Activation of G12/G13 Results in Shape Change and Rho/Rho-Kinase–mediated Myosin Light Chain Phosphorylation in Mouse Platelets

Platelets respond to various stimuli with rapid changes in shape followed by aggregation and secretion of their granule contents. Platelets lacking the α-subunit of the heterotrimeric G protein Gq do not aggregate and degranulate but still undergo shape change after activation through thromboxane-A2 (TXA2) or thrombin receptors. In contrast to thrombin, the TXA2 mimetic U46619 led to the selective activation of G12 and G13 in Gαq-deficient platelets indicating that these G proteins mediate TXA2 receptor-induced shape change. TXA2 receptor-mediated activation of G12/G13 resulted in tyrosine phosphorylation of pp72syk and stimulation of pp60c-src as well as in phosphorylation of myosin light chain (MLC) in Gαq-deficient platelets. Both MLC phosphorylation and shape change induced through G12/G13 in the absence of Gαq were inhibited by the C3 exoenzyme from Clostridium botulinum, by the Rho-kinase inhibitor Y-27632 and by cAMP-analogue Sp-5,6-DCl-cBIMPS. These data indicate that G12/G13 couple receptors to tyrosine kinases as well as to the Rho/Rho-kinase–mediated regulation of MLC phosphorylation. We provide evidence that G12/G13-mediated Rho/Rho-kinase–dependent regulation of MLC phosphorylation participates in receptor-induced platelet shape change

Expression of TLR 2, -4 and -9 in peripheral mononuclear cells of Patients with atopic dermatitis

Titelblatt und Inhaltsverzeichnis Einleitung Fragestellung MaterialundMethoden Ergebnisse Diskussion Zusammenfassung Abkürzungsverzeichnis Materialien LiteraturverzeichnisDie atopische Dermatitis (AD) gehört neben der allergischen Rhinokonjunktivitis und dem allergischen Asthma bronchiale zu den Erkrankungen des atopischen Formenkreises. Die pathophysiologischen Mechanismen, die zu dieser chronisch-persistierenden oder chronisch-rezidivierenden entzündlichen Hauterkrankung führen, sind bisher noch nicht vollständig aufgeklärt. Eine bisher noch ungeklärte initiale Prädisposition führt zunächst zu einer durch Th2-Zytokine dominierten Immunantwort. Im weiteren Verlauf tragen IFN-γ-produzierende Th1-Zellen zur Chronifizierung der Erkrankung bei. Die Mitglieder der Toll-like Rezeptor-Familie spielen eine entscheidende Rolle in der Erkennung mikrobieller Pathogen-assoziierter molekularer Muster und stellen ein wichtiges Verbindungsglied zwischen der angeborenen und der adaptiven Immunantwort dar. Ihre Stimulation induziert über die Produktion von Zytokinen wie IL-12 vorwiegend eine Immunantwort vom Th1-Typ. Aufgrund ihres immunmodulierenden Potentials nehmen sie dadurch möglicherweise eine Schlüsselstellung in der Pathogenese von Erkrankungen ein, die durch eine Th1/Th2-Dysbalance gekennzeichnet sind, wie dies bei der AD der Fall ist. Um zu untersuchen, ob erwachsene AD-Patienten gegenüber einer gesunden Kontrollgruppe Unterschiede im Expressionsmuster der TLR2, -4 und -9 in Zellen des peripheren Bluts aufweisen, wurde in dieser Arbeit die TLR-Expression auf mRNA- und Proteinebene mittels real-time PCR und Durchflusszytometrie untersucht. Die Ergebnisse der vorliegenden Arbeit zeigen, dass Patienten mit atopischer Dermatitis eine stärkere TLR2-Expression in PBMC und Monozyten aufweisen als eine gesunde Kontrollgruppe. Dieser Expressionsunterschied lässt sich anhand der Untersuchungsergebnisse sowohl auf mRNA- als auch auf Proteinebene nachvollziehen. Um die funktionelle Relevanz der verstärkten TLR- Expression zu prüfen, wurden Monozyten mit TLR2-Liganden (Tri-Acyl Lipopeptide (Pam3Cys) und Lipoteichonsäure (LTA)) stimuliert und die Induktion der TNF-a-Sekretion analysiert. Sowohl nach Stimulation mit Pam3Cys als auch mit LTA konnte in der Gruppe der AD-Patienten eine höhere Induktion der TNF-a-Sekretion gegenüber der Kontrollgruppe festgestellt werden. Zusammengefasst zeigen die Ergebnisse der vorliegenden Arbeit eine gesteigerte TLR2-Expression bei AD-Patienten, die sich funktionell in einer erhöhten Sekretionsbereitschaft des pro-inflammatorischen Zytokins TNF-a widerspiegelt. Dass eine erhöhte TNF-a-Sekretionsbereitschaft eine Rolle im Verlauf der Erkrankung zu spielen scheint, belegen auch aktuelle Untersuchungen, in denen gezeigt werden konnte, dass Patienten mit schweren, chronischen Hautläsionen von einer systemischen Therapie mit TNF-a-Antagonisten profitieren.Background: Recent studies suggest that a decreased exposure to microbial burden in early infancy is a risk factor in the development of allergic disorders. Toll-like receptors play an important role in innate immunity by recognizing microbial components. Their activation leads predominately to the induction of an adaptive immune response of the T-helper 1 (Th1) type. Th2 cell derived cytokines and enhanced IgE responses contribute to the pathogenesis of atopic dermatitis (AD). Therefore, the aim of this study was to analyze the expression profiles of TLR2, -4 and -9 in patients with extrinsic atopic dermatitis and to investigate functional implications. Methods: mRNA-expression profiles of TLR2, -4 and -9 in peripheral mononuclear blood cells from AD patients and healthy controls were measured using quantitative real-time PCR. Surface expression of TLR2 and -4 on monocytes was determined by flow cytometry while TNF-a-secretion after stimulation with TLR2-ligands was measured by ELISA. Results: These data show that the expression of TLR2 but not TLR4 and TLR9 is increased in patients with AD compared to healthy controls. A detailed analysis of TLR-expression in different cell types reveal that monocytes from atopic patients exhibit an increased TLR2 surface expression. The enhanced TLR2 expression has an important implication since stimulation of monocytes with TLR2 specific ligands leads to an 2-fold higher increase of TNF-a production in monocytes from AD patients. Conclusion: Our results indicate an increased monocytic TLR2 expression in AD patients which may contribute to the chronic inflammatory state of the disease by promoting TNF-a production

Goethite Nanorods: Synthesis and Investigation of the Size Effect on Their Orientation within a Magnetic Field by SAXS

Goethite is a naturally anisotropic, antiferromagnetic iron oxide. Following its atomic structure, crystals grow into a fine needle shape that has interesting properties in a magnetic field. The needles align parallel to weak magnetic fields and perpendicular when subjected to high fields. We synthesized goethite nanorods with lengths between 200 nm and 650 nm in a two-step process. In a first step we synthesized precursor particles made of akaganeite (β-FeOOH) rods from iron(III)chloride. The precursors were then treated in a hydrothermal reactor under alkaline conditions with NaOH and polyvinylpyrrolidone (PVP) to form goethite needles. The aspect ratio was tunable between 8 and 15, based on the conditions during hydrothermal treatment. The orientation of these particles in a magnetic field was investigated by small angle X-ray scattering (SAXS). We observed that the field strength required to trigger a reorientation is dependent on the length and aspect ratio of the particles and could be shifted from 85 mT for the small particles to about 147 mT for the large particles. These particles could provide highly interesting magnetic properties to nanocomposites, that could then be used for sensing applications or membranes

Recurrent mutation of the ID3 gene in Burkitt lymphoma identified by integrated genome, exome and transcriptome sequencing

Burkitt lymphoma is a mature aggressive B-cell lymphoma derived from germinal center B cells. Its cytogenetic hallmark is the Burkitt translocation t(8;14)(q24;q32) and its variants, which juxtapose the MYC oncogene with one of the three immunoglobulin loci. Consequently, MYC is deregulated, resulting in massive perturbation of gene expression. Nevertheless, MYC deregulation alone seems not to be sufficient to drive Burkitt lymphomagenesis. By whole-genome, whole-exome and transcriptome sequencing of four prototypical Burkitt lymphomas with immunoglobulin gene (IG)-MYC translocation, we identified seven recurrently mutated genes. One of these genes, ID3, mapped to a region of focal homozygous loss in Burkitt lymphoma. In an extended cohort, 36 of 53 molecularly defined Burkitt lymphomas (68%) carried potentially damaging mutations of ID3. These were strongly enriched at somatic hypermutation motifs. Only 6 of 47 other B-cell lymphomas with the IG-MYC translocation (13%) carried ID3 mutations. These findings suggest that cooperation between ID3 inactivation and IG-MYC translocation is a hallmark of Burkitt lymphomagenesis