Die Rolle der Aktin-regulierenden Proteine Coronin 1A und Coronin 1B in der Entwicklung und Funktion natürlicher Killerzellen

NK cells play an important role in the clearance of viral and intracellular bacterial infections as well as in the elimination of tumor cells and have also been implicated in the regulation of other immune cells. Recent work demonstrates a crucial role of actin cytoskeletal-activities in controlling NK cell function. In eukaryotic cells, the maintenance of the actin cytoskeleton and its quick remodeling in response to various stimuli is orchestrated by a cohort of actin-associated proteins. Coronin proteins are part of this complex network of actin-regulatory proteins and, as such, have been implicated in various actin-mediated cellular activities in different cellular systems. However, the role of coronins in the control of NK cell functions is yet unknown. In this thesis, I provide genetic evidence for an important role of two mammalian coronins, coronin 1a (Coro1a) and coronin 1b (Coro1b), in the regulation of NK cell development and function. By utilizing gene deficient mice I could demonstrate that the loss of Coro1a-function was directly linked to dysregulated F-actin organization in NK cells. While Coro1a-/- mice had normal numbers of peripheral NK cells, Coro1a-/- NK cells generally were of a more immature phenotype compared to wild type counterparts. The impaired NK cell maturation in Coro1a-/- mice was additionally associated with developmental alterations in the bone marrow. Although the single loss of Coro1b-function did not affect NK cell development and maturation, the immature phenotype of Coro1a-/- NK cells was further augmented by the additional loss of Coro1b. In vitro, IL-15 expanded Coro1a-/- and Coro1a-/-Coro1b-/- NK cells exhibited a comparable maturation status as wild type NK cells, but showed clear defects in central NK cell functions like killing of tumor cells, chemokine induced migration and cytokine secretion. The decreased cytotoxicity of Coro1a-/- and Coro1a-/-Coro1b-/- NK cells was associated with an impaired polarization of cytolytic granules and a reduced NK cell degranulation, whereas no major defects were found in early steps of the cytolytic response, including the formation of the cytolytic synapse and the consequent activation of NK cells. Thus, distinct actin associated processes involved in NK cell activities have different requirements on coronin proteins and the actin cytoskeleton. In conclusion, this study expands our knowledge on the function of actin-regulatory coronin proteins, by demonstrating that they do not only regulate known actin-dependent processes in NK cells, such as cell migration and cytotoxic granule release, but also control NK cell development. As coronin proteins and actin regulation are highly conserved between mice and man, the results obtained from this study are relevant for the human system. Thus, defective NK cell responses may contribute to the clinical symptoms observed in SCID patients with an inborn defect of Coro1a.NK-Zellen übernehmen wichtige Aufgaben in der Abwehr viraler und intrazellulärer bakterieller Infektionen sowie bei der Eliminierung von Tumorzellen. Des Weiteren tragen sie zur Regulation anderer Immunzellen bei. In aktuellen Publikationen konnte gezeigt werden, dass das Aktin-Zytoskelett eine entscheidende Funktion bei der Kontrolle von NK Zell-Aktivitäten übernimmt. In eukaryotischen Zellen werden die Aufrechterhaltung und der Umbau des Aktin-Zytoskeletts durch eine Vielzahl von Aktin-assoziierten Proteinen gesteuert. Einen wichtigen Bestandteil dieses komplexen Netzwerkes Aktin-regulierender Proteine bilden die Coronine. Diese werden mit der Regulation einer Vielzahl Aktin-vermittelter Prozesse in unterschiedlichen Zellsystemen in Verbindung gebracht. Eine Beteiligung von Coroninen an der Steuerung von NK Zell-Funktionen ist bisher jedoch unbekannt. In dieser Arbeit konnte ich durch die Verwendung von Gen-defizienten Mausstämmen eine wichtige regulative Rolle zweier Säugetier Coronine, genauer Coronin 1a (Coro1a) und Coronin 1b (Coro1b), für die Entwicklung und Funktion von NK Zellen nachweisen. Des Weiteren konnte ich zeigen, dass der Verlust der Funktion von Coro1a in direkter Verbindung mit einer fehlgesteuerten Aktin-Zytoskelett-Organisation in NK Zellen steht. Coro1a-defiziente Mäuse wiesen eine normale Anzahl peripherer NK Zellen auf, diese waren jedoch im Vergleich zu den Wildtyp-Kontrollen durch einen generell unreiferen Phänotyp gekennzeichnet. Ein vergleichbarer Effekt bezüglich des Reifegrades von Coro1a-/- NK Zellen wurde auch im Knochenmark beobachtet. Dieser war jedoch zusätzlich mit einer Steigerung der Anzahl der Coro1a-defizienten NK Zellen und somit einer veränderten NK Zell-Entwicklung im Knochenmark assoziiert. Der alleinige Verlust von Coro1b hatte hingegen keine Auswirkung auf die Entwicklung und Reifung der NK Zellen, jedoch wurde der unreife Phänotyp Coro1a-defizienter NK Zellen durch den zusätzlichen Verlust von Coro1b weiter verstärkt. IL-15-expandierte Coro1a-/- und Coro1a-/-Coro1b-/- NK Zellen zeigten im Vergleich zu Wildtyp-Zellen einen ähnlichen Reifegrad, wiesen allerdings in in vitro Studien klare Defizite bezüglich zentraler NK Zell-Funktionen wie der Abtötung von Tumorzellen, der Chemokin-induzierten Migration und der Zytokinfreisetzung auf. Die geminderte Zytotoxizität der Coro1a-/- und Coro1a-/-Coro1b-/- NK Zellen war assoziiert mit einer verringerten Polarisierung zytolytischer Granula sowie einer reduzierten NK Zell-Degranulation. Hingegen konnten keine nennenswerten Defekte im Bereich der frühen zytolytischen Antwort bezüglich einer gestörten Ausbildung der zytolytischen Synapse und einer daraus resultierenden verminderten Aktivierung der NK Zellen nachgewiesen werden. Daraus lässt sich schließen, dass Coronine und das Aktin-Zytoskelett differentiell bei unterschiedlichen Aktin-vermittelten Prozessen beansprucht werden. Zusammenfassend lässt sich sagen, dass die Untersuchungen im Rahmen dieser Arbeit das Wissen um Aktin-regulierende Coronine erweitern, indem gezeigt wird, dass nicht nur nachweislich Aktin-abhängige Prozesse wie die Zellmigration und die Freisetzung zytotoxischer Granula durch diese Proteine in NK Zellen reguliert werden sondern zusätzlich auch die Entwicklung von NK Zellen durch Coronine gesteuert wird. Da die untersuchten Coronin-Proteine sowie generelle Aktin-regulierende Prozesse zwischen Maus und Mensch stark konserviert sind, sind die Ergebnisse dieser Arbeit ebenfalls für das humane System relevant. Fehlerhafte NK Zell-Antworten könnten somit als weitere Ursache für die klinischen Symptome in SCID Patienten, die einen angeborenen Coro1a-Defekt aufweisen, verantwortlich sein

Das Wesen des Transformationspunktes

Bestimmung der Viskosität und des Volumens nach hinreichend langem Warten an glasigem Selen und Kolophonium. Fehlen eines Knicks für den Transformationspunkt. Geschwindigkeit der Volumenänderung bei verschiedenen Temperaturen. Der Transformationspunkt ist eine Einfriertemperatur. Atomistische Vorstellungen. Das Volumen der Schrumpfungsrisse. Bemerkungen über die Verbindungsbildung in Gläsern und über ihren Nachweis

The Stokes-Einstein Relation in Supercooled Aqueous Solutions of Glycerol

The diffusion of glycerol molecules decreases with decreasing temperature as its viscosity increases in a manner simply described by the Stokes-Einstein(SE) relation. Approaching the glass transition, this relation breaks down as it does with a number of other pure liquid glass formers. We have measured the diffusion coefficient for binary mixtures of glycerol and water and find that the Stokes-Einstein relation is restored with increasing water concentration. Our comparison with theory suggests that addition of water postpones the formation of frustration domainsComment: 4 Pages and 3 Figure

Transformationspunktbestimmungen an zusammengesetzten Gläsern durch Messung der Viskosität.

Bestimmung der Viskosität in der Umgebung der Einfriertemperatur. - Messungen an Gläsern, deren „Transformationspunkte" besonders gut bekannt sind; bei diesen Temperaturen haben alle Gläser dieselbe Viskosität. - Messungen an Gläsern der Reihe Na2O-SiO2; Störungen durch die Kristallisation bei geringeren Na2O-Gehalten. - Messungen an Biboraten. - Beziehungen zwischen den Temperatur-Koeffizienten der Viskosität und des Volumens. - Elastisches Verhalten

A phylogenomic analysis of Marek's disease virus reveals independent paths to virulence in Eurasia and North America

Virulence determines the impact a pathogen has on the fitness of its host, yet current understanding of the evolutionary origins and causes of virulence of many pathogens is surprisingly incomplete. Here, we explore the evolution of Marek's disease virus (MDV), a herpesvirus commonly afflicting chickens and rarely other avian species. The history of MDV in the 20th century represents an important case study in the evolution of virulence. The severity of MDV infection in chickens has been rising steadily since the adoption of intensive farming techniques and vaccination programs in the 1950s and 1970s, respectively. It has remained uncertain, however, which of these factors is causally more responsible for the observed increase in virulence of circulating viruses. We conducted a phylogenomic study to understand the evolution of MDV in the context of dramatic changes to poultry farming and disease control. Our analysis reveals evidence of geographical structuring of MDV strains, with reconstructions supporting the emergence of virulent viruses independently in North America and Eurasia. Of note, the emergence of virulent viruses appears to coincide approximately with the introduction of comprehensive vaccination on both continents. The time-dated phylogeny also indicated that MDV has a mean evolutionary rate of ~1.6 × 10−5 substitutions per site per year. An examination of gene-linked mutations did not identify a strong association between mutational variation and virulence phenotypes, indicating that MDV may evolve readily and rapidly under strong selective pressures and that multiple genotypic pathways may underlie virulence adaptation in MDV

Comparison of Porcine Epidemic Diarrhea Viruses from Germany and the United States, 2014

Since 2013, highly virulent porcine epidemic diarrhea virus has caused considerable economic losses in the United States. To determine the relation of US strains to those recently causing disease in Germany, we compared genomes and found that the strain from Germany is closely related to variants in the United States

Biological effects of carbon black nanoparticles are changed by surface coating with polycyclic aromatic hydrocarbons

BACKGROUND: Carbon black nanoparticles (CBNP) are mainly composed of carbon, with a small amount of other elements (including hydrogen and oxygen). The toxicity of CBNP has been attributed to their large surface area, and through adsorbing intrinsically toxic substances, such as polycyclic aromatic hydrocarbons (PAH). It is not clear whether a PAH surface coating changes the toxicological properties of CBNP by influencing their physicochemical properties, through the specific toxicity of the surface-bound PAH, or by a combination of both. METHODS: Printex(R)90 (P90) was used as CBNP; the comparators were P90 coated with either benzo[a]pyrene (BaP) or 9-nitroanthracene (9NA), and soot from acetylene combustion that bears various PAHs on the surface (AS-PAH). Oxidative stress and IL-8/KC mRNA expression were determined in A549 and bronchial epithelial cells (16HBE14o-, Calu-3), mouse intrapulmonary airways and tracheal epithelial cells. Overall toxicity was tested in a rat inhalation study according to Organization for Economic Co-operation and Development (OECD) criteria. Effects on cytochrome monooxygenase (Cyp) mRNA expression, cell viability and mucociliary clearance were determined in acute exposure models using explanted murine trachea. RESULTS: All particles had similar primary particle size, shape, hydrodynamic diameter and zeta-potential. All PAH-containing particles had a comparable specific surface area that was approximately one third that of P90. AS-PAH contained a mixture of PAH with expected higher toxicity than BaP or 9NA. PAH-coating reduced some effects of P90 such as IL-8 mRNA expression and oxidative stress in A549 cells, granulocyte influx in the in vivo OECD experiment, and agglomeration of P90 and mucus release in the murine trachea ex vivo. Furthermore, P90-BaP decreased particle transport speed compared to P90 at 10 mug/ml. In contrast, PAH-coating induced IL-8 mRNA expression in bronchial epithelial cell lines, and Cyp mRNA expression and apoptosis in tracheal epithelial cells. In line with the higher toxicity compared to P90-BaP and P90-9NA, AS-PAH had the strongest biological effects both ex vivo and in vivo. CONCLUSIONS: Our results demonstrate that the biological effect of CBNP is determined by a combination of specific surface area and surface-bound PAH, and varies in different target cells

Differential regulation of mast cell degranulation versus cytokine secretion by the actin regulatory proteins Coronin1a and Coronin1b

Coronin1a inhibits mast cell degranulation through actin cytoskeletal dynamics while augmenting cytokine secretion, an effect exacerbated by additional loss of Coronin1b

Development and evaluation of real time RT-PCR assays for detection and typing of Bluetongue virus

Bluetongue virus is the type species of the genus Orbivirus, family Reoviridae. Bluetongue viruses (BTV) are transmitted between their vertebrate hosts primarily by biting midges (Culicoides spp.) in which they also replicate. Consequently BTV distribution is dependent on the activity, geographic distribution, and seasonal abundance of Culicoides spp. The virus can also be transmitted vertically in vertebrate hosts, and some strains/serotypes can be transmitted horizontally in the absence of insect vectors. The BTV genome is composed of ten linear segments of double-stranded (ds) RNA, numbered in order of decreasing size (Seg-1 to Seg-10). Genome segment 2 (Seg-2) encodes outer-capsid protein VP2, the most variable BTV protein and the primary target for neutralising antibodies. Consequently VP2 (and Seg-2) determine the identity of the twenty seven serotypes and two additional putative BTV serotypes that have been recognised so far. Current BTV vaccines are serotype specific and typing of outbreak strains is required in order to deploy appropriate vaccines. We report development and evaluation of multiple ‘TaqMan’ fluorescence-probe based quantitative real-time type-specific RT-PCR assays targeting Seg-2 of the 27+1 BTV types. The assays were evaluated using orbivirus isolates from the ‘Orbivirus Reference Collection’ (ORC) held at The Pirbright Institute. The assays are BTV-type specific and can be used for rapid, sensitive and reliable detection / identification (typing) of BTV RNA from samples of infected blood, tissues, homogenised Culicoides, or tissue culture supernatants. None of the assays amplified cDNAs from closely related but heterologous orbiviruses, or from uninfected host animals or cell cultures