<p>Ambrosia artemisiifolia L., invazivna korovsko-ruderalna biljka, poslednjih<br />godina u na&scaron;oj zemlji predstavlja izuzetno veliki problem, ne samo za poljoprivredu<br />nego i za stanovni&scaron;tvo, izazivajući svojim polenom jake alergijske i druge zdravstvene<br />komplikacije. Shvatajući probleme koje ona izaziva, sve zemlje u kojima ambrozija<br />predstavlja veliki, pre svega zdravstveni problem, ulažu ogromne napore, izdvajaju<br />velika finansijska sredstva, organizuju stanovni&scaron;tvo, &scaron;kole, vojsku, javne službe,<br />sredstva informisanja, donose zakone, uredbe i deklaracije, da bi ograničili njeno<br />&scaron;irenje i osigurali njeno suzbijanje, dok se u na&scaron;oj zemlji jo&scaron; uvek nedovoljno ozbiljno<br />shvata ovaj problem.<br />Kako se radi o biljci koja se razmnožava samo generativno (semenom), istraživanja<br />&#39;&#39;banke semena&#39;&#39; pelenaste ambrozije u određenim zemlji&scaron;nim profilima omogućavaju<br />preciznu prognozu njene pojave na određenim stani&scaron;tima, &scaron;to kasnije znatno olak&scaron;ava<br />suzbijanje kako mehaničkim putem tako i primenom herbicida. Zemlji&scaron;ni uzorci za<br />analizu rezervi semena su uzeti, sa karakterističnih ruderalnih stani&scaron;ta, na području<br />Novi Sad, Zrenjanin i Ruma. Obzirom da se radi o ruderalnim stani&scaron;tima, na svim<br />istraživanim lokalitetima utvrđena je relativno visoka brojnost semena pelenaste<br />ambrozije. Prikazano istraživanje je prvo ovog tipa sprovedeno na teritoriji Republike<br />Srbije.<br />Ova proučavanja su od izuzetnog naučnog značaja budući da obuhvataju poznavanje<br />osnovnih biolo&scaron;kih osobina semena ambrozije, kao &scaron;to su morfolo&scaron;ke osobine,<br />karakteristike klijanja, nicanja i načina rasprostiranja.<br />Za potrebe ispitivanja klijavosti semena A. artemisifolia 2007. godine sa teritorije 15<br />gradskih i prigradskih naselja Grada Novog Sada prikupljeni su plodovi pelenaste<br />ambrozije. Od ukupne količine prikupljenog semena pelenaste ambrozije, polovina je<br />odložena u hladnu komoru sa temperaturom od +4&ordm;C a drugi deo materijala je odložen<br />u komoru sa temperaturom od - 8&ordm;C da bi se simulirali zimski uslovi, odnosno kako bi<br />seme bilo izloženo jarovizaciji. Naklijavanje izdvojenog semena vr&scaron;eno je u klimakomori,<br />pod uslovima povoljnim za nicanje semena. Nakon 9, 14, 21 i 28 dana<br />ocenjivana je klijavost semena. Utvrđena je dobra klijavost semena pelenaste<br />ambrozije, koja je bila značajno veća kod stratifikovanog semena.<br />Na osnovu rezultata dobijenih tokom ispitivanja rasprostranjenosti i suzbijanja<br />korovsko-ruderalne alergene biljke Ambrosia artemisifolia L. u regiji gradova Novi<br />Sad, Zrenjanin i Ruma, izvedeni su sledeći zaključci: Obavljena proučavanja<br />rasprostranjenosti i suzbijanja korovsko-ruderalne alergene biljke Ambrosia<br />artemisifolia L. u 15 zona grada Novog Sada, 10 zona u Zrenjaninu i 7 u Rumi,<br />ukazuju na izuzetnu zastupljenost ove biljke. Na osnovu proučavanja rasprostranjenosti i praćenja feno-faza razvoja pelenaste ambrozije obavljeno je<br />mehaničko ko&scaron;enje ručnim ili motornim kosilicama. Imajući u vidu stalne napore u<br />pogledu za&scaron;tite životne sredine koja podrazumeva na prvom mestu pozitivan uticaj na<br />zdravlje ljudi, na gradskim lokacijama tretman herbicidom glifosat nije primenjen, već<br />samo u nenastanjenim zonama i na zapu&scaron;tenim poljoprivrednim povr&scaron;inama i<br />utrinama. Prilikom suzbijanja pelenaste ambrozije mehaničkim putem, istaknuto je da<br />je ko&scaron;enje potrebno obaviti 3-4 puta tokom jedne vegetacione sezone, usled relativno<br />brze regeneracije poko&scaron;enih biljaka. Hemijsko suzbijanje ambrozije primenom<br />herbicida glifosat, pokazalo se kao znatno efikasnije jer je za njeno suzbijanje bio<br />dovoljan uglavnom jedan tretman.</p> / <p>Ambrosia artemisiifolia L., an invasive weed-ruderal plant in recent years in<br />our country is a very big problem, not only for agriculture but also for the human<br />population, causing her severe pollen allergy and other health complications.<br />Recognizing the problems that it causes, all countries where ragweed is a large,<br />primarily health problem, invest huge efforts, allocate significant financial resources,<br />organized residents, schools, the military, the media, legislate, to limit its spread and<br />ensure its suppression, while our country does not yet have a serious approach to this<br />problem.<br />Since this is a plant that reproduces a generative (seed), &#39;&#39;seed bank&#39;&#39; research in some<br />soil profiles allow to forecast its occurrence at some sites, which greatly facilitates<br />the later contol of both mechanically and by herbicides. Soil samples for analysis of<br />seed reserves were taken with typical ruderal habitats, in Novi Sad, Zrenjanin and<br />Ruma. Since these are ruderal habitats, at all study sites, a relatively high number of<br />ragweed seeds apears. Our study is the first of this type that was conducted in the<br />Republic of Serbia.<br />These studies are of great scientific importance as it includes knowing of basic<br />biological properties of ragweed seeds, such as morphological characteristics, the<br />characteristics of germination, emergence and control methods.<br />For the purposes of testing seed germination A. artemisifolia in year 2007. samples<br />of ambrosia seeds were collected from 15 different localities in the territory of the<br />city and suburbs of Novi Sad. Collected seeds were splited on two halfs: one part was<br />delayed in the cold chamber with a temperature of +4 &deg; C and the second part of the<br />material is disposed in the chamber with a temperature of - 8 &deg; C to simulate winter<br />conditions, in purpose to achieve seed vernalization. Seed germination was<br />performed in air chamber under optimal conditions for seed germination. After 9, 14,<br />21 and 28 days seed germination was evaluated. Germination of stratified seeds was<br />significantly higher than germination of not stratified seeds.<br />Based on the obtained results of distribution and controll of weed-ruderal allergenic<br />plant Ambrosia artemisiifolia L. in the cities Novi Sad, Zrenjanin and Ruma, the<br />following conclusions were made: Results mapping and control of Ambrosia<br />artemisiifolia L. in 15 zones in the city of Novi Sad, 10 zones in Zrenjanin, and 7 in<br />Ruma, indicating significant presence of this plant. Based on the study of the<br />distribution and monitoring of ragweed growth stages, mechanical controll mesures<br />were performed manually, by trimmers or by tractor. Because of ongoing efforts to<br />protect the environment, in the urban locations herbicide treatment was not applied.<br />Glyphosate treatment was applied only in uninhabited areas, agricultural land and<br />wasteland. If ragweed is controlled mechanically, the cutting should be done 3-4<br />times per year due to the relatively rapid regeneration of plants. Chemical control of<br />ragweed (herbicide glyphosate), has proven much more effective. One treatment per<br />year was sufficient.</p>

Ng Sin Man. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 120-135). / Abstracts in English and Chinese. / Acknowledgements --- p.i / Abbreviations --- p.iii / Abstract --- p.vi / 撮要 --- p.ix / Publications --- p.xi / Table of contents --- p.xiii / Chapter Chapter 1 --- General Introduction / Chapter 1.1 --- Allergic Diseases and Allergic Inflammation --- p.1 / Chapter 1.1.1 --- Prevalence of Allergic Diseases --- p.1 / Chapter 1.1.2 --- Common Allergic Diseases: Allergic Asthma --- p.1 / Chapter 1.1.3 --- Common Allergic Diseases: Atopic Dermatitis --- p.2 / Chapter 1.1.4 --- Allergic Inflammation --- p.3 / Chapter 1.2 --- The Inflammatory Leukocytes: Mast Cells and Eosinophils --- p.6 / Chapter 1.2.1 --- Characteristics of Mast Cells --- p.6 / Chapter 1.2.2 --- Mast Cells Distribution --- p.8 / Chapter 1.2.3 --- Mast Cells Subtypes --- p.8 / Chapter 1.2.4 --- HMC-1 Cells --- p.9 / Chapter 1.2.5 --- Characteristics of Eosinophils --- p.12 / Chapter 1.3 --- Adhesion Molecules in Allergic Diseases --- p.15 / Chapter 1.3.1 --- Adhesion Molecules and Leukocyte Migration --- p.15 / Chapter 1.3.2 --- Selectin --- p.17 / Chapter 1.3.3 --- Intermolecular Adhesion Molecules --- p.17 / Chapter 1.3.4 --- Integrin --- p.18 / Chapter 1.4 --- Cytokines and Chemokines in Allergic Diseases --- p.18 / Chapter 1.4.1 --- IL-6 --- p.20 / Chapter 1.4.2 --- CXCL1 --- p.21 / Chapter 1.4.3 --- CXCL8 --- p.21 / Chapter 1.4.3 --- CCL2 --- p.22 / Chapter 1.5 --- Intercellular Signal Transduction Pathways in Inflammation --- p.24 / Chapter 1.5.1 --- RAS-RAF-mitogen-activated Protein Kinases --- p.24 / Chapter 1.5.2 --- Janus Kinase/ Signal Transducers and Activators of Transcriptions Pathway --- p.27 / Chapter 1.5.3 --- Nuclear Factor-KB Pathway --- p.29 / Chapter 1.5.4 --- Phosphoinositide 3-Kinase Pathway --- p.31 / Chapter 1.6 --- Aims and Scope of the Study --- p.33 / Chapter Chapter 2 --- Materials and Methods / Chapter 2.1 --- Materials --- p.35 / Chapter 2.1.1 --- HMC-1 Cell Line --- p.35 / Chapter 2.1.2 --- Human Buffer Coat --- p.35 / Chapter 2.1.3 --- Human Mast Cell Chymase and TLR ligands --- p.35 / Chapter 2.1.4 --- Media and Reagents for Cell Culture --- p.36 / Chapter 2.1.5 --- Reagents and Buffers for Purification of Human Eosinophils --- p.37 / Chapter 2.1.6 --- Reagents and Buffers for Flow Cytmetry --- p.38 / Chapter 2.1.7 --- Reagents and Buffers for Total RNA Extraction --- p.41 / Chapter 2.1.8 --- Reagents and Buffers for Reverse Transcription-Polymerase Chain Reaction (RT-PCR) --- p.42 / Chapter 2.1.9 --- Reagents and Buffers for Agarose Gel Electrophoresis --- p.45 / Chapter 2.1.10 --- Reagents and Buffers for Sodium Dodecyl Sulfate -polyacrylamide Gel Electrophoresis (SDS-PAGE) --- p.46 / Chapter 2.1.11 --- Reagents and Buffers for Western Blot Analysis --- p.48 / Chapter 2.1.12 --- Chemotactic Migration --- p.51 / Chapter 2.1.13 --- Signaling Transduction Inhibitors and Protein Synthesis Inhibitors --- p.51 / Chapter 2.2 --- Methods --- p.52 / Chapter 2.2.1 --- HMC-1 Cell Cultures --- p.52 / Chapter 2.2.2 --- Purification of Buffy Coat Eosinophils by MACS and Eosinophil Culture --- p.52 / Chapter 2.2.3 --- Total Cellular RNA Extraction --- p.53 / Chapter 2.2.4 --- RT-PCR --- p.54 / Chapter 2.2.5 --- Agarose Gel Electrophoresis --- p.55 / Chapter 2.2.6 --- Flow Cytometry Analysis --- p.55 / Chapter 2.2.7 --- Protein Array Analysis of Cytokine Release --- p.57 / Chapter 2.2.8 --- Quantitative Analysis ofCXCLl --- p.58 / Chapter 2.2.9 --- Total Protein Extraction --- p.58 / Chapter 2.2.10 --- SDS-PAGE --- p.58 / Chapter 2.2.11 --- Western Blot Analysis --- p.59 / Chapter 2.2.12 --- Chemotactic Migration Analysis --- p.60 / Chapter 2.2.13 --- Statistical Analysis --- p.60 / Chapter Chapter 3 --- Effects of Mast Cell Derived Chymase on Human Eosinophils and the Signaling Mechanisms: Implication in Allergic Inflammation / Chapter 3.1 --- Introduction --- p.61 / Chapter 3.2 --- Results --- p.65 / Chapter 3.2.1 --- Effects of Chymase on Eosinophil Survival --- p.65 / Chapter 3.2.2 --- Effects of Chymase on the Adhesion Molecule Expression of Eosinophils --- p.68 / Chapter 3.2.3 --- Effects of Chymase on the Chemokinetic Properties on Eosinophils --- p.71 / Chapter 3.2.4 --- Effects of Chymase on the Release of Chemokines and IL-6 from Eosinophils --- p.73 / Chapter 3.2.5 --- Signal Transduction Mechanism Involved in Regulating Chymase-induced Effects on Eosinophils --- p.78 / Chapter 3.3 --- Discussion --- p.71 / Chapter Chapter 4 --- TLR-mediated Effects and Signal Transduction Mechanism of HMC-1 Cells / Chapter 4.1 --- Introduction --- p.92 / Chapter 4.2 --- Results --- p.97 / Chapter 4.2.1 --- Expression of Adhesion Molecules on HMC-1 Cells --- p.95 / Chapter 4.2.2 --- TLR Expression Profile on HMC-1 Cells --- p.97 / Chapter 4.2.3 --- Effects of TLR ligands on HMC-1 Cell Adhesion Molecule Expressions --- p.99 / Chapter 4.2.4 --- TLR7-induced Phosphorylation of ERK and Effects of PD98059 on TLR7-induced ERK Phosphorylation --- p.104 / Chapter 4.2.5 --- Effect of TLR7 Ligand on HMC-1 Cells Cytokine Release --- p.108 / Chapter 4.3 --- Discussion --- p.110 / Chapter Chapter 5 --- Conclusions and Future Perspectives / Chapter 5.1 --- Conclusions --- p.115 / Chapter 5.2 --- Future Perspectives --- p.117 / References --- p.120 / Appendix --- p.136

The prevalence of food allergies is a growing concern in the United States. Approximately 8% of the pediatric population has some form of food allergy. Many of these children are either in the preschool and primary school setting, which is where the majority of allergic reactions occur. If the symptoms of a food allergy reaction are not treated within minutes of exposure, the results can be damaging or fatal. Evidence continues to demonstrate that preschool and school personnel do not feel trained or prepared should a severe reaction arise. The purpose of this evidence-based project was to determine if the implementation and instruction of food allergy guidelines and an educational in-service program on the treatment of food allergies would increase the knowledge and ability of preschool personnel to respond should a reaction occur. The adult learning theory of Knowles and Bandura's theory of self-efficacy were the theoretical frameworks for this project. This project incorporated a 40-minute educational in-service along with the introduction of food allergy guidelines including an emergency action plan and epinephrine auto-injector training. A pretest and posttest were administered prior to and following the educational in-service, respectively. A paired sample t test revealed there was a dramatic increase in knowledge following the educational in-service about food allergy management, recognition, and treatment. Preschool personnel felt more empowered to react should a food allergy reaction occur. By teaching preschool-personnel about food allergies, they will have the necessary resources that will support the creation of a safer environment for children challenged with food allergies.

Anhand der Daten aus der "Multizentrische Allergie Studie" (MAS) wird der natürliche Verlauf allergischer Erkrankungen und die Zusammenhänge unterschiedlicher Expositionen auf die Entwicklung von Allergien zu untersucht, um daraus verbesserte therapeutische und präventive Maßnahmen ableiten zu können. Methode MAS ist eine prospektive multizentrische Geburtskohortenstudie. 1990 wurden in fünf deutschen Städten 1314 Neugeborenen und deren Familien eingeschlossen (499 Atopie-Risikokinder und 815 zufällig ausgewählte Kinder ohne Atopierisiko). Symptome und Erkrankungen, die Lebenssituation der Familie, Risikofaktoren und Labordaten wurden jährlich über 7 Jahre erhoben. Die Datenanalyse erfolgte mittels multivariater Regressionsmodelle. Ergebnisse und Fazit Für das Serum-Gesamt-IgE wurden populationsbasierte Perzentilen für die ersten sechs Lebensjahre berechnet, die als "Normwerte-Tabellen" interpretiert werden können. Eine allergische Sensibilisierung entwickelt sich hauptsächlich während der ersten Lebensjahre. Am frühesten bilden sich Sensibilisierungen gegen Nahrungsmittelallergene aus, nach dem dritten Geburtstag überwiegen Sensibilisierungen gegen Inhalationsallergene mit Prävalenzen von 10% bis 20%. Als signifikante Risikofaktoren für eine Sensibilisierung erwiesen sich die Exposition gegenüber Umwelt(schad)stoffen wie dem Tabakrauch (OR=2,3), latexhaltigen Materialien in Krankenhäusern (OR=2,4) und verschiedenen Allergenen. Der stärkste Zusammenhang zeigte sich zwischen einer Exposition mit Innenraumallergenen und einer Sensibilisierung gegen diese Allergentypen. Weitere Risikofaktoren waren die Bereitschaft des Organismus, sehr früh in den ersten 2 Lebensjahren erhöhte Niveaus von IgE im Serum auszubilden, und eine atopische Prädisposition. Als wichtigste Risikofaktoren für die Entwicklung einer allergischen Rhinitis erwiesen sich das männliche Geschlecht (OR=2,4), keine weiteren Geschwister (OR=2,0), eine Sensibilisierung gegen Nahrungsmittel (OR=3,3), eine atopischen Dermatitis (OR=2,5) und eine atopische Familienanamnese (OR=3,0). Die prädiktive Wertigkeit einzelner Faktoren wurde gegeneinander abgegrenzt und das Atopierisiko bei unterschiedlichem Auftreten einzelner Prädiktoren berechnet. Trotz der guten prädiktiven Eigenschaften der Prädiktoren "hohes spezifisches IgE gegen Hühnerei" oder "dauerhafter Nachweis von IgE gegen Nahrungsmittel im Kleinkindalter" bedeutet die Verwendung dieser Parameter, dass eine Prädiktion frühestens 1-2 Jahre nach Geburt und erst nach wiederholter Bestimmung der IgE-Antikörper möglich ist. Auch wenn weiterhin Forschungsbedarf zur Allergieprävention besteht und die Wirksamkeit von einigen präventiven Maßnahmen noch systematisch evaluiert werden muss, sind auf einzelnen Feldern konkrete Handlungsanweisungen zur Prävention möglich. / Using data from the German Multicenter Allergy Study (MAS), we investigated the natural course of atopic diseases and the relationship of varied exposures to the development of allergies in order to develop more effective therapeutic and preventative treatment strategies. Methods The MAS is a prospective multicenter birth cohort study. In the year 1990, a total of 1314 newborns and their families from five German cities were included in the study, including 499 infants at high risk for atopy and 815 randomly selected infants with no known risk factors for atopy. Over a period of seven years, data was collected annually regarding symptoms, illnesses, family living situation and environment, risk factors, and laboratory tests. For data analysis we used multivariate regression models. Results and Conclusions From birth to the age of six, total serum IgE levels were calculated annually as population-based percentiles, and can thus be interpreted as a table of standard values. Allergic sensitization generally occurred within the first few years of life. Sensitization to food allergens occurred earliest, whereas sensitization to aeroallergens predominated after the age of three (with a prevalence of 10-20%). Significant risk factors for allergic sensitization included exposure to environmental pollutants/substances such as tobacco smoke (OR=2.3) or latex-containing medical products (OR=2.4). With regard to the relationship between exposure and subsequent sensitization to a specific allergen, the strongest correlation was observed with indoor allergens. Other risk factors included the early development of elevated serum IgE levels within the first year of life and an atopic predisposition. The most important risk factor for the development of allergic rhinitis was male gender (OR=2.4), no siblings (OR=2.0), sensitization to a food allergen (OR=3.3), atopic dermatitis (OR=2.5), and a family history of allergic disease (OR=3.0). The predictive value of individual factors was determined and the risk of developing atopic diseases calculated for individual predictors. In spite of the good predictive qualities of the factors " high values of specific IgE to hen's egg" and "long-lasting sensitization to food allergens during infancy", reliable prediction using these parameters can only be made 1-2 years after birth at the earliest and following a repeated measurement of IgE antibodies. There is still need for further research in allergy prevention, and the effectiveness of a number of preventative measures has yet to be evaluated in a systematic manner. Nevertheless, the results of this and other studies can already help physicians develop clearer, evidence-based strategies in the prevention of allergic disease.

Les effets anti-allergiques des bactéries lactiques sont suggérés par plusieurs études épidémiologiques, des essais cliniques et des modèles expérimentaux d’allergie. Cependant, les propriétés immunomodulatrices des bactéries lactiques sont sous-exploitées par les stratégies vaccinales développées pour combattre l’allergie et les mécanismes empruntés par ces bactéries pour moduler l’allergie restent peu caractérisés. Dès lors, nous avons caractérisé les propriétés immunomodulatrices qu’exerce Lactobacillus plantarum NCIMB8826, une bactérie lactique modèle, sur la cellule dendritique étant donné le rôle déterminant de cette cellule sur la réponse allergique. Nous montrons que L. plantarum induit une forte sécrétion d’IL-12 p40, d’IL-12 p70, de TNF-a mais une faible production d’IL-10. Cette faculté à induire la sécrétion de cytokines polarisantes dépend de TLR2, de TLR9, de MyD88, de NF-kB, des MAPKs (en particulier JNK, p38 et ERK 1/2), de la composition de l’acide lipotéichoïque de L. plantarum et de CD14. Nous montrons aussi que l’ADN génomique de L. plantarum est un agoniste de TLR9 et que CD14 et CD36 facilitent la liaison de la cellule dendritique avec L. plantarum. Ensuite, nous avons évalué le potentiel vaccinal d’une coadministration L. plantarum + Der p 1 dans un modèle murin d’allergie à Der p 1. Cette formulation vaccinale prévient la production d’IgE Der p 1-spécifique et atténue l’éosinophilie pulmonaire tout en stimulant une forte production d’anticorps IgG2a Der p 1-spécifiques et d’IFN-g par les cellules spléniques. Ces effets bénéfiques nous ont conduit à élaborer une bactérie lactique recombinante dérivée de L. plantarum produisant Der p 1 pour la vaccination contre l’allergie aux acariens. La forme antigénique que nous avons réussi à faire produire par L. plantarum correspond à une protéine de fusion entre la Maltose Binding Protein de E. coli et ProDer p 1 (le zymogène de Der p 1), la présence de ce partenaire de fusion étant indispensable à la production de ProDer p 1. En prophylaxie, la vaccination par cette bactérie recombinante prévient la production d’anticorps IgE-Der p 1-spécifiques et stimule la production d’anticorps IgG2a spécifiques, reproduisant les effets de la coadministration L. plantarum + Der p 1. Elle réduit de manière drastique la production d’IL-5 des cellules spléniques et des cellules ganglionnaires médiastinales et prévient l’éosinophilie pulmonaire mais n’a pas d’effet sur l’hyperréactivité bronchique. Der p 1 étant un des allergènes d’acarien les plus immunodominants, cet ensemble de données montre donc que cette bactérie recombinante constitue un vaccin prophylactique prometteur pour la prévention de l’allergie aux acariens. Des résultats préliminaires obtenus à partir de cellules dendritiques humaines et lymphocytes T autologues montrent la forte capacité de cette bactérie recombinante à induire le développement d’une réponse Th1 fortement polarisée (production d’IFN-g en l’absence de production d’IL-4 et d’IL-5), ce qui suggère que l’utilisation de cette bactérie recombinante pourrait être envisagée pour le traitement de l’allergie chez l’homme.

The major allergen Der p 1 of the house dust mite Dermatophagoides pteronyssinus is a papain-like cysteine protease (CA1) associated to the development of allergic diseases such as asthma, rhinitis or atopic dermatitis. This allergen is expressed as an inactive precursor, called proDer p 1, formed by a 25 kDa catalytic domain downstream to an 10 kDa N-terminal propeptide, which blocks the active site cleft. The propeptide of Der p 1 exhibits a specific fold, which makes it unique in the CA1 propeptide family as it is characterised by the presence of four alpha helices and the absence of ERFNIN motif. In this study, we investigated the activation steps involved in the maturation of recombinant proDer p 1 expressed in Pichia pastoris under acidic conditions and we studied the influence of acidic pH on the structure of both propeptide and catalytic domain. Therefore, we characterized the interaction between the propeptide and mature Der p 1 at different pH values in terms of activity inhibition, structural stability and proteolytic susceptibility. According to our results, the auto-activation of proDer p 1 is a multistep mechanism, characterized by at least two intermediates (ATFE- and SNGG-) corresponding to the loss of the first and second propeptide alpha helices, respectively. The propeptide strongly inhibits unglycosylated and glycosylated recombinant Der p 1 (KD= 7 nM) at neutral pH. This inhibition is pH dependent, decreasing from pH 7 to pH 4 and can be related to structural changes of the propeptide initiated by the protonation of the aspartate residue of Lys17-Asp51-Tyr19 structural triad presents within the propeptide N-terminal domain. This protonation triggers conformational changes of the first propeptide alpha helix leading to an increase of the propeptide flexibility, an increase of its proteolytic sensitivity and the formation of a molten globule state. In addition, we compare mature protease, zymogen and propeptide pH unfolding and stability and highlights that the presence of the propeptide does not influence the catalytic domain pH unfolding and stability as the propeptide displays a weaker pH stability than the protease domain. These results confirmed that the propeptide unfolding is the key event of the activation process. Finally, we unravel the intermolecular contribution of mature Der p 1 in the activation process and highlights that activation of the precursor can be achieved, under acidic conditions, by intermolecular process but initial auto-activation most probably occurs through an intramolecular process or by the proteolysis by the catalytic domain of another zymogen in which the propeptide is unfolded. According to our results, we proposed that activation of the zymogen at pH 4 reflects a compromise between activity preservation and propeptide unfolding and that the location of the activation sites on the propeptide structure is a compromise between sequence recognition specificity and proteolytic susceptibility of the corresponding area.

Allergic diseases are influenced by genetics and the environment. Maternal allergy appears to confer a higher risk for allergic sensitization than paternal allergy, suggesting an in utero influence. A decrease in particular infections or a lower exposure to microbial components during infancy is suggested to contribute to the high allergy prevalence in affluent societies. Toll-like receptors (TLR) 2 and 4 recognize peptidoglycan (PGN) and LPS respectively, are expressed on e.g. monocytes, and have been implicated in modulating the risk of IgE-sensitization. This thesis aimed to study the influence of maternal allergy and early microbial exposure on monocyte function and allergic sensitization during childhood. Blood samples from children participating in a prospective allergy cohort were used. Two-year old infants with allergic mothers had lower IL-6 production and reduced activation of the TLR-signalling intermediate p38-MAPK in response to PGN than children with non-allergic mothers. In 5-year old children, allergic disease and not maternal allergy influenced monocytic TLR2-regulation. Five-year olds who were seropositive for Epstein-Barr virus (EBV) at 2-years of age had a lower risk of persistent IgE-sensitization while EBV contraction after 2-years of age related to a higher risk of IgE-sensitization. Upon in vitro stimulation, NK cells from EBV+ 2-year olds produced lower IFN-g levels. EBV+ 2-year olds had also lower systemic IFN-g. In comparison to CD14++CD16- monocytes, CD14+CD16+ cells induced NK-cell IFN-g more potently in vitro, and EBV+ infants tended to have lower proportions of these CD14+CD16+ monocytes. This thesis highlights the importance of early-life microbial (EBV) exposure for a proper allergy-protective immunity. Also, maternal allergic heredity appears to influence monocytic microbial responses in early infancy. All these aspects relate to altered monocyte functionality, which suggest that they could have a role in allergic sensitization.

Atopic allergy is the most common chronic disease among children in the developed world. This high prevalence could be associated with low microbial exposure. The early gut microbiota appears to be important for immune maturation. Immunomodulatory components in human milk might differ between mothers and could therefore explain the contradictory results seen regarding breastfeeding and allergy development. The aim of this thesis was to investigate whether early colonization with certain gut microbiota species influences childhood immune responses and allergy development up to age five. Also, as human milk oligosaccharides (HMOs) might stimulate the growth of certain gut microbiota species, the consumption of neutral colostrum HMOs was investigated for their role in allergy development up to 18 months. The concentrations of neutral colostrum HMOs varied considerably between women; however this variation could not be explained by their allergic status. Neither was the consumption of neutral colostrum HMOs related to allergy development in their children up to 18 months. Infants who harboured lactobacilli group I and Bifidobacterium adolescentis one week after birth developed allergic disease less frequently during their first five years than infants who did not harbour these bacteria at the same time. Also, colonization with several Bifidobacterium species was associated with higher levels of house dust endotoxin and larger family size. The early Bifidobacterium flora influenced levels of salivary secretory IgA at six and 12 months but not during later childhood. Moreover, the intensity of early Bacteroides fragilis colonization was inversely associated with spontaneous Toll-like receptor 4 mRNA expression in peripheral blood cells collected 12 months after birth. In conclusion, these results indicate that the early infant gut microbiota influences systemic and mucosal immune maturation during infancy, and that it might be altered in infants developing allergic disease.

In the first part of the thesis, protein-ligand interactions were investigated using the chaperone LcrH, from Yersinia as target protein. The structure of a peptide encompassing the amphipathic domain (residue 278-300) of the protein YopD from Yersinia was determined by NMR in 40% TFE. The structure of YopD278-300 is a well defined α-helix with a β-turn at the C-terminus of the helix capping the structure. This turn is crucial for the structure as peptides lacking the residues involved in the turn are unstructured. NMR relaxation indicates that the peptide is not monomeric. This is supported by intermolecular NOEs found from residue Phe280 to Ile288 and Val292 indicative of a multimeric structure with the helical structures oriented in an antiparallel manner with hydrophobic residues forming the oligomer. The interaction with the chaperone LcrH was confirmed by 1H relaxation experiments and induced chemical shift changes in the peptide Protein-ligand interactions were investigated further in the second paper using a different approach. A wide range of substances were used in screening for affinity against the chaperones PapD and FimC from uropathogenic Escherichia coli using 1H relaxation NMR experiments, surface plasmon resonance and 19F NMR. Fluorine NMR proved to be advantageous as compared to proton NMR as it is straight forward to identify binding ligands due to the well resolved 19F NMR spectra. Several compounds were found to interact with PapD and FimC through induced line-broadening and chemical shift changes for the ligands. Data corroborate well with surface plasmon resonance and proton NMR experiments. However, our results indicate the substances used in this study to have poor specificity for PapD and FimC as the induced chemical shift is minor and hardly no competitive binding is observed. Paper III and IV is an investigation of the structural features of the allergenic 2S albumin Ber e 1 from Brazil nut. Ber e 1 is a 2S albumin previously identified as the major allergen of Brazil nut. Recent studies have demonstrated that endogenous Brazil nut lipids are required for an immune response to occur in vivo. The structure was obtained from 3D heteronuclear NMR experiments followed by simulated annealing using the software ARIA. Interestingly, the common fold of the 2S albumin family, described as a right-handed super helix with the core composed of a helix bundle, is not found in Ber e 1. Instead the C-terminal region is participating in the formation of the core between helix 3, 4 and 5. The dynamic properties of Ber e 1 were investigated using 15N relaxation experiments and data was analyzed using the model-free approach. The analysis showed that a few residues in the loop between helix 2 and 3 experience decreased mobility, compared to the rest of the loop. This is consistent with NOE data as long range NOEs were found from the loop to the core region of the protein. The anchoring of this loop is a unique feature of Ber e 1, as it is not found in any other structures of 2S albumins. Chemical shift mapping of Ber e 1 upon the addition of lipid extract from Brazil nut identified 4 regions in the protein where chemical shift perturbations were detected. Interestingly, all four structural clusters align along a cleft in the structure formed by helix 1-3 on one side and helix 4-5 on the other. This cleft is big enough to encompass a lipid molecule. It is therefore tempting to speculate whether this cleft is the lipid binding epitope in Ber e 1.

Regulatorische T-Zellen (Tregs) spielen eine entscheidende Rolle bei der Regulation atopischer Erkrankungen. Die Voraussetzungen für eine allergische Reaktionslage werden schon während der intrauterinen Entwicklung geschaffen. Über den Einfluss der intrauterinen Umgebung auf die Tregs zur Geburt ist bisher wenig bekannt. In der vorliegenden Arbeit sollte in der prospektiven Geburtskohorten-Studie LINA (Einfluss von Lebensstil und Umweltfaktoren auf das Allergierisiko Neugeborener) geklärt werden, inwiefern der Immunstatus der werdenden Mutter, eine atopische Familienanamnese sowie Umweltexpositionen während der Schwangerschaft den Immunstatus der Neugeborenen beeinflussen. Ein besonderer Schwerpunkt wurde dabei auf Tregs gelegt. Weiterhin sollte die Relevanz der Tregs zur Geburt für das Allergierisiko im ersten Lebensjahr des Kindes analysiert werden. Die Messung der Anzahl und Funktionalität der Tregs im Blut der werdenden Mutter in der 34. Schwangerschaftswoche und im Nabelschnurblut erfolgte sowohl durchflusszytometrisch in einer Subkohorte (n=24 Mutter-Kind Paare), als auch durch eine methylspezifische qPCR in der gesamten Kohorte der LINA-Studie (n=346 Mutter-Kind Paare). Die Ergebnisse dieser Arbeit deuten erstmals darauf hin, dass mütterliche Tregs möglicherweise einen regulatorischen Einfluss hinsichtlich der Programmierung des fötalen Immunsystems haben (Hinz et al., Clin Exp Allergy 2010). Die durchflusszytometrische Charakterisierung der Tregs der Mutter-Kind Paare zeigte beim Vergleich der Expression von CD4, CD25, CD127 und FOXP3, dass der Anteil der CD4+CD25high Tregs im Nabelschnurblut deutlich höher war, der Anteil FOXP3 positiver Zellen innerhalb der CD4+CD25high Tregs Population war zur Geburt jedoch signifikant geringer, verglichen mit den werdenden Müttern. Weiterhin war eine geringe Anzahl mütterlicher Tregs während der Schwangerschaft und eine erhöhte Produktion der TH2-Zytokine IL-4, IL-5 und IL-13 mit erhöhten Gesamt-IgE-Spiegeln im Nabelschnurblut verbunden (Hinz et al., 2010). Durch die Quantifizierung der Tregs auf Basis des TSDR-Methylierungsstatus` im FOXP3 Gen, einer spezifischen und zuverlässigen Methode zum Nachweis stabiler Tregs, konnte der Zusammenhang zwischen einer Vielzahl pränataler Faktoren, Tregs zur Geburt und dem Allergierisiko in der gesamten Geburtskohorte geklärt werden (Hinz et al., Allergy 2011). Das männliche Geschlecht des Kindes, die Atopie der Eltern, Rauchen und Desinfektionsmittel-Exposition während der Schwangerschaft sowie eine erhöhte mütterliche Produktion von IFN-γ, IL-13 und IL-17E war mit einer geringeren Treg-Anzahl im Nabelschnurblut assoziiert. Für Kinder mit einer geringeren Treg-Anzahl im Nabelschnurblut war das Risiko für eine atopische Dermatitis und einer Sensibilisierung gegen Nahrungsmittelallergene im ersten Lebensjahr signifikant höher (Hinz et al., 2011).