Mimotopes for therapy of grass pollen induced asthma

Die Immuntherapie mit Allergenen ist heute noch immer mit unerwünschten Nebenwirkungen assoziiert. Zum Teil ist dies bedingt durch die Induktion von anaphylaktogenen Antikörpern, die gegen irrelevante Moleküle oder gar irrelevante Epitope der eingesetzten Allergene gerichtet sind. Dies könnte durch epitopspezifische Immuntherapie verhindert werden, bei der Mimotope oder anti-idiotypische Antikörper mit Mimikry-Potential (Review in Chapter I) anstelle von Allergenen eingesetzt werden. Das Ziel dieser Doktorarbeit war es, die Mimotoptherapie gegen Gräserpollenallergie weiter zu optimieren und die hierfür notwendigen proof of concept Experimente zu etablieren und durchzuführen. Im ersten experimentellen Ansatz (Chapter II), wurde ein Mausmodell für allergisches Asthma für die Sensibilisierung mit dem Graspollen-Hauptallergen Phl p 5 adaptiert. Akutes Asthma wurde durch systemische Sensibilisierung, gefolgt von inhalativer Exposition mit dem Allergen induziert, wodurch Mukussekretion, Lymphfollikelbildung und eosinophile Entzündung der Atemwege ausgelöst wurden. Um zwischen allergischem Asthma und un-spezifisch getriggerter Hyperreagibilität zu unterscheiden, fügten wir noch Ovalbumin (OVA) als ein irrelevantes Kontrollallergen für die Aerosolisierung von Phl p 5-allergischen Mäusen hinzu. In einem neuartigen Ansatz führten wir Analysen des Austausches der Atemgase mithilfe indirekter Kalorimetrie vor und nach Provokationen mit aerosolisierten Antigenen durch. Verstärkte Atmung konnte nach 48 Stunden nur in der mit Phl p 5 nebulisierten Mausgruppe festgestellt werden, charakterisiert durch erhöhte O2 Konsumation und CO2 Freisetzung in die Ausatemluft. Obwohl die Atemwegsentzündung der mit OVA provozierten Gruppe generell schwächer als in Mäusen mit allergischem Asthma war, zeigten beide Gruppen eine signifikante Abnahme der motorischen Aktivität und wiesen Azidose kombiniert mit einem prägnanten Abfall von roten und weißen Blutkörperchen in der Peripherie auf, gepaart mit deren Umverteilung in die Lunge. Daher ist die pathophysiologische Relevanz dieses Mausmodells erwiesen. Es dient aus diesem Grund nicht nur der Erforschung der Mechanismen von allergeninduziertem Asthma versus nicht-spezifischer Atemwegsprovokation, sondern eignet sich auch für proof of concept Studien von Mimotopvakzinen. Folglich zeigt Chapter III einen Ansatz, bei dem solchermaßen sensibilisierte Phl p 5-asthmatische Mäuse zur therapeutischen Vakzinierung mit einem synthetisch hergestellten Mimotop gekoppelt an KLH, eingesetzt wurden. Als Mimotop-behandelte Mäuse mit dem aerosolisierten Phl p 5 re-exponiert wurden um die Allergenexposition in der Pollensaison beim Menschen nachzuahmen, wiesen diese eine signifikant geringere Zahl an Eosinophilen in der Bronchoalveolarlavage und im peribronchiolaren Gewebe, sowie eine beeindruckende Herunterregulation der Mukusproduktion im Vergleich zu den „sham“-behandelten Tieren oder zu Mäusen, die nur mit KLH behandelt wurden, auf. Um die Mimotoptechnologie weiter zu verbessern, konstruierten wir in Chapter IV eine Minigen Vakzine, indem wir das im Asthmamodell verwendete Peptidmimotop in eine DNA Vakzine übersetzten. Wir verglichen parallel zwei verschiedene Verabreichungswege für die Immunisierung mit der Genvakzine. Die Applikation mit der Gene gun war mehr mit einer Th2 –ausgerichteten Immunantwort assoziiert. Im Gegensatz dazu wurde durch intradermale Injektion des Minigenkonstruktes keine IgE Antwort induziert, sondern die Produktion blockierender IgG Antikörper ausgelöst, welche das allergenvermittelte Crosslinking von Effektorzell-gebundenem IgE inhibieren konnten. Darüber hinaus konnte keine Proliferation und Zytokinproduktion von allergen-spezifischen T-Zellen beobachtet werden. Die präklinischen Befunde welche in dieser Doktorarbeit erhoben wurden deuten darauf hin, dass Mimotope in Peptid oder DNA Form therapeutisches Potential für die Behandlung von allergischen Erkrankungen, im besonderen Asthma, besitzen. Dies ist wahrscheinlich auf die Tatsache zurückzuführen, dass Mimotope ausschließlich Allergen- B-Zellepitope repräsentieren, jegliche Aktivierung pro-inflammatorischer T-Lymphozyten jedoch ausgeschlossen werden kann.Allergen immunotherapy is today still associated with undesired side effects. This is in part due to the induction of anaphylactogenic antibodies directed against irrelevant molecules or even irrelevant epitopes within the applied allergens. This could be avoided by epitope-specific immunotherapy using mimotope peptides or anti-idiotypic Fab fragments with mimicry potential (reviewed in Chapter I) instead of allergens. This thesis aimed to further optimize mimotope immunotherapy against grass pollen allergy, and to set up and perform the necessary proof of concept studies. Therefore, in the first experimental approach (Chapter II), a mouse model of allergic asthma was adapted to sensitization with grass pollen major allergen Phl p 5. Acute asthma was induced via systemic sensitization followed by inhalative allergen challenge rendering mucus secretion, lymphoid follicle formation and eosinophilic inflammation of the airways. To discriminate allergic asthma from non-specifically triggered airway hyperreactivity, we added ovalbumin (OVA) being an irrelevant control antigen for aerosolization of Phl p 5-allergic mice. In an innovative approach we performed respiratory gas exchange measurements by indirect calorimetry before and after challenges with aerosolized antigens. Enhanced respiration was found up to 48 hrs after provocation in the Phl p 5-nebulized mouse group only, characterized by significantly increased consumption of O2 and release of CO2 into exhaled air. Although airway inflammation of the OVA challenged group was overall milder than in mice with allergic asthma, both groups showed significantly less motor activity and exhibited acidosis combined with a sharp decline of white and red blood cells shown to be redistributed to the lung. This mouse asthma model therefore proved to be of pathophysiological relevance, being suitable not only for exploring the mechanisms of allergen- induced asthma versus non-specific airway provocation, but importantly also for proof of concept studies for mimotope vaccines. Consequently, Chapter III shows an approach using in such a way sensitized Phl p 5-asthmatic mice for therapeutical vaccination with a synthetic peptide mimotope linked to carrier KLH. When mimotope-treated mice were then re-exposed with the aerosolized Phl p 5 to mimic allergen re-exposure of humans in the pollen season, they exhibited significantly lower eosinophil numbers in the bronchoalveolar lavage and peribronchiolar tissue as well as an impressive downregulation of mucus production as compared to sham-treated animals or animals treated with KLH only. To further improve mimotope technology, we constructed in Chapter IV a minigene vaccine by translating the same peptide mimotope as used in the asthma model into a DNA vaccine. We compared head to head two different administration routes for immunization with the gene vaccine. Whereas gene gun bombardment was found to be associated with a Th2 biased immune response, intradermal injection did not induce IgE production but rendered blocking IgG antibodies able to inhibit allergen-mediated crosslinking of effector cell-bound IgE. In addition, no proliferation and cytokine production of allergen-specific T cells were observed. The pre-clinical evidence collected in this thesis work suggests that mimotopes in peptide or DNA form have therapeutic potency for the treatment of allergic diseases. This is possibly due to the fact that they represent pure B cell-epitope mimics and completely avoid the activation of any pro-inflammatory T-lymphocytes

Pilot Study on Potential Impacts of Fisheries-Induced Changes in Zooplankton Mortality on Marine Biogeochemistry

In this pilot study we link the yield of industrial fisheries to changes in the zooplankton mortality in an idealized way accounting for different target species (planktivorous fish—decreased zooplankton mortality; large predators—increased zooplankton mortality). This indirect approach is used in a global coupled biogeochemistry circulation model to estimate the range of the potential impact of industrial fisheries on marine biogeochemistry. The simulated globally integrated response on phytoplankton and primary production is in line with expectations—a high (low) zooplankton mortality results in a decrease (increase) of zooplankton and an increase (decrease) of phytoplankton. In contrast, the local response of zooplankton and phytoplankton depends on the region under consideration: In nutrient-limited regions, an increase (decrease) in zooplankton mortality leads to a decrease (increase) in both zooplankton and phytoplankton biomass. In contrast, in nutrient-replete regions, such as upwelling regions, we find an opposing response: an increase (decrease) of the zooplankton mortality leads to an increase (decrease) in both zooplankton and phytoplankton biomass. The results are further evaluated by relating the potential fisheries-induced changes in zooplankton mortality to those driven by CO2 emissions in a business-as-usual 21st century emission scenario. In our idealized case, the potential fisheries-induced impact can be of similar size as warming-induced changes in marine biogeochemistry

Factors of Reading Ability and their Contribution to Reading : A Comparison of Children with Different Levels of Reading Ability,in Relation to The Simple View of Reading

The simple view of reading is a theory in which reading is divided into decoding and linguistic comprehension. The theory has been used to explain and to subgroup reading disabilities. Research has also investigated if additional factors are of relevance to reading. In the present study, decoding was divided into the factors orthographic decoding and phonological decoding, while comprehension was divided into word comprehension and grammatical comprehension. The purpose was to analyze these factors and their contribution to reading ability in children in fourth grade: one group with reading difficulties (n=36) and one group with typical reading ability (n=36). Results were analyzed using multiple regression and the two groups were compared. The contribution of non-verbal ability and general processing speed was also studied. The results showed differences between the groups. Decoding made the largest contribution to reading ability for the children with reading difficulties, while linguistic comprehension contributed the most for the children with typical reading ability. Differences were also found in how much the underlying factors contributed to decoding and linguistic comprehension, respectively. Non-verbal ability and general processing speed made small contributions, for both groups, to reading ability, decoding and linguistic comprehension, respectively. However, the two factors increased the explained variance in reading ability for the children with reading difficulties. In this group the two factors in The simple view of reading explained somewhat less of the variance in reading ability than for the children with typical reading ability. The study shows that decoding and comprehension are useful predictors of reading ability, and also indicates that The simple view of reading is less appropriate for children with reading difficulties.The simple view of reading är en teori om läsning där läsförmåga delas in i vkodning och språkförståelse. Teorin har bland annat använts för att kartlägga lässvårigheter. Forskning har undersökt om ytterligare faktorer har betydelse för läsning. I föreliggande studie delades avkodning in i faktorerna ortografisk avkodning och fonologisk avkodning, medan språkförståelse delades in i ordförståelse och satsförståelse. Syftet var att undersöka faktorernas bidrag till läsförmåga hos barn i skolår fyra: en grupp med lässvårigheter (n=36) och en grupp med typisk läsförmåga (n=36). Testresultaten analyserades med multipel regressionsanalys och de båda grupperna jämfördes. Bidragen från icke-verbal förmåga och generell processhastighet studerades också. Resultaten visade att profilerna skilde sig åt mellan grupperna. För barnen med lässvårigheter gav avkodning störst bidrag till läsförmåga, medan språkförståelse var den mest bidragande faktorn för barnen med typisk läsförmåga. Skillnader fanns även i hur underliggande faktorer bidrog till avkodning respektive språkförståelse. Icke-verbal förmåga och generell processhastighet visade sig ge små bidrag till läsförmåga, avkodning respektive språkförståelse, för båda grupperna. När dessa två faktorer lades till framkom dock en relativt stor ökning av den förklarade variansen i läsförmåga hos barnen med lässvårigheter. Hos denna grupp var den förklarade variansen i läsförmåga av de två faktorerna i The simple view of reading något mindre än hos barnen med typisk läsförmåga. Sammantaget visar studien att avkodning och förståelse är användbara prediktorer till läsförmåga, samtidigt som resultaten antyder att The simple view of reading är mindre tillämpbar för barn med lässvårigheter

Design, implementation and evaluation online environment regarding the distance education of teachers regarding the teaching of greek language as a second foreign language based on the programs of EDIAMME for the Greek language education abroad

Στα πλαίσια της παρούσας διπλωματικής εργασίας, δημιουργήθηκε εκπαιδευτικό υλικό για την εξ αποστάσεως επιμόρφωση εκπαιδευτικών σχετικά με τη διδασκαλία της Ελληνικής ως δεύτερης/ξένης γλώσσας στα προγράμματα του Ε.ΔΙΑ.Μ.ΜΕ. για την ελληνόγλωσση εκπαίδευση στο εξωτερικό. Το υλικό, αφού σχεδιάστηκε στο διαδικτυακό λογισμικό του H5P και τοποθετήθηκε στην πλατφόρμα του Chamilo, δόθηκε και αξιολογήθηκε από πτυχιούχους παιδαγωγικών τμημάτων και τμημάτων φιλολογίας. Το εκπαιδευτικό υλικό αποτελείται από οκτώ επιμέρους ενότητες, με την κάθε μια να εστιάζει σε διαφορετικό θεματικό άξονα για την καλύτερη κατανόηση του εγχειρήματος. Το εκπαιδευτικό υλικό αξιολογήθηκε ως προς τα χαρακτηριστικά του από οκτώ αξιολογήτριες. Συγκεκριμένα, οι συμμετέχουσες στην έρευνα κλήθηκαν να απαντήσουν σε ένα ερωτηματολόγιο αξιολόγησης του εκπαιδευτικού υλικού ως προς την επιστημονική συνοχή και τεκμηρίωση του εκπαιδευτικού υλικού, την απλή και κατανοητή παρουσίαση του γνωστικού αντικειμένου, την ευχρηστία του εκπαιδευτικού υλικού, την υποστήριξη και την καθοδήγηση στη μελέτη του, την αλληλεπίδραση ανάμεσα σε εκπαιδευτικό υλικό και εκπαιδευόμενο, τη δυνατότητα αναστοχασμού και αυτοαξιολόγησης από το εκπαιδευτικό υλικό στον εκπαιδευόμενο, τη διατύπωση του σκοπού και των προσδοκώμενων αποτελεσμάτων κάθε διδακτικής ενότητας και τη δημιουργία του εκπαιδευτικού υλικού σύμφωνα με τις αρχές της Πολυμεσικής Μάθησης. Τα αποτελέσματα της έρευνας έδειξαν ότι στην πλειοψηφία τους οι αξιολογήτριες σχολίασαν πολύ θετικά το εκπαιδευτικό υλικό. ενώ έκαναν και ενδιαφέρουσες παρατηρήσεις σε επιμέρους σημεία. Συμπερασματικά, το υλικό κρίθηκε εξαιρετικά κατανοητό και χρήσιμο από την πλειοψηφία των συμμετεχουσών και κρίθηκε ικανό να πραγματώσει τον σκοπό του.In the context of this master thesis, an educational material was established regarding the distance education of teachers regarding the teaching of Greek language as a second / foreign language based on the programs of E.DIA.M.ME. for Greek language’s education abroad. The educational material was firstly designed in the H5P online software, and then implemented on the Chamilo platform. Then it was given and evaluated by experienced teachers in this specific matter. The educational material consists of eight sub-sections, each of which presents a specific topic for the best and most accurate understanding of the project. The educational material was evaluated in terms of its characteristics by eight teachers. Specifically, the teachers participating in the research were asked to answer a questionnaire regarding the evaluation of the educational material in terms of scientific coherence and documentation, the simplicity and understanding of the presentation of the Cognitive Object, the usability, the support and guidance in studying terms, the interaction between the material and the learner, the possibility of self-reflection and self-evaluation to the learner, the formulation of the purpose and the expected results of each teaching chapter and the evaluation of the material according to the principles of Multimedia Learning. The results of the research showed that the majority of the teachers were excited, and confident on the educational material, and their comments were, at the majority of the cases, very positive. On the other hand, there were various negative comments in some special cases. In conclusion, the material was considered extremely understandable and useful by the majority of participants and is assessed as capable for its purpose

Anti-Ids in Allergy: Timeliness of a Classic Concept

Anti-idiotypic antibodies (anti-ids) are part of natural immune responses with regulatory capacity. Their effect on an antigen-specific, so-called Ab1 antibody response, is dependent on 1) the original antigen, which they mirror, being Ab2 antibodies, and 2) their isotype. In the case of IgE-mediated allergy, natural anti-ids against allergen-specific IgE represent internal images of allergen molecules. A key biologic feature of allergens is that they can crosslink IgE, expressed by B-lymphocytes or passively bound via high affinity receptors to effector cells, which renders cellular activation. Therefore, the IgE cross linking capability of anti-ids determines whether they dampen or enhance immediate-type hypersensitivity. Correspondingly to classic antiallergen blocking IgG antibodies, anti-ids may also interact with inhibitory FcγRIIb receptors and, thereby, down-regulate TH2-type inflammation. Anti-ids and other B-cell epitope mimetics, like mimotopes and DARPins, represent antigen surrogates, which can be used for vaccination. Intriguingly, they may induce antibody responses without activating potentially proinflammatory, antiallergen T-lymphocytes. Taken together, collective evidence suggests that anti-ids, although representing immunologic classics, are a timeless concept in allergology. Keywords: anti-idiotypes, allergy, vaccination, FcεRI, FcγRIIb, blockin

Anti-idiotypic Fab Fragments Image a Conserved N-terminal Epitope Patch of Grass Pollen Allergen Phl p 1

BACKGROUND AND AIMS: Naturally occurring anti-idiotypic antibodies structurally mimic the original antibody epitope. Anti-idiotypes, therefore, are interesting tools for the portrayal of conformational B-cell epitopes of allergens. In this study we used this strategy particularly for major timothy grass pollen (Phleum pratense) allergen Phl p 1. METHODS AND RESULTS: We used a combinatorial phage display library constructed from the peripheral IgG repertoire of a grass pollen allergic patient which was supposed to contain anti-idiotypic Fab specificities. Using purified anti-Phl p 1 IgG for biopanning, several Fab displaying phage clones could be isolated. 100 amplified colonies were screened for their binding capacity to anti-Phl p 1-specific antibodies, finally resulting in four distinct Fab clones according to sequence analysis. Interestingly, heavy chains of all clones derived from the same germ line sequence and showed high homology in their CDRs. Projecting their sequence information on the surface of the natural allergen Phl p 1 (PDB ID: 1N10) indicated matches on the N-terminal domain of the homo-dimeric allergen, including the bridging region between the two monomers. The resulting epitope patches were formed by spatially distant sections of the primary allergen sequence. CONCLUSION: In this study we report that anti-idiotypic specificities towards anti-Phl p 1 IgG, selected from a Fab library of a grass pollen allergic patient, mimic a conformational epitope patch being distinct from a previously reported IgE epitope area

Peripheral Erythrocytes Decrease upon Specific Respiratory Challenge with Grass Pollen Allergen in Sensitized Mice and in Human Subjects

<div><p>Background and Aims</p><p>Specific hyper-responsiveness towards an allergen and non-specific airway hyperreactivity both impair quality of life in patients with respiratory allergic diseases. We aimed to investigate cellular responses following specific and non-specific airway challenges locally and systemically in i) sensitized BALB/c mice challenged with grass pollen allergen Phl p 5, and in ii) grass pollen sensitized allergic rhinitis subjects undergoing specific airway challenge in the Vienna Challenge Chamber (VCC).</p><p>Methods and Results</p><p>BALB/c mice (n = 20) were intraperitoneally immunized with grass pollen allergen Phl p 5 and afterwards aerosol challenged with either the specific allergen Phl p 5 (n = 10) or the non-specific antigen ovalbumin (OVA) (n = 10). A protocol for inducing allergic asthma as well as allergic rhinitis, according to the united airway concept, was used. Both groups of exposed mice showed significantly reduced physical activity after airway challenge. Specific airway challenge further resulted in goblet cell hyperplasia, enhanced mucous secretion, intrapulmonary leukocyte infiltration and lymphoid follicle formation, associated with significant expression of IL-4, IL-5 and IL-13 in splenocytes and also partially in lung tissue. Concerning circulating blood cell dynamics, we observed a significant drop of erythrocyte counts, hemoglobin and hematocrit levels in both mouse groups, challenged with allergen or OVA. A significant decrease in circulating erythrocytes and hematocrit levels after airway challenges with grass pollen allergen was also found in grass pollen sensitized human rhinitis subjects (n = 42) at the VCC. The effects on peripheral leukocyte counts in mice and humans however were opposed, possibly due to the different primary inflammation sites.</p><p>Conclusion</p><p>Our data revealed that, besides significant leukocyte dynamics, particularly erythrocytes are involved in acute hypersensitivity reactions to respiratory allergens. A rapid recruitment of erythrocytes to the lungs to compensate for hypoxia is a possible explanation for these findings.</p></div

Aerosol allergen challenge specifically attracts leukocytes to lungs.

<p>(<b>A</b>) Volume of lymphoid follicles V/S (fol, epi) per total surface area of airway epithelial basal membrane [µm] (<b>B</b>) and percentage of airway walls containing eosinophils in fields of view (FOVs). Lung histology representatives with respect to lymphoid follicles from Phl p 5 sensitized and (<b>C</b>) OVA challenged or (<b>D</b>) Phl p 5 challenged and (<b>E</b>) naïve mice. Data are presented by boxplots (median, quartiles, minimum and maximum) and n = 5 mice/group. *, p<0.05, **, p<0.01 and n.s. means not significant.</p

Physical activity after airway challenge.

<p>Rearing of Phl p 5-allergic in allergen (red squares) or nonspecific antigen (black points) challenged mice. Data are mean ± SD and n = 5 mice/group. ***, p<0.001.</p