Tinea pedis is a chronic fungal infection of the feet, very often observed in patients who are immuno-suppressed or have diabetes mellitus. The practicing allergist may be called upon to treat this disease for various reasons. Sometimes tinea infection may be mistaken for atopic dermatitis or allergic eczema. In other patients, tinea pedis may complicate allergy and asthma and may contribute to refractory atopic disease. Patients with recurrent cellulitis may be referred to the allergist/immunologist for an immune evaluation and discovered to have tinea pedis as a predisposing factor. From a molecular standpoint, superficial fungal infections may induce a type2 T helper cell response (Th2) that can aggravate atopy. Th2 cytokines may induce eosinophil recruitment and immunoglobulin E (IgE) class switching by B cells, thereby leading to exacerbation of atopic conditions. Three groups of fungal pathogens, referred to as dermatophytes, have been shown to cause tinea pedis: Trychophyton sp, Epidermophyton sp, and Microsporum sp. The disease manifests as a pruritic, erythematous, scaly eruption on the foot and depending on its location, three variants have been described: interdigital type, moccasin type, and vesiculobullous type. Tinea pedis may be associated with recurrent cellulitis, as the fungal pathogens provide a portal for bacterial invasion of subcutaneous tissues. In some cases of refractory asthma, treatment of the associated tinea pedis infection may induce remission in airway disease. Very often, protracted topical and/or oral antifungal agents are required to treat this often frustrating and morbid disease. An evaluation for underlying immuno-suppression or diabetes may be indicated in patients with refractory disease.

Peanut, a major allergenic food, has life-threatening potential and is difficult to be totally avoided due to its common use in processed foods. Thermal processing can influence the allergenic properties of peanuts. However, the kinetics of the reactions caused by thermal processing has not been characterized. In our study, kinetics of the commonly used thermal processing methods on a commercial peanut cultivar (Virginia) using five time intervals was conducted. Water-soluble and SDS-sample buffer soluble proteins were extracted sequentially, and analyzed by sodium-dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and western-blot using human plasma containing IgE antibodies. The relationships between thermal processing (time) and log transformed water-soluble/total extractable major allergen content could be explained by a simple linear regression kinetic model for most of the processing methods (except high-pressure steaming). Among all the methods with optimal processing point, frying for 6 min had relatively lower IgE binding (linear epitopes) ratio may be due to the fact that this processing condition causing break down, cross-linking and aggregation of Ara h 2, and relatively lower solubility. Besides thermal processing, enzymatic processing also is considered to be an effective method in the allergenicity of peanuts. Eleven peanut lines (Coded MS-1~MS-11, MS-9 is the check and a common cultivar namely Valencia) were pre-screened from 122 peanut lines harvested in 2015 for allergen levels. These pre-screened lines were re-planted in 2016 for further analysis. One line, MS-7, was selected for lower Ara h 1 (8.5-9.5% of total protein) and Ara h 2 (4.2-6.6% of total protein) content in 2015 and 2016. Roasted MS-9 (check) peanut powders were used for enzymatic treatment for enzyme selection. A first order kinetic reaction model was conducted to describe the relationship between enzyme concentration (0-400AzU/g) and IgE-binding property reduction. Among eight food-grade enzymes, bromelain, papain and ficin hydrolysates had lower IgE-binding properties in terms of high IgE-binding property reducing rate (K, ≥ 0.4) and were selected for the following study. MS-7 (selected) & MS-9 (at level of 200AzU/g) hydrolyzed by three selected enzymes (200AzU/g) were used for IgE binding property comparison, TGase crosslinking and functional properties study. After hydrolyzed by the selected enzymes (200 AzU/g), the emulsion and foaming stabilities were decreased. Emulsion and foaming stabilities were increased in TGase (5U/g protein) crosslinked hydrolysates, which were even higher than soy protein isolate (SPI). The IgE-binding properties of TGase treated hydrolysates were similar to the hydrolysates without TGase treatment. MS-7 hydrolysates (with/without TGase) possessed less IgE-binding properties and similar functionality as compared with MS-9 hydrolysates.

Grund der Studie: Landwirte tragen eines der höchsten Risiken an berufsbedingtem Asthma zu erkranken, wobei die Allergene vom Rind eine entscheidende Rolle spielen. Das Wissen bezüglich Risikoprofile und Prävention ist sehr gering. Es herrscht aber eine Diskrepanz zwischen klinischer und Labordiagnostik, d.h. eindeutige klinische Reaktionen finden keine Bestätigung durch Allergietestung mit kommerziellen Extrakten. Patienten berichten auch, unterschiedliche Reaktionen auf unterschiedliche Rassen zu zeigen. Noch gibt es aber keine Studien zu rassespezifischen Unterschieden.Ziel der vorliegenden Arbeit: Vergleich verschiedener Extrakte (kommerzielle und eigene) mittels SDS-Page und dem Vergleich der erhaltenen Proteinmuster. Die eigenen Extrakte berücksichtigen die verschiedenen Rinderrassen der betroffenen Landwirte in Süd- und Norddeutschland. Und Nachweis eines möglichen individuellen Sensibilisierungsmusters der betroffenen Landwirte unter Berücksichtigung dieser beschwerdeauslösenden Rinderrassen.Klinische Aspekte: Untersuchung mit den Seren von 42 symptomatischen Landwirten (16 weibliche und 26 männliche Landwirte im Alter zwischen 25 und 74 Jahren), aus dem norddeutschen (Niedersachsen) sowie dem süddeutschen (Bayern und Baden-Württemberg) Raum kommend. Alle hatten eindeutigen klinischen Symptomen der Rinderhaarallergie.Material und Methoden: Die verwendeten Rinderhaare für diese Untersuchungen stammen von eigenen Tieren der erkrankten Landwirte oder, wenn die Rinderhaltung bereits aufgegeben war, von Haaren reinrassiger Rinder aus Zuchthöfen verwendet. Insgesamt wurden Haare von 8 Rinderrassen verwendet.Durchführung: In durchgeführten Voruntersuchungen wurde die Spezifität dieser Immunblotexperimente durch Negativkontrollen nachgewiesen (Probanden mit RAST-Klasse 0, ohne keine klinische Allergiesymptomatik, immer in der Stadt gelebt, keine Tierhaltung) Die Proteine wurden von Rinderhaaren extrahiert, der Proteingehalt der Lösung bestimmt und anschließend erfolgte die Durchführung von SDS-Page und Immunblot. Die Reaktivität zwischen kommerziellen Extrakten und den selbst hergestellten Extrakten konnte so verglichen und mit auch dem Proteinangebot verglichen werden. Dieses Procedere für jeden Landwirt durchgeführt.Ergebnisse: Die Auswertung der SDS-Polyacrylamid-Gele ergab keine relevanten Unterschiede innerhalb der kommerziellen Extrakte und nur geringe Differenzen zwischen den einzelnen Extrakten I bis IV. Die Färbungen fanden sich eher im Nierdermolekularbereich und bei 20 kDa (Bos d2). Es konnten auch keine relevanten Unterschiede innerhalb der selbst hergestellten Extrakte dargestellt werden, aber zum Teil deutliche Differenzen zwischen den unterschiedlichen Rassen. Die am intensivsten gefärbten Banden für alle Extrakte waren auch bei 20 kDa (Bos d 2). Ebenfalls intensive Färbungen für alle Extrakte lagen im Molekulargewichtsbereich von 20 bis 28 kDa. Deutliche Unterschiede zwischen den einzelnen Rassen konnte im Molekulargewichtsbereich von << 14 und < 14 kDa und zwischen 30 und 67 kDa (also im niedrigen und mittleren MW) dargestellt werden. Ein wichtiges Ergebnis dieser Studie ist der Nachweis, dass 32 % der Landwirte mit eindeutigen Symptomen einer Rinderhaarallergie, aber negativer klinischer Diagnostik mittels kommerzieller Extrakte im Immunblot Reaktionen ihres Serums mit den selbst hergestellten Extrakten zeigen. Des Weiteren bestätigen die erlangten Ergebnisse auch die hohe allergologische Relevanz der in der Literatur beschriebenen Proteine bei 20 und 22 kDa, zeigen aber auch für die selbst hergestellten Extrakte eine immunologische Reaktion in anderen Molekulargewichtsbereichen. Die eingangs aufgestellte Hypothese eines möglicherweise rassespezifischen Sensibilisierungsmusters lässt sich dagegen nicht aufrechterhalten. Rassespezifische Unterschiede konnten sich keiner bestimmten Systematik zuordnen lassen, eher individuelle Sensibilisiereungsmuster ohne Bezug zur Rasse. Hypothese: In den kommerziellen Extrakten sind nicht mehr alle für eine Sensibilisierung relevanten Proteine repräsentiert oder haben durch eine mögliche Procezierung bei der Herstellung ihre immunologische Reagibilität eingebüßt.

La détection précoce de l’athérosclérose, avant le développement de ses séquellespathologiques, comme l’infarctus du myocarde, l’angine ou l’accident cérébrauxvasculaire(ACV), représente un important défi au niveau de la médecine diagnostiqueactuelle. Malgré les récentes avances technologiques, les maladies cardiovasculairesdemeurent la principale cause de décès dans les pays occidentaux et la détection à unstage plus précoce s’avère nécessaire pour permettre une intervention thérapeutiqueadéquate. Notre étude se concentre sur la détection de l’athérosclérose, plusspécifiquement la vulnérabilité de la plaque, grâce à l’imagerie moléculaire combinée àl’observation pathologique. Afin de prédire la rupture de la plaque, l’imageriemoléculaire a émergé comme outil diagnostique puissant suite au développementcroissant de sondes ayant de l’affinité pour les molécules cibles du processusd’athérosclérose. Comme résultantes, ces molécules sélectives possédant une forteaffinité pour des cibles surexprimées durant le processus de formation de la plaque,comme l’αvβ3 par exemple, devrait représentées des sondes prometteuses pour ladétection de l’athérosclérose.Objectif L’objectif global de notre étude était d’évaluer et de prédire lavulnérabilité de la plaque d’athérome à l’aide de différents marqueurs moléculaires. Leprincipal objectif de notre recherche était d’évaluer la possibilité de détecter précocementla plaque en utilisant une ITOP (integrin targeted optical probe). Cette sonde synthétiquenouvellement développée et ciblant l’intégrine αvβ3 avait déjà démontré une affinité etspécificité particulièrement élevée pour le récepteur de l’αvβ3 dans le cancer. Nousavons également exploré la relation entre cette sonde et l’observation pathologique desplaques d’athéromes sur le modèle animale WHHL et sur des plaques humainesprovenant de différents patients.Procédure et Résultats Les expériences ont été réalisées sur un total de 12 lapinsWatanabe hyperlipidémiques de souche WHHL (Watanabe heritable hyperlipidemic) et 1lapin contrôle NZW (New Zealand White). Premièrement, notre ITOP, marquée avec lafluorescéine isothiocyanate (FITC), a été utilisée pour détecter in vitro et ex vivo laprésence du récepteur de l’αvβ3. La microscopie à fluorescence a révélé un importantmarquage de la plaque d’athérome, lequel était absent dans les tissus provenant des lapinscontrôles NZW. Le marquage a été détecté au niveau de segments de plaques provenantde deux régions distinctes de l’aorte ascendante et descendante dans chaque lapin. Lesignal a été détecté principalement au niveau de l’adventitia et de l’intima proximale desvaisseaux aortiques, correspondant directement à l’expression de l’intégrine αvβ3,déterminée par essai immunochimique avec un anticorps contre l’αvβ3. De plus, uneforte association s’est révélée entre le niveau de marquage de la sonde ciblant l’αvβ3 etl’épaisseur de l’adventitia. Deuxièmement, nous avons évalué notre sonde sur deséchantillons humains affectés par l’athérosclérose et comparé les résultats avec uneévaluation morphologique. Nous avons remarqué la même tendance que chez le lapin, soiun marquage plus important lorsque l’adventitia s’épaissi. Finalement, nous avons testé lasonde sur des artères coronaires provenant d’une autopsie d’un patient affecté par le ADHIESet comparé les résultats avec l’évaluation morphologique de leurs artèrescoronaires. Nous avons trouvé un lien entre la morphologie de la plaque et la prévalenced’anévrysmes coronaires chez ces patients.Conclusion L’expression de l’αvβ3 est reliée à la foi aux processus inflammatoires età la sténose. Notre ITOP à marqué efficacement in vitro le premier type de plaqued’athérome classé comme avancé (type IV) et pouvant produire des manifestationscliniques. En combinaison avec l’imagerie noninvasive détectant la sténose, il pourraits’avéré utile dans la détection de la plaque vulnérable. / Purpose The detection of early atherosclerosis, before the development of its later sequelae of myocardial infarction, angina or stroke, constitutes an important challenge in current diagnostic medicine. Despite all the recent technological advances, cardiovascular disease remains the leading cause of death in the Western World and needs to be detected at an earlier stage to allow for more timely therapeutic intervention. This study is focusing on the detection of atherosclerosis or more specifically plaque vulnerability with the help of molecular imaging and pathological observation. Effectively, to predict plaque rupture, molecular imaging has emerged as a powerful diagnostic tool, consequent to the development of a growing number of new probes with affinity for key molecular targets. As a result, such selective molecule with high affinity for overexpressed target in plaque formation, as αvβ3 integrin, should have promise as a probe for imaging atherosclerosis. With the help of molecular imaging combined with pathological observations, we can better comprehend, predict, and detect plaque vulnerability and rupture. Objectives The overall objective of this study is to evaluate different molecular tools to predict the vulnerability of the atheromatous plaque. The major objective of the research was to investigate the possibility of detecting atherosclerotic plaque by using a newly developed synthetic αvβ3 integrin targeted optical probe (ITOP) showing particularly high affinity and specificity for the αvβ3 receptor. We also investigate the relation between this probe and pathological observation of atherosclerotic plaques from WHHL animal model and different human samples. Procedures and Results For this study, experiments were performed on 12 Watanabe heritable hyperlipidemic (WHHL) rabbits and 1 New Zealand White (NZW) rabbits for control. First, our ITOP labeled with fluorescein isothiocyanate was used for detecting the presence of αvβ3 receptors in vitro and ex vivo on a Watanabe rabbit model. Fluorescence microscopy demonstrated a strong labeling of atherosclerotic plaques, which was absent in tissue from normal NZW rabbits. Segments of plaque accumulation from two distinct regions of ascending and descending aortas were labeled in each rabbit. The signal was found principally in the adventitia and proximal intima of the aortic vessel, corresponding directly to the expression of integrin αvβ3 as determined by antibody assay. Moreover, there was a close association between the level of labeling with the αvβ3 targeted probe and the thickness of the adventitia. Secondly, the ITOP was evaluated on human atherosclerotic samples, and was found to efficiently labeled atherosclerotic plaques. Moreover, we observed the same tendency as in the Watanabe rabbit: the ITOP intensity correlated with the degree of adventitial thickening. Finally, we tested the ITOP on Job's Syndrome coronary arteries, and have been able to detect a plaque corresponding to the first type of advanced atherosclerosis (type IV). We also found a relationship between plaque morphology and predisposition to aneurysms in Job's syndrome. Conclusions αvβ3 expression is related to inflammatory and stenotic processes. Our ITOP can efficiently label in vitro the first type of advanced atherosclerotic plaque. In combination with noninvasive imaging techniques that evaluate stenosis, it has great potential for the detection of vulnerable plaque.

La prévalence des allergies alimentaires IgE-médiées aurait triplé au cours de la dernière décennie avec des études Nord-Américaines atteignant les 8% chez les enfants. Quoiqu’il n’y ait à ce jour aucun traitement curatif pour les allergies alimentaires, l’immunothérapie oral (OIT) constitue une nouvelle approche expérimentale prometteuse. Cette dernière consiste en l’administration de doses progressive d’allergènes par voie orale sur une période prolongée dans le but d’instaurer un état de désensibilisation et possiblement une tolérance orale soutenue. Cette approche a été démontrée sécuritaire et permettrait la désensibilisation à haute dose de plus de 80% des participants allergiques aux arachides, lait ou œufs. Dans cette thèse, nous présentons 2 études de phase 1 portant sur des protocoles d’OIT, destinés à optimiser l’efficience du traitement chez les sujets avec allergies alimentaires multiples. Près de 30% des enfants avec allergie alimentaire sont allergiques à plus d’un aliment, une proportion qui augmente à 70% lorsqu’on considère les cas les plus sévères. Ces enfants sont à risque augmenté de réactions accidentelles et souffrent d’un impact plus grand sur leur qualité de vie. Dans la première étude, en créant un mélange individualisé avec un ratio stochiométrique 1:1 entre les protéines des aliments allergiques de l’enfant, nous démontrons qu’il est possible de désensibiliser jusqu’à 5 aliments simultanément avec un profil d’innocuité similaire à une monothérapie. Dans la seconde étude, nous utilisons un traitement à l’omalizumab, un anticorps monoclonal anti-IgE, pour permettre une désensibilisation orale multi-allergénique fortement accélérée. Lorsque comparé à l’approche sans omalizumab, ce protocole s’associe à une nette diminution du temps requis pour atteindre les doses d’entretien, passant d’une médiane de 21 à 4 mois, sans affecter le profil d’innocuité. Alors que ces études fournissent des approches cliniques raisonnables pour désensibiliser la population multi-allergique, plusieurs questions persistent, notamment en ce qui a trait à l’induction de tolérance permanente. Une barrière majeure à cet égard réside dans notre piètre compréhension des mécanismes sous-jacents à l’immunothérapie. Prenant avantage d’échantillons cliniques bien caractérisés provenant des essais cliniques ci-haut mentionnés, nous utilisons les nouvelles technologies de séquençage TCR pour suivre la distribution clonale des lymphocytes T spécifiques aux arachides durant une immunothérapie orale. Nous démontrons que l’OIT s’associe à des changements significatifs dans les fréquences des clones spécifiques, suggérant un processus d’épuisement clonal et de remplacement. Nous démontrons par ailleurs que le test de prolifération lymphocytaire, traditionnellement utilisé pour évaluer la réponse cellulaire allergique, est dominé par une distribution polyclonale hautement non-spécifique. Cette observation a des implications majeures considérant que la plupart de la littérature actuelle sur la réponse T se base sur cette technique. En somme, cette thèse jette les bases pour des programmes de recherche translationnelle pour optimiser et personnaliser les protocoles cliniques actuels et développer de nouvelles avenues d’investigation et de traitement pour améliorer la prise en charge des sujets avec allergies alimentaires. / The prevalence of IgE-mediated food allergies has tripled over the last decade with prospective studies indicating that up to 8% of children may be affected in North America. There is currently no cure for food allergy but oral immunotherapy (OIT) is an experimental approach to treat food allergies. It consists in the progressive administration from minute to large amounts of the allergenic food by the mouth over a prolonged period of time to induce a state of desensitization and possibly sustained tolerance. This approach has been shown to be safe and to allow desensitization to high doses in over 80% of participants allergic to peanuts, milk or egg. In this thesis, we present two phase 1 trials on OIT protocols designed to efficiently treat multiple foods allergies. About 30% of children with food allergy are allergic to more than one food. This proportion increases to 70% when considering the most severe cases. Children with multiple food allergies are at higher risk of accidental reactions and suffer from greater impact on quality of life than those with single food allergies. By creating a customized treatment mix with a 1:1 stoichiometric ratio for the child’s relevant food proteins, we were first able to safely desensitize up to 5 foods simultaneously with a safety profile similar to single allergen therapy and a minimal increase in time to maintenance. Then, taking advantage of recent evidence showing that omalizumab, an anti-IgE receptor monoclonal antibody, can significantly raise reaction thresholds in food allergic subjects, we used short courses of omalizumab to allow very rapid oral desensitization to various foods in a second phase 1 study. When compared to “standard” multi-OIT, the omalizumab-enabled rush protocol resulted in a decreased time to maintenance from a median of 21 to 4 months. While these studies provide reasonable clinical approaches to this population, many questions remain, especially with regards to long term tolerance. A major limit to our progress in improving these protocols stems from our lack of understanding of the underlying immune mechanisms of oral immunotherapy. Taking advantage of well phenotyped samples from the afore-mentioned trials, we used next-generation high-throughput TCR sequencing to follow clonal distribution of peanut specific T cells during oral immunotherapy. We found that OIT is associated with significant changes in food-specific clonal frequencies, suggesting clonal exhaustion and replacement as an underlying mechanism of OIT. In addition, we show that the proliferation assay which is traditionally used to assess the cellular response is dominated by a highly non-specific polyclonal distribution. This observation has important implications considering most of the current literature on T cell response to immunotherapy is based on this assay. This highlights the need for the development of new tools to assess the cellular allergic response. Overall this thesis lays the ground for further comprehensive translational research programs on the treatment of food allergy.

Abstract There are eight foods that contribute to food allergies in the western world and peanut is the most common. Currently, there are no medical treatments that can cure an individual of food allergy, so avoidance of the allergic food is the only option. In the United States, there are three immunodominant allergic proteins accountable for patient sensitization to peanut, Arachis hypogea 1, 2, and 3 (Ara h 1, Ara h 2, Ara h 3). Therefore, research into why peanuts are more allergic than other foods that have homologous proteins is critical and may be obtained by studying the structural and allergenic properties of individual allergens and the changes that occur due to food processing. In this study, the basic and acidic subunits of Ara h 3 were cloned, expressed, and purified, and compared with each other and with the native Ara h 3 purified from peanut for differences in binding to IgE from peanut allergic individuals. Also, an in vitro Maillard reaction was performed on purified native raw Ara h 3 and patient serum IgE western blots were performed. This study concluded that an in vitro Maillard reaction enhanced IgE binding to Ara h 3, IgE binding to native Ara h 3 was in most cases higher than to the recombinant Ara h 3 subunits, and recognition of the acidic subunit was much higher than the and basic subunits in both the recombinant and native forms of the protein were investigated. Keywords:

Introduction: The pathogenesis and treatment of allergic bronchopulmonary aspergillosis (ABPA), severe asthma-non fungal sensitised (SANFS) and severe asthma with fungal sensitization (SAFS) is poorly understood. IL-17A, IgE and microbiome may be associated with pathogenesis of asthma, but their role in fungal-associated asthma is uncertain. Further, the efficacy of voriconazole, posaconazole and nebulised amphotericin B (NAB) in ABPA and SAFS has not been fully studied. Aims and objectives: The aim of this PhD thesis was to evaluate the role of IL-17A, IgE and lung microbiome in patients with SANFS, SAFS and ABPA. We also studied the efficacy and safety of NAB, voriconazole and posaconazole. Methods: Airway lymphocytes and peripheral blood mononuclear cells (PBMC) from patients with ABPA (n=16), SAFS (n=15), SANFS (n=11), mild asthma (MA) (n=6) and NH (n=11) were characterized by flow cytometric analysis (FACS) to determine the % of CD (+) IL-17A expressing cells. We also evaluated microbiome population using culture and PCR plus sequencing from BAL of these patients. In chapter 3, we analysed total and specific IgE in blood from adult cohorts of SAFS (n=34) and ABPA (n=48) using ImmunoCAP 100. In chapter 5 we studied the efficacy of voriconazole and posaconazole and in chapter 6; we studied the efficacy of NAB.Results: %CD4+IL-17A expressing cells were significantly higher in patients with severe asthma and correlated positively with serum neutrophil and presence of fungi in the airways. ABPA, SAFS and SANFS were similar but all were significantly higher than MA and NH. There were no differences in IL-17A expression between blood and the lung. Fungi were more frequently associated with severe asthma and low FEV1. Steroid treatment significantly increased airway fungal load. IgE against staphylococcal aureus (SE-IgE) correlated positively with FEV1 and OCS dose. Voriconazole and posaconazole improved asthma severity and radiological abnormalities. NAB was associated bronchospasm, but was extrely effective in the few patients (n=3) that took treatment for >12 months. These responders had unique characteristics. Conclusions: IL-17A, SE-IgE, and lung microbiome are associated with asthma severity. Steroid use in these patients may increase airway fungal load. Whereas voriconazole and posaconazole are efficacious, the use of NAB is associated with significant bronchospasm. SE-IgE -high asthma patients may be a distinct asthma phenotype. Larger studies are needed.

<p>IgG, IgM and IgE in complex with antigen have the capacity to regulate specific immune responses. In this investigation, the role of Fc receptors for IgG (FcγRI, FcγRII and FcγRIII) and complement receptors 1 and 2 (CR1/2) for antibody-mediated enhancement of antibody responses are investigated.</p><p>IgM is known to efficiently activate complement and thereby enhance specific antibody responses but it is not known if this involves binding to CR1/2. Using CR1/2 deficient mice, immunized with sheep erythrocytes alone or together with specific IgM, we present evidence that IgM-mediated enhancement is completely dependent on CR1/2 expression, whereas IgG or IgE in complex with bovine serum albumin (BSA) induce strong antibody responses in CR1/2-deficient mice. Enhancement by IgE is mediated via the low affinity receptor for IgE (FcεRII, CD23). However, the receptors which are involved in IgG-mediated enhancement are not known. We find that γ-chain-deficient mice (lacking FcγRI and FcγRIII) have impaired antibody responses to IgG/BSA complexes. In contrast, FcγRIII deficient mice have normal responses, suggesting that FcγRI mediates the effect. Furthermore, IgG/BSA complexes induce up to 189-fold stronger antibody responses in FcγRIIB-deficient mice than in wild-type mice. The threshold dose of IgG/BSA required was lower, the response was sustained for longer and initiated earlier in FcγRIIB-deficient than in wild-type animals. The findings suggest that FcγRIIB acts as a "safety-valve" preventing excessive antibody production during an immune response. We show for the first time that IgG3/BSA complexes can mediate enhancement of specific antibody responses. Their effect does not involve known Fcγ receptors.</p>

IgG, IgM and IgE in complex with antigen have the capacity to regulate specific immune responses. In this investigation, the role of Fc receptors for IgG (FcγRI, FcγRII and FcγRIII) and complement receptors 1 and 2 (CR1/2) for antibody-mediated enhancement of antibody responses are investigated. IgM is known to efficiently activate complement and thereby enhance specific antibody responses but it is not known if this involves binding to CR1/2. Using CR1/2 deficient mice, immunized with sheep erythrocytes alone or together with specific IgM, we present evidence that IgM-mediated enhancement is completely dependent on CR1/2 expression, whereas IgG or IgE in complex with bovine serum albumin (BSA) induce strong antibody responses in CR1/2-deficient mice. Enhancement by IgE is mediated via the low affinity receptor for IgE (FcεRII, CD23). However, the receptors which are involved in IgG-mediated enhancement are not known. We find that γ-chain-deficient mice (lacking FcγRI and FcγRIII) have impaired antibody responses to IgG/BSA complexes. In contrast, FcγRIII deficient mice have normal responses, suggesting that FcγRI mediates the effect. Furthermore, IgG/BSA complexes induce up to 189-fold stronger antibody responses in FcγRIIB-deficient mice than in wild-type mice. The threshold dose of IgG/BSA required was lower, the response was sustained for longer and initiated earlier in FcγRIIB-deficient than in wild-type animals. The findings suggest that FcγRIIB acts as a "safety-valve" preventing excessive antibody production during an immune response. We show for the first time that IgG3/BSA complexes can mediate enhancement of specific antibody responses. Their effect does not involve known Fcγ receptors.

L’asthme est répandu chez 250 millions de personnes dans le monde, dont la majorité souffre d’asthme allergique à médiation IgE. La diaphonie neuro-immune a récemment fait l’objet d’études approfondies. La présente thèse comprend des études sur des modèles de souris, y compris nos travaux publiés portant sur l’interaction neurone sensoriel-cellule B, un manuscrit soumis sur l’interaction neurones pruricepteurs vagaux-basophiles, ainsi que nos méthodes de recherche publiées sous forme de chapitre de livre. Dans le chapitre 1, nous avons démontré que la perte de fonction des neurones sensoriels atténuait l’inflammation allergique des voies respiratoires induite par les acariens et l’activation des cellules IgE+ B. Les données in vitro suggèrent que l’activation des lymphocytes B et la production d’anticorps étaient améliorées par la substance P. Dans le chapitre 2, nous avons démontré que les neurones sensoriels vagaux répondent aux pruritogènes, à l’analogue du LPA xy-17, à la β-alanine et à la chloroquine, suggérant que le pruricepteur-comme le comportement de certains neurones sensoriels vagaux, comme le prédisent les récents résultats de séquençage d’ARN unicellulaire. Plus important encore, nous avons démontré que l’oncostatine M est produite par les basophiles sous stimulation basée sur FcεRI et peut sensibiliser plusieurs populations sensorielles vagales, y compris les sous-ensembles jugulaires TRPV1+Tac1+ et MrgprA3+. Enfin, dans le chapitre 3, nous illustrons les méthodes détaillées d’étude des neurones sensoriels vagaux, y compris les approches génétiques et pharmacologiques de perte de fonction, et l’analyse transcriptomique des neurones sensoriels vagaux issus du tri cellulaire activé par fluorescence (FACS). Prises ensemble, nos données suggèrent que les neurones sensoriels peptidergiques participent à l’établissement de l’immunité humorale et que les neurones pruricepteurs vagaux répondent aux médiateurs inflammatoires allergiques, y compris la cytokine amplificatrice des démangeaisons, l’oncostatine M, produite par les basophiles activés par FcεRI. Des études supplémentaires sont nécessaires pour déchiffrer le rôle des neurones pruricepteurs vagaux dans la transmission du signal du tronc cérébral, l’inflammation périphérique et la mécanique pulmonaire globale de l’asthme. / Asthma is prevalent among 250 million people globally, the majority of which feature IgE-mediated allergic asthma. The neuro-immune crosstalk has been under intense study recently. The present thesis comprises studies of mouse models, including our published work focusing on sensory neuron-B cell interaction, a submitted manuscript on vagal pruriceptor neurons-basophil interaction, as well as our research methods published as a book chapter. In chapter 1, we demonstrated that loss of function of sensory neurons dampened house dust mite-induced allergic airway inflammation, IgE+ B cell activation. In vitro data suggests that the B cell activation and antibody production were enhanced by substance P. In chapter 2, we demonstrated that vagal sensory neurons respond to pruritogens, the LPA analog xy-17, β-alanine, and chloroquine, suggesting the pruriceptor-like behavior of some vagal sensory neurons, as predicted by recent singlecell RNA sequencing results. Most importantly, we demonstrated that oncostatin M is produced by basophils under FcεRI-based stimulation, and can sensitize multiple vagal sensory populations, including the jugular TRPV1+Tac1+ and MrgprA3+ subsets. Finally, in chapter 3, we illustrate the detailed methods for studying vagal sensory neurons, including the genetic and pharmacological loss-of-function approaches, and transcriptomic analysis of vagal sensory neurons yield from fluorescence-activated cell sorting (FACS). Taken together, our data suggests that peptidergic sensory neurons participate in the humoral immunity establishment and vagal pruriceptor neurons respond to allergic inflammatory mediators, including the itch-amplifying cytokine, oncostatin M, produced by FcεRI-activated basophils. Further studies are needed to decipher the role of vagal pruriceptor neurons in brainstem signal transmission, peripheral inflammation, and overall asthmatic lung mechanics.