sized vesicles, so called extracellular vesicles (EVs), from regenerative cardiac cells represent a promising new therapeutic approach to treat cardiovascular diseases. However, it is not yet sufficiently understood how cardiac-derived EVs facilitate their protective effects. Therefore, we investigated the immune modulating capabilities of EVs from human cardiac-derived adherent proliferating (CardAP) cells, which are a unique cell type with proven cardioprotective features. Results: Differential centrifugation was used to isolate EVs from conditioned medium of unstimulated or cytokinestimulated (IFNγ, TNFα, IL-1β) CardAP cells. The derived EVs exhibited typical EV-enriched proteins, such as tetraspanins, and diameters mostly of exosomes (< 100 nm). The cytokine stimulation caused CardAP cells to release smaller EVs with a lower integrin ß1 surface expression, while the concentration between both CardAP-EV variants was unaffected. An exposure of either CardAP-EV variant to unstimulated human peripheral blood mononuclear cells (PBMCs) did not induce any T cell proliferation, which indicates a general low immunogenicity. In order to evaluate immune modulating properties, PBMC cultures were stimulated with either Phytohemagglutin or anti-CD3. The treatment of those PBMC cultures with either CardAP-EV variant led to a significant reduction of T cell proliferation, pro-inflammatory cytokine release (IFNγ, TNFα) and increased levels of active TGFβ. Further investigations identified CD14+ cells as major recipient cell subset of CardAP–EVs. This interaction caused a significant lower surface expression of HLA-DR, CD86, and increased expression levels of CD206 and PD-L1. Additionally, EV-primed CD14+ cells released significantly more IL-1RA. Notably, CardAP-EVs failed to modulate anti-CD3 triggered T cell proliferation and pro-inflammatory cytokine release in monocultures of purified CD3+ T cells. Subsequently, the immunosuppressive feature of CardAPEVs was restored when anti-CD3 stimulated purified CD3+ T cells were co-cultured with EV-primed CD14+ cells. Beside attenuated T cell proliferation, those cultures also exhibited a significant increased proportion of regulatory T cells. Conclusions: CardAP-EVs have useful characteristics that could contribute to enhanced regeneration in damaged cardiac tissue by limiting unwanted inflammatory processes. It was shown that the priming of CD14+ immune cells by CardAP-EVs towards a regulatory type is an essential step to attenuate significantly T cell proliferation and proinflammatory cytokine release in vitro

Human Cardiac-Derived Adherent Proliferating Cells Reduce Murine Acute Coxsackievirus B3-Induced Myocarditis

BACKGROUND: Under conventional heart failure therapy, inflammatory cardiomyopathy typically has a progressive course, indicating a need for alternative therapeutic strategies to improve long-term outcomes. We recently isolated and identified novel cardiac-derived cells from human cardiac biopsies: cardiac-derived adherent proliferating cells (CAPs). They have similarities with mesenchymal stromal cells, which are known for their anti-apoptotic and immunomodulatory properties. We explored whether CAPs application could be a novel strategy to improve acute Coxsackievirus B3 (CVB3)-induced myocarditis. METHODOLOGY/PRINCIPAL FINDINGS: To evaluate the safety of our approach, we first analyzed the expression of the coxsackie- and adenovirus receptor (CAR) and the co-receptor CD55 on CAPs, which are both required for effective CVB3 infectivity. We could demonstrate that CAPs only minimally express both receptors, which translates to minimal CVB3 copy numbers, and without viral particle release after CVB3 infection. Co-culture of CAPs with CVB3-infected HL-1 cardiomyocytes resulted in a reduction of CVB3-induced HL-1 apoptosis and viral progeny release. In addition, CAPs reduced CD4 and CD8 T cell proliferation. All CAPs-mediated protective effects were nitric oxide- and interleukin-10-dependent and required interferon-γ. In an acute murine model of CVB3-induced myocarditis, application of CAPs led to a decrease of cardiac apoptosis, cardiac CVB3 viral load and improved left ventricular contractility parameters. This was associated with a decline in cardiac mononuclear cell activity, an increase in T regulatory cells and T cell apoptosis, and an increase in left ventricular interleukin-10 and interferon-γ mRNA expression. CONCLUSIONS: We conclude that CAPs are a unique type of cardiac-derived cells and promising tools to improve acute CVB3-induced myocarditis

Molecular simulations and visualization: introduction and overview

Here we provide an introduction and overview of current progress in the field of molecular simulation and visualization, touching on the following topics: (1) virtual and augmented reality for immersive molecular simulations; (2) advanced visualization and visual analytic techniques; (3) new developments in high performance computing; and (4) applications and model building

Evidence-based Kernels: Fundamental Units of Behavioral Influence

This paper describes evidence-based kernels, fundamental units of behavioral influence that appear to underlie effective prevention and treatment for children, adults, and families. A kernel is a behavior–influence procedure shown through experimental analysis to affect a specific behavior and that is indivisible in the sense that removing any of its components would render it inert. Existing evidence shows that a variety of kernels can influence behavior in context, and some evidence suggests that frequent use or sufficient use of some kernels may produce longer lasting behavioral shifts. The analysis of kernels could contribute to an empirically based theory of behavioral influence, augment existing prevention or treatment efforts, facilitate the dissemination of effective prevention and treatment practices, clarify the active ingredients in existing interventions, and contribute to efficiently developing interventions that are more effective. Kernels involve one or more of the following mechanisms of behavior influence: reinforcement, altering antecedents, changing verbal relational responding, or changing physiological states directly. The paper describes 52 of these kernels, and details practical, theoretical, and research implications, including calling for a national database of kernels that influence human behavior

Interacting Partners of Pro-Interleukin-16

1\. Titelblatt und Inhaltsverzeichnis 2\. Abkürzungsverzeichnis 3\. Einleitung 4\. Material und Methoden 5\. Ergebnisse 6\. Diskussion 7\. Zusammenfassung 8\. Summary 9\. Literaturverzeichnis 10\. AnhangDas Zytokin IL-16 ist ein CD4-Ligand mit proinflammatorischen, immunoregulatorischen und antiretroviralen Eigenschaften. Ähnlich wie einige andere Zytokine wird IL-16 über ein Vorläuferprotein, Pro-IL-16 synthetisiert und durch Caspase-3 prozessiert. Pro-IL-16 ist ein 67 kDa großes Protein, das in Zellen des Immunsystems exprimiert wird. Es enthält konservierte SH3-Bindungsdomänen in der amino-terminalen Hälfte und drei PDZ-Domänen in seiner C-terminalen Region, wovon ein Fragment das sezernierte Interleukin 16 darstellt. Ziel der Arbeit war es, weitere potenzielle Wechselwirkungspartner für Pro-IL-16 zu identifizieren. Diese sollten Hinweise über die Funktion von Pro-IL-16 bzw. IL-16 liefern. Durch YTH-Screens wurden die eng miteinander verwandten F-Aktin Bindungsproteine HS1, Abp1, Lasp1 und Cortactin als Bindungspartner für die Prolin-reiche Region des Pro-IL-16 identifiziert. Ebenso das Mikrotubuli-bindende Protein Hook3, dessen Wechselwirkung mit Pro- IL-16 jedoch nicht weiter untersucht wurde. Die Interaktion von HS1, Abp1, Lasp1 und Cortactin mit Pro-IL-16 wurde durch YTH-Spezifitätstests verifiziert. Koimmunopräzipitationsexperimente bestätigten die Wechselwirkung zwischen HS1 und Pro-IL-16. Durch die Substitution von Prolin durch Alanin in den beiden PXXP-Motiven der Prolin-reichen Region des Pro-IL-16 und die Klonierung von SH3-Deletionsmutanten wurde durch in vitro-Bindungs Assays bestätigt, dass die SH3-Domäne des HS1-Proteins an das N-terminale PXXP-Motiv des Pro-IL-16 bindet. Des Weiteren wurde die Bindung von endogenem IL-16 an HS1 durch Immunopräzipitation bestätigt. Eine Kolokalisation von HS1 mit Pro- IL-16 und molekularen Komplexen des Zytoskeletts konnte durch Immunfluoreszenz-Aufnahmen gezeigt werden. Die gefundenen Interaktionspartner HS1, Abp1, Lasp1 und Cortactin gehören zu den F-Aktin-Bindungsproteinen und sind in die Modulation des Aktin-Assembly involviert. Eine Beteiligung von Pro-IL-16 bei der Organisation der Immunologischen Synapse wird diskutiert.The cytokine IL-16 is a CD4 ligand with proinflamatory, immunoregulatory and antiretroviral properties. Like many other cytokines, IL-16 is synthesised as a precursor protein (pro-IL-16) and processed by caspase-3. Pro-IL-16 is a 67 kDa protein expressed in cells of the immune system. Part of the amino terminal region comprises conserved SH3-binding domains and the C-terminal region contains three PDZ domains, one fragment of which forms the secreted IL-16. The aim of this work was to identify additional interaction partners of pro-IL-16, knowledge of which would allow the functions of pro-IL-16 and IL-16 to be further elucidated. Using YTH-screening, the closely related F-actin binding proteins HS1, Abp1, Lasp1 and cortactin were shown to interact with the proline rich region of pro-IL-16. The interaction between pro-IL-16 and a further binding partner, the microtubulin binding protein Hook3, was not investigated further. The interaction of HS1, Abp1, Lasp1 and cortactin with pro-IL-16 was verified using YTH specificity tests and co-immunoprecipitation experiments confirmed the interaction between HS1 and pro-IL-16. By substituting proline with alanine in both PXXP-motifs of the pro-IL-16 proline-rich region and by cloning SH3 deletion mutants, it was possible using in vitro binding assays to confirm the binding of the HS1 protein SH3 domain to the N-terminal PXXP motif of pro-IL-16. The binding of endogenous IL-16 to HS1 was also confirmed by immunoprecipitation. The colocalisation of HS1 and pro-IL-16 and molecular complexes of the cytoskeleton was demonstrated by immunofluorescence microscopy. Because the interacting partners identified, HS1, Abp1, Lasp1 and cortactin, are F-actin binding proteins involved in the modulation of actin assembly, the possible involvement of pro-IL-16 in the organisation of the immunological synapse is under investigation