Migräne im Kindes- und Jugendalter — Ausblick auf innovative Behandlungsansätze im Rahmen multimodaler Therapiekonzepte

Although migraine is a~relevant health issue in children and adolescents, clinical care and research are still underrepresented and underfunded in this field. Quality of life can be significantly reduced when living with frequent episodes of pain. Due to the high level of vulnerability of the developing brain during adolescence, the risk of chronification and persistence into adulthood is high. In this narrative review, we describe the corner stones of a~patient-centered, multimodular treatment regimen. Further, an update on the pathophysiology of migraine is given considering the concept of a~periodically oscillating functional state of the brain in migraine patients (\textquotedblmigraine is a~brain state\textquotedbl). Besides central mechanisms, muscular structures with the symptoms of muscular pain, tenderness, or myofascial trigger points play an important role. Against this background, the currently available nonpharmacological and innovative neuromodulating approaches are presented focusing on the method of repetitive peripheral magnetic stimulation.Die Migräne ist auch im Kindes- und Jugendalter ein häufiges, aber in klinischer Versorgung und Wissenschaft oft unterrepräsentiertes Krankheitsbild. Gerade im Kindes- und Jugendalter bestehen relevante Einschränkungen der Lebensqualität durch das (häufige) Schmerzerfahren. Bedingt durch die entwicklungsspezifisch hohe Vulnerabilität des adoleszenten Gehirns besteht ein hohes Chronifizierungs- und Persistenzrisiko bis ins Erwachsenenalter hinein. In diesem Beitrag werden die Bestandteile eines patientenzentrierten, multimodalen Therapiekonzepts dargestellt. Darüber hinaus werden die aktuellsten Erkenntnisse zu den pathophysiologischen Grundlagen der Migräneerkrankung beleuchtet, nach denen Migräne durch einen sich phasenweise verändernden Funktionszustand des Gehirns entsteht (Stichwort: „migraine is a brain state“). Auch periphere Komponenten wie Muskelschmerzen, -verspannungen und -triggerpunkte spielen eine wichtige Rolle. Vor diesem Hintergrund werden nichtpharmakologische innovative Therapieansätze vorgestellt, die auf dem Prinzip der Neuromodulation beruhen, mit Fokus auf der repetitiven peripheren Magnetstimulation

Adoptive transfer of allergen-expressing B cells prevents IgE-mediated allergy

IntroductionProphylactic strategies to prevent the development of allergies by establishing tolerance remain an unmet medical need. We previously reported that the transfer of autologous hematopoietic stem cells (HSC) expressing the major timothy grass pollen allergen, Phl p 5, on their cell surface induced allergen-specific tolerance in mice. In this study, we investigated the ability of allergen-expressing immune cells (dendritic cells, CD4+ T cells, CD8+ T cells, and CD19+ B cells) to induce allergen-specific tolerance in naive mice and identified CD19+ B cells as promising candidates for allergen-specific cell therapy.MethodsFor this purpose, CD19+ B cells were isolated from Phl p 5-transgenic BALB/c mice and transferred to naive BALB/c mice, pre-treated with a short course of rapamycin and an anti-CD40L antibody. Subsequently, the mice were subcutaneously sensitized three times at 4-week intervals to Phl p 5 and Bet v 1 as an unrelated control allergen. Allergen-expressing cells were followed in the blood to monitor molecular chimerism, and sera were analyzed for Phl p 5- and Bet v 1-specific IgE and IgG1 levels by RBL assay and ELISA, respectively. In vivo allergen-induced lung inflammation was measured by whole-body plethysmography, and mast cell degranulation was determined by skin testing.ResultsThe transfer of purified Phl p 5-expressing CD19+ B cells to naive BALB/c mice induced B cell chimerism for up to three months and prevented the development of Phl p 5-specific IgE and IgG1 antibody responses for a follow-up period of 26 weeks. Since Bet v 1 but not Phl p 5-specific antibodies were detected, the induction of tolerance was specific for Phl p 5. Whole-body plethysmography revealed preserved lung function in CD19+ B cell-treated mice in contrast to sensitized mice, and there was no Phl p 5-induced mast cell degranulation in treated mice.DiscussionThus, we demonstrated that the transfer of Phl p 5-expressing CD19+ B cells induces allergen-specific tolerance in a mouse model of grass pollen allergy. This approach could be further translated into a prophylactic regimen for the prevention of IgE-mediated allergy in humans

Fluorescent S-layer fusion proteins

Diese Arbeit beschreibt die Herstellung und Charakterisierung von fluoreszierenden S-Schicht Fusionsproteinen als Bausteine für die Entwicklung von nano-strukturierten monokristallinen Proteinbeschichtungen auf Siliziumpartikel mit kontrollierten Fluoreszenzeigenschaften. Das S-Schichtprotein SgsE vom Geobacillus stearothermophilus NRS 2004/3a wurde mit den pH-Wert abhängigen cyanen, grünen und gelben Fluoreszenzproteinen (Varianten des grünen Fluoreszenzproteins GFP) und dem roten Fluoreszenzprotein mRFP1 fusioniert. Diese fluoreszierenden S-Schicht Fusionsproteine, die gleichzeitig als Matrix und optische Sensoren dienen, re-assemblieren in Lösung und auf Siliziumpartikel indem sie 2D Nanostrukturen mit p2 Gittersymmetrie ausbilden (a=11 0.5 nm, b=14 0.4 nm, g=80 1). Fluorimetrie, konfokal Mikroskopie und Durchflusszytometrie wurden für die Messung der pH-Wert Abhängigkeit herangezogen. Die fluoreszierenden S-Schicht Fusionsproteine können als pH-Sensor verwendet werden wobei am berechneten pKa Wert (pH6 pH5) die Fluoreszenzintensität um 50 % sinkt. Die GFP Varianten zeigen zwischen pH4 und pH3 keine Fluoreszenz, während der Chromophor vom mRFP1 in sauren Bedingungen nur wenig beeinträchtigt wird. Am isoelektrischen Punkt der beschichteten Partikel (pH4.6 0.2) konnte man eine Zunahme der Partikelaggregation im Durchflusszytometer beobachten. Das cyane und gelbe Fluoreszenzprotein wurden herangezogen, um so ein bi-fluoreszierendes S-Schicht Tandemfusionsprotein mit der Möglichkeit zum Resonanz Energietransfer herzustellen. Eine Transfereffizienz von 20% und ein molekularer Abstand zwischen Donor (cyan) und Akzeptor (gelb) Chromophor von ca. 6.2 nm wurden nachgewiesen. Dieses Tandemprotein re-assemblierte auf festen Trägern. Die außerordentliche Kombination von Fluoreszenz und Selbst-Assemblierung von bi-funktionellen S-Schicht Tandemfusionsproteinen macht sie zu einem vielversprechenden Werkzeug in der Nanobiotechnologie.This work describes the construction and characterisation of fluorescent S-layer fusion proteins used as building blocks for the fabrication of nanostructured monomolecular biocoatings on silica particles with defined fluorescence properties. The S-layer protein SgsE of Geobacillus stearothermophilus NRS 2004/3a was fused with the pH-dependant cyan, green and yellow variant of the green fluorescent protein (GFP) and the red fluorescent protein mRFP1. These fluorescent S-layer fusion proteins, acting as scaffold and optical sensing element simultaneously, were able to reassemble in solution and on silica particles forming 2D nanostructures with p2 lattice symmetry (a=11 0.5 nm, b=14 0.4 nm, g=80 1). The pH-dependant fluorescence behaviour was studied with fluorimetry, confocal microscopy and flow cytometry. These fluorescent S-layer fusion proteins can be used as pH-sensor. 50% of the fluorescence intensity decreases at their calculated pKa values (pH6 pH5). The fluorescence intensity of the GFP variants vanished completely between pH4 and pH3 whereas the chromophore of the red protein mRFP1 was only slightly affected in acidic conditions. At the isoelectric point of the S-layer coated silica particles (pH4.6 0.2) an increase in particle aggregation was detected by flow cytometry. The cyan and yellow fluorescent proteins were chosen to create a bi-fluorescent S-layer tandem fusion protein with the possibility for resonance energy transfer (FRET). A transfer efficiency of 20% and a molecular distance between the donor (ECFP) and acceptor (YFP) chromophores of around 6.2 nm could be shown. This bi-fluorescent ECFP-SgsE-YFP tandem fusion protein was able to reassemble on solid surfaces. The remarkable combination of fluorescence and self-assembly and the design of bi-functional S-layer tandem fusion protein matrices makes them to a promising tool in nanobiotechnology.Autorin: Birgit KainzAbweichender Titel laut Übersetzung der Verfasserin/des VerfassersZsfassung in dt. SpracheWien, Univ. für Bodenkultur, Diss., 2010OeBB(VLID)193139

Coupling of a Major Allergen to the Surface of Immune Cells for Use in Prophylactic Cell Therapy for the Prevention of IgE-Mediated Allergy

Up to a third of the world’s population suffers from allergies, yet the effectiveness of available preventative measures remains, at large, poor. Consequently, the development of successful prophylactic strategies for the induction of tolerance against allergens is crucial. In proof-of-concept studies, our laboratory has previously shown that the transfer of autologous hematopoietic stem cells (HSC) or autologous B cells expressing a major grass pollen allergen, Phl p 5, induces robust tolerance in mice. However, eventual clinical translation would require safe allergen expression without the need for retroviral transduction. Therefore, we aimed to chemically couple Phl p 5 to the surface of leukocytes and tested their ability to induce tolerance. Phl p 5 was coupled by two separate techniques, either by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) or by linkage via a lipophilic anchor, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-poly(ethylene glycol)-maleimide (DSPE-PEG-Mal). The effectiveness was assessed in fresh and cultured Phl p 5-coupled cells by flow cytometry, image cytometry, and immunofluorescence microscopy. Chemical coupling of Phl p 5 using EDC was robust but was followed by rapid apoptosis. DSPE-PEG-Mal-mediated linkage was also strong, but antigen levels declined due to antigen internalization. Cells coupled with Phl p 5 by either method were transferred into autologous mice. While administration of EDC-coupled splenocytes together with short course immunosuppression initially reduced Phl p 5-specific antibody levels to a moderate degree, both methods did not induce sustained tolerance towards Phl p 5 upon several subcutaneous immunizations with the allergen. Overall, our results demonstrate the successful chemical linkage of an allergen to leukocytes using two separate techniques, eliminating the risks of genetic modifications. More durable surface expression still needs to be achieved for use in prophylactic cell therapy protocols