Dissertations / Theses on the topic 'Allergic response'

Allergic manifestations are increasingly common in infants and children. Accumulating evidence suggests that the ‘epidemic’ increase of childhood allergy may be associated with environmental factors such as stress. Although the impact of stress on the manifestation and exacerbation of allergy has been demonstrated, the underlying mechanisms of stress-induced exacerbation are still obscure. A growing number of studies have suggested an altered hypothalamus-pituitary-adrenal (HPA) axis function to stress in allergic children. It is speculated that a dysfunctional HPA axis in response to stress may facilitate and/or consolidate immunological aberrations and thus, may increase the risk for allergic sensitization and exacerbation especially under stressful conditions. In the present review the potential impact of a hyporesponsive as well as a hyperresponsive HPA axis on the onset and chronification of childhood allergy is summarized. Moreover, potential factors that may contribute to the development of an aberrant HPA axis responsiveness in allergy are discussed
Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich

Allergic manifestations are increasingly common in infants and children. Accumulating evidence suggests that the ‘epidemic’ increase of childhood allergy may be associated with environmental factors such as stress. Although the impact of stress on the manifestation and exacerbation of allergy has been demonstrated, the underlying mechanisms of stress-induced exacerbation are still obscure. A growing number of studies have suggested an altered hypothalamus-pituitary-adrenal (HPA) axis function to stress in allergic children. It is speculated that a dysfunctional HPA axis in response to stress may facilitate and/or consolidate immunological aberrations and thus, may increase the risk for allergic sensitization and exacerbation especially under stressful conditions. In the present review the potential impact of a hyporesponsive as well as a hyperresponsive HPA axis on the onset and chronification of childhood allergy is summarized. Moreover, potential factors that may contribute to the development of an aberrant HPA axis responsiveness in allergy are discussed.
Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

The purpose of this thesis was to show a relationship between muscle strength and allergic reactions. Clinics have for many years relied on what have become traditional methods for testing for allergic reactions. These include such tests as the Patch test and the Skin-Prick test. Both these tests cause discomfort and irritation to sensitive patients. The historical development of allergies is outlined, showing that scientists and clinicians in the late 18th and early 19th century were aware of certain reactions which people showed to apparently normal non-reactive foods. The development of methods of diagnosing allergies such as the elimination diet, which dates back to approx 1940, are surveyed. Current practices are also studied with the use of photographs. The reaction of the skin to modern day testing practices is shown. In some cases these reactions are quite violent. The Applied Kinesiology Test is examined as a universally used test which when carried out by an experienced Kinesiologist can give a true but highly subjective response to a patients allergic condition. A system has been developed based on the relationship between muscle strength and allergies, the components of this system were readily available at little cost. Once a basic system was built it was possible to perform trials on patients at an established allergy clinic. With the aid of a data printer it became possible to prove first graphically and second statistically that there was a statistically valid relationship between the measured muscle strength and allergies. The conclusion shows that the final version of the operating system is capable of equaling the performance of an experienced Kinesiologist, showing its results both digitally and graphically rather than subjectively. The various deductions and recommendations which are detailed, point towards a readily marketable device which could represent a potential break through in modern allergy testing. The system will also open the way for clinicians to make an objective assessment of the relationship between muscle strength and allergic sensitivity via further research.

Mast cells (MCs) play a central role in inflammation by releasing mediators following activation. S100A8 and S100A9 are abundantly expressed in inflammatory sites such as asthmatic lung, sunburnt skin and atherosclerosis where MCs are involved in pathogenesis; roles of S100A8 in MC function are undetermined. The aims of this thesis were to determine effects of S100A8 on MC activation, particularly provoked by IgE and UVB. Initially, effects of UVB on MC activation were investigated as detailed functions were unclear. Cord blood-derived human mast cells (CBMCs) were treated in vitro with varying doses of UVB and production of multiple cytokines and viability investigated. UVB exposure selectively increased levels of IL-8 (CXCL8), and to a less extent IL-1β, but not eight other cytokines tested. New protein synthesis partially contributed and IL-8 production was p38 MAPK-dependent. UVB dose-dependently induced MC apoptosis indicating a potential regulatory mechanism of MC function. The ability of recombinant S100A8, S100A9 or S100A8/9 heterodimer to modulate IgE/antigen (DNP/anti-DNP)-mediated activation of a murine MC line, and of bone marrow-derived (mBM) MC activation was determined. The S100s did not directly induce degranulation or induce IL-6. S100A8 significantly inhibited DNP/anti-DNP-provoked degranulation, and IL-6 and TNF mRNA and protein induction. S100A8 did not alter FcεRIα expression. S100A9 was less effective; and the S100A8/9 complex was also suppressive. S100A8 only weakly suppressed non-specific MC degranulation. Mutation of Cys41 in S100A8 negated its suppressive activity. Because S100A8 scavenges oxidants via this reactive Cys residue, we propose that this may mediate its ability to downmodulate IgE-dependent MC responses. Similar to the thiol scavenger N-acetyl-L-cysteine, S100A8 but not the Ala41 mutant, attenuated DNP/anti-DNP-provoked LAT phosphorylation. However, the disulfide-bonded S100A8 dimer and S100A8 containing a sulfinamide bond between Cys41 and Lys34/35 also reduced MC activation, indicating an additional pathway(s). S100A8 did not suppress antigen/IgE-induced responses of CBMC possibly because these may not truly reflect fullymature human tissue MCs. S100A8 did not alter UVB-induced IL-8 release by CBMCs, or affect apoptosis. Murine S100A8 may have anti-inflammatory properties by regulating MC activation in an activator-specific manner, at least partially by scavenging ROS to suppress intracellular signalling.

L'asthme allergique est caractérisé par une réponse prédominante de typeTh2, mais d'autres profils tels que Th17 et Th22 sont aussi observés dans l'asthme plus sévère. La pollution atmosphérique et notamment les hydrocarbures aromatiques polycycliques (HAP) contenus dans les particules d'échappement diesel (DEP) contribuent à l'augmentation de la prévalence de l'asthme et jouent un rôle adjuvant dans le développement et l'exacerbation de l'inflammation allergique en orientant la réponse immune vers un profil Th2Dans la première partie de la thèse, nous avons évalué les effets des HAP sur la polarisation Th17/Th22 de PBMC de sujets allergiques asthmatiques et sujets non-allergiques. Les PBMC de patients asthmatiques présentent une augmentation des profils Th17/Th22 par rapport aux sujets non-allergiques. La stimulation par DEP-HAP et le benzo [a] pyrène (B[a]P) induit une augmentation de l’IL-22 mais de façon étonnante diminue la production d'IL-17A dans les deux groupes. Les facteurs de transcription associés aux cellules Th17: RORA et RORC, sont diminués, alors que le gène cible d’AhR, CYP1A1, est augmenté dans les deux groupes. Notch est diminué uniquement chez les patients asthmatiques. La production d'IL-22 induite par les HAP provient principalement de cellules Th22. L'antagoniste d’AhR reverse presque complètement les effets des DEP, mais seulement partiellement les effets de B[a]P, sur la regulation réciproque entre IL-22/IL-17. Les kinases PI3K, JNK et ERK participent à l’augmentation de l’IL-22 induite par le B[a]P, alors que la p38 MAP kinase a un effet inhibiteur. La deuxième partie de la thèse a évalué l'effet combiné des PRRs et des allergènes sur l’activation et leur capacité à induire une polarisation Th17/Th22 chez des sujets sains et des sujets allergiques asthmatiques. Les DCs stimulées par les allergènes de chien induisent une faible production d'IL-22 par les cellules T. L’activation supplémentaire par les ligands de TLR3, TLR9 et NOD2 conduit à une augmentation de la production augmentée de chimiokines pro-Th2 uniquement par les DCs de patients asthmatiques allergiques aux chiens. 7 A l’inverse, un rôle adjuvant est observé sur la maturation et la production de cytokines pro-Th17/Th22 par les DCs de sujets asthmatiques et de sujets non-allergiques. Parmi les cellules T co-cultivées avec des DC stimulées par l'allergène de chien et les ligands de PRR, nous observons un mélange de cellules de type Th2/Th17 et Th22 chez les patients asthmatiques et un profil Th1/Th22 chez les sujets non-allergiques. L’IL-22 est principalement produite par les Th22 dans les deux groupes avec plus de cellules Th22 observés chez les sujets asthmatiques. L’IL-17 et l’IL-22 sont régulées différemment entre les sujets asthmatiques et les sujets sains, le TGF-β ayant un rôle pro-Th17 tandis que l'IL-23 a un rôle pro-Th22. In vivo, un modèle d'asthme chronique induite par l’allergène de chien a été développé et conduit à une augmentation de la résistance des voies aériennes, une production de chimiokines Th2 et de cytokines Th2/Th17/Th22 ainsi qu’au recrutement de neutrophiles mais peu d’éosinophiles dans le poumon. L'expression du gène de l'IL-22 intervient de façon précoce alors que la protéine apparait plus tard. Le ligand de NOD2 induit une augmentation de la résistance des voies aériennes, ainsi que de la production de protéines et l'expression des gènes induits par l'allergène de chien, mais réduit le recrutement des éosinophiles dans les poumons. Ces résultats montrent que chez l'homme, les productions d’IL-22 et d'IL-17 sont régulées différemment entre les sujets asthmatiques allergiques et les sujets sains. Au total, la pollution et certaines infections bactériennes ou virales pourraient participer chez les patients asthmatiques à sévérité de la maladie et la progression du remodelage des voies aériennes. La modèle in vivo développée permettra de mieux disséquer les mécanismes qui participent à la sévérité de l'asthme
Allergic asthma is characterized by a predominant Th2 response, but additional profiles such as Th17 and Th22 are observed in more severe asthma. Components of the air pollution such as polycyclic aromatic hydrocarbons (PAH) contained in diesel exhausts particles (DEP) contributes to increased prevalence of asthma and play an adjuvant role in the development and exacerbation of allergic inflammation through the skewing of the immune response towards a Th2 profile. In the first part of the thesis, we evaluated the effects of PAH on the Th17/Th22 polarization of PBMCs from healthy and allergic asthmatic subjects, PBMCs from athmatic patients exhibited an increased Th17/Th22 profile compared with non-allergic subjects. DEP-PAH and Benzo[a]Pyrene (B[a]P) stimulation further increased IL-22 but surprisingly decreased IL-17A production in both groups. Th17 transcription factors RORA and RORC were down regulated, whereas AhR target gene CYP1A1 was up-regulated in both groups. Notch was decreased only in asthmatic patients. PAH-induced IL-22 production originated mainly from Th22 cells. The AhR antagonist reversed almost completely the effects of DEP-PAH, but only partially the effects of B[a]P, on IL-22/IL-17 reciprocal regulation. The kinases PI3K, JNK and ERK participated to the enhancing effect of B[a]P on IL-22 production, whereas p38 MAP kinase had an inhibitory effect.The second part of the thesis evaluated the co-stimulatory effect of combined PRR- and allergen-activated DCs on Th17/Th22 polarization in healthy and asthmatic subjects. Dog allergen stimulated DCs induced a small production of IL-22 in T cells. Additional activation by TLR3, TLR9 and NOD2 ligands led to increased production of pro-Th2 chemokines by DCs only from asthmatic patients allergic to dogs. In contrast, an adjuvant role was observed on the maturation and pro-Th17/Th22 cytokines production by DCs from both asthmatic and non-allergic subjects. In T cells co-cultured with DCs stimulated by dog allergen and PRR ligands, we found a mixed Th2/Th17/Th22 profile in asthmatic patients and a Th1/Th22 profile in non-allergic subjects. IL-22 producing cells were mainly Th22 in both groups with more Th22 cells were observed in asthmatic subjects. IL-17 and IL-22 production was9differently regulated between asthmatic subjects and non-allergic subjects, TGF-β having a pro-Th17 role while IL-23 having a pro-Th22 role.In vivo, a model of chronic dog-induced asthma was developed leading increased airway resistance, Th2 chemokine and Th2/Th17/Th22 cytokine production as well as neutrophil but little eosinophil recruitment in the lung. Gene expression of IL-22 was observed at early time points whereas IL-22 protein appeared later on. NOD2 ligand further increased airway resistance, protein production and gene expression induced by dog allergen challenge but inhibited the small eosinophil recruitment in the lung.These results show that in humans, IL-17 and IL-22 productions are regulated differently between allergic asthmatic and non-allergic subjects. Altogether, pollution and some bacterial or viral infections may contribute in asthmatic patients to the severity of the disease and to progression of airway remodeling. The developed in vivo model will allow dissecting the mechanisms participating to the severity of asthma

Over one third of the human population is currently infected by one or more species of parasitic helminth, but the immune responses elicited by these infections remain poorly defined. Studies in helminth-exposed human populations and laboratory models suggest that helminth infection elicits a range of different effector cell types and that protective immunity and resistance to immune-mediated pathology depends on the balance between these responses. The aim of this thesis was to investigate how cytokines, the molecular mediators of the immune system, can be used to characterise human immune phenotype during natural and experimental helminth infection. Cytokines associated with innate inflammatory (TNFα, IL-6 and IL-9), Thl (IFNγ, IL-2 and IL-12p70), Th2 (IL-4, IL-5 and IL-13), Th17 (IL-17A, IL-21 and IL-23) and regulatory (IL-10 and TGFβ)immune phenotypes were analysed to provide the most comprehensive analysis of cytokine responses in human helminth infection conducted to-date. Using a multivariate statistical approach cytokines were analysed as combined immune profiles to reflect their complex interactions in vivo. In the first part of the study venous blood samples collected from a cross-sectional cohort of 284 Zimbabweans (age range: 3 -86 years) endemically-exposed to Schistosoma haematobium were cultured with antigens from different stages of the parasite's life-cycle(cercariae, adult worms and eggs) and the anti-schistosome vaccine candidate antigen glutathionine-S-transferase (GST). Cytokines responses were quantified in culture supernatants via enzyme-linked immunosorbent assay (ELISA). These assays were repeated 6 weeks after clearance of infection by anti-helminthic treatment. Parasitological and demographic characterisation of the cohort before, 6 weeks, 6 and 18 months after treatment allowed cytokine responses to be related to epidemiological patterns of infection before treatment and the risk of re-infection after treatment. The main findings of this study were:Cytokine responses to the antigens of S. haematobium cercariae are more proinflammatory than those elicited by adult worms and eggs prior to treatment, reflecting the distinct proteomes and exposure patterns of the 3 life-cycle stages Young children (5-10 years old) have a more regulatory and Th17-polarised cytokine response to S. haematobium antigens than older children and adults. These responses are significantly associated with schistosome infection intensity and may contribute to the development of resistance to schistosomiasis with age and exposure to infection Anti-helminthic treatment leads to a shift in S. haematobium cercariae, egg and GST specific cytokine responses towards a more pro-inflammatory phenotype The magnitude of change in S. haematobium-specific cytokine profiles after treatment is dependent on schistosome infection intensity at the time of treatment Individuals who remain un-infected up to 18 months after treatment to clear schistosome infection have a more pro-inflammatory and IL-21-polarised response to S. haematobium antigens 6 weeks after treatment than those who become re-infected, suggesting that post-treatment cytokine profiles promote resistance to re-infection. The second part of the study assayed systemic, parasite and allergen-specific cytokine responses in 45 adults with seasonally exacerbated allergy to grass pollen who were experimentally exposed to Trichuris suis. Cytokine responses in infected individuals were compared to those of 44 un-infected controls. This aspect of the study showed that: Exposure to T. suis promotes systemic and parasite-specific Th2 and regulatory cytokine responses, but does not alter cytokine responses to environmental allergens.

L'immunothérapie spécifique (ou désensibilisation) aux venins d'hyménoptères est un traitement qui permet de prévenir la récidive d’une anaphylaxie chez les patients allergiques au venin de guêpe ou d’abeille. Une augmentation très rapide des doses est souvent utilisée lors de la phase initiale de ce traitement dont la bonne tolérance n’est pas bien expliquée. Le but de ce travail était de décrire les changements précoces du système immunitaire pendant l’initiation de l’immunothérapie aux venins d’hyménoptères pouvant expliquer cette bonne tolérance. Nous avons inclus 29 patients traités pour une allergie au venin d’hyménoptères avec une initiation de traitement par « ultra-rush » en 3h30. Des prélèvements sanguins ont été pratiqués avant le début du traitement, à 1h30 et juste avant la dernière injection de venin. L’évolution de la tryptase sanguine a été analysée. L'activation des polynucléaires basophiles ainsi que l'expression FcεRI à leur surface ont été analysées par intensité moyenne de fluorescence par cytométrie en flux. Pour évaluer l'évolution de la réactivité des polynucléaires basophiles, un test d'activation des basophiles (TAB) a été réalisé à chaque temps. L’évolution des populations lymphocytaires T et myéloïdes a été également analysée par cytométrie en flux. Nous avons montré une diminution significative de la tryptase sérique pendant l’ultra-rush, de même qu’une diminution significative de l’activation des polynucléaires basophiles et une diminution de l’expression de FcεRI à leur surface. Etonnamment, le TAB a montré une réponse in vitro des basophiles significativement plus élevée à l'extrait de venin à la fin de « l'ultra-rush » par rapport à avant le début du traitement. Nous avons également montré une augmentation significative des cellules dendritiques et une diminution significative des lymphocytes « Natural Killer » (NK) dans le sang. Concernant les populations lymphocytaires T, nous avons montré une ugmentation significative des populations lymphocytaires T dans le sang, sauf pour les Lymphocytes T CD4+et CD8+ naïfs. En conclusion, l’augmentation des doses de venin par « ultra-rush » est bien tolérée grâce à une inhibition des polynucléaires basophiles impliquant une diminution de l’expression de FcεRI à leur surface. L'ultra-rush entraîne également des modifications précoces dans la réponse immunitaire innée et adaptative
Hymenoptera venom immunotherapy (VIT) is a treatment that prevents sting inducing anaphylaxis in allergic patient. Fast-up dosing schedule are often used at the initial phase of VIT. This fast dosing schedule well tolerated, but the mechanisms behind this good tolerance have not yet been elucidated, as well as its consequences on the rest of the immune systems. The aim of this study is to describe early immune system change during initial phase of VIT We included 29 patients undergoing VIT by 3h30 ultra-rush up dosing phase. Blood puncture was performed before the beginning of the treatment, at 1h30 and just before the last venom injection. Blood tryptase evolution was measured. Basophils phenotype and FcεRI surface expression were analyzed by flow cytometry at each step of the ultra-rush. To assess basophils responsiveness evolution, basophils activation test (BAT) was also perform. Myeloid and T lymphocytes population’s evolution were analyzed by flow cytometry. We have shown a significantly lower concentration of blood tryptase at the end of ultra-rush, and a significantly lower basophils activation and FcεRI expression. Surprisingly, BAT has shown a significantly higher in vitro response to venom extract at the end of ultra-rush. We also found significantly increase in blood dendritic cells concentration and lower blood Natural Killer (NK) Cells. We observed higher lymphocytes population in blood except for naïve CD4+ and CD8+ T cells. In conclusion, ultra-rush fast up dosing is well tolerated thanks to a basophils inhibition involving lower FcεRI surface expression. Ultra-rush also leads to early change in innate and adaptive immune response

Mast cells play a critical role in both acute and chronic inflammation and mature in peripheral tissues from bone marrow-derived progenitors that circulate in the blood as immature precursors. Mast cell progenitors are likely to encounter the serum-borne bioactive sphingolipid metabolite, sphingosine-1-phosphate (S1P), during migration to target tissues. Mast cells developed from human cord blood-derived progenitors cultured with stem cell factor (SCF) alone express intragranular tryptase (MCT), the phenotype predominant in the lung. S1P accelerated the development of cord blood-derived mast cells (CB-MCs) and strikingly increased the numbers of mast cells expressing chymase. These mast cells have functional FcepsilonRI, and similar to skin mast cells that express both tryptase and chymase (MCTC), also express CD88, the receptor for C5a, and are activated by anaphylatoxin C5a and the secretagogue compound 48/80. S1P induced release of IL-6, a cytokine known to promote development of functionally mature MCTC, from cord blood cultures containing adherent macrophages, and from highly purified macrophages, but not from macrophage-depleted CB-MCs. In contrast, S1P stimulated secretion of the chemokine, monocyte chemoattractant protein 1 (MCP-1/CCL2), from these macrophage-depleted and purified CB-MCs.

The mechanisms of allergen induced airway narrowing, during allergen induced early (ER) and late (LR) responses, and airway hyperresponsiveness were investigated in vivo using the ovalbumin (OA) sensitized Brown Norway (BN) rat. ER and LR were induced by aerosolized OA. The lungs were fixed using a quick-freezing technique to maintain the pathological alterations as close as possible to their in vivo conditions. The results from both morphologic and morphometric studies showed that during the ER and LR, airway narrowing was mainly caused by airway smooth muscle contraction, but not by airway wall thickening or mucus. These findings were supported by intravenous administration of Evans blue which was used as a measure of vascular leakage. The degree of airway narrowing during the early response was greater in the large intrapulmonary airways than in the small intrapulmonary airways. Airway narrowing during the late response was limited to the large intrapulmonary airways. These findings were closely related to the mast cell distribution which progressively increased from small to large intrapulmonary airways in normal BN rats. These findings suggests that mast cells may play an important role in both early or late responses.
In order to study structural mechanisms of airway hyperresponsiveness, increased airway responses to inhaled methacholine were induced by repeated inhalational challenge of sensitized BN rats with OA. Quantitation of airway smooth muscle, airway wall area and epithelial area demonstrated that increased airway hyperresponsiveness was associated with the increased amount of airway smooth muscle but not with airway wall thickening. There was a correlation between the quantity of airway smooth muscle in large airways and the change in airway hyperresponsiveness. Hence, it is possible that an altered quantity of airway smooth muscle is an important mechanical contributor to airway narrowing in human allergic asthma.

The early-life microbiota is important for postnatal immune maturation and implied in immune mediated diseases. The aim of this work was to study specific species of bacteria in the gut microbiota and relate them to immune function and allergic disease during childhood. In paper I we investigated gut bacteria in feces from infants included in a prospective allergy cohort. We found that children with non-allergic parents were more likely to be colonized with a group of lactobacilli. Further, lactobacilli colonization was more prevalent in children remaining non-allergic, regardless of allergic heredity. In paper II we related the infant gut bacteria to immune function at two years of age. Infant Staphylococcus (S.) aureus colonization associated with increased immune responsiveness, whereas co-colonization with S. aureus and lactobacilli associated with reduced responses. In paper III we investigated T regulatory (Treg) cell phenotype and cytokine production during childhood, and related S. aureus and lactobacilli colonization to Treg phenotype at the age of two. The Treg population matured with age, regarding phenotype and cytokine production. Furthermore, infant S. aureus colonization associated with Treg phenotype at the age of two. In paper IV we investigated the in vitro peripheral blood mononuclear cells responses to soluble factors produced by lactobacilli and S. aureus. Both T- and natural killer cells responded with cytokine production, degranulation and proliferation after S. aureus and simultaneous culture with lactobacilli could dampen the S. aureus-induced responses. Taken together this thesis shows that the gut microbiota is altered in children who develop allergies, and that early life bacteria associate with immune function. Our in vitro findings support that lactobacilli modulate immune maturation and responses, and that early lactobacilli-colonization may be important for a properly regulated maturation of the immune system.

At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 3: Manuscript. Paper 4: Manuscript.

Dendritic cells (DCs) are key players in the induction and re-elicitation of T helper (Th) 2 immune responses to allergens. Recent data by our group have shown that different C-type lectin receptors (CLRs) on DCs play a major role in allergen recognition and uptake. Particularly, mannose receptor (MR), through modulation of Toll-like receptor (TLR) 4 signalling pathway, can regulate indoleamine 2,3 dioxygenase (IDO) activity favouring Th2 responses. IDO is the rate limiting enzyme involved in tryptophan (TRP) catabolism and it is well known for its role in modulating immune responses through TRP depletion and generation of immune-regulatory metabolites known as kynurenines (KYN). Interestingly, another CLR named dendritic cell-specific intracellular adhesion molecule 3-grabbing non-integrin (DC-SIGN) has been suggested to support Th1 responses; however, the mechanisms involved are unknown. Therefore, the main objective of this project was to evaluate the role of CLRs, with focus on MR and DC-SIGN, and TLRs in modulating the IDO pathway in order to understand their involvement in regulating Th2 immune responses. In addition, it was aimed to study how regulation of IDO levels could impact immune responses in order to understand the signalling pathways involved in this process. The data show that mannosylated allergens can down-regulate TLR4-induced IDO1 and IDO2 expressions as well as IDO activity in human DCs, most likely through binding to the MR. This correlates with data showing increased IDO activity in MRlow-DCs (generated by gene silencing) stimulated with mannan compared with control-DCs (CT-DCs). Conversely, DC-SIGN could exert a differential regulation on IDO levels depending on the antigen’s sugar moieties. Mannosylated ligands could up-regulate TLR4-induced IDO activity through DC-SIGN as evidence by a significant reduction in IDO activity in DC-SIGNlow-DCs upon stimulation with mannan compared with CT-DCs. In contrast, DC-SIGN-specific fucosylated ligands, such as Lewis-X (LeX), can down-regulate IDO activity in a TLR4-dependent manner. It was also found that allergens from diverse source can down-regulate IL-12p70 production by DC, which can further impact T helper cell polarization by reducing IFN-γ production by T cells in autologous co-cultures. Moreover, mannan was also shown to decrease CD86 expression in human DCs, which might impact the IDO pathway, as CD86 is one of the B7 family molecules involved in IDO induction. Furthermore, it was found that the LeX can potentially down-regulate IL-6 production by DCs. In addition, DCs stimulated with LeX and LPS induce lower levels of IFN-γ production by autologous T cells than DCs stimulated with LPS only. This effect was reversed in the presence of KYN, showing that the IDO pathway can regulates T helper cell polarization. TLR4 signalling and LPS exposure have been shown to play a pivotal role in Th2-mediated inflammation and asthma. In addition, it is known that CLRs can modulate TLR-induced responses in an allergy context. Therefore, we further study the role of the TLR signalling pathway in modulating IDO levels in human DCs. TLR4 engagement on human DCs was shown to induce high levels of both IDO isoforms. DCs primed with LPS induce much higher levels of IDO than single LPS controls, inducing a cellular re-programing. This was characterized by an induction of anti-inflammatory mediators such as IL-10 and the transcription factor aryl-hydrocarbon receptor (AhR); the last has been previously linked with the IDO pathway in mouse DCs. Intriguingly, TLR9 engagement with synthetic cytosine-phosphate guanosine (CpG) A motifs was able to down-regulate IDO and induce a pro-inflammatory phenotype in human DCs. Future studies should aim at evaluating their implications in immune responses under tolerance and during infection. Finally, the study was aimed at elucidating the intracellular molecules involved in IDO regulation in human DCs. The analysis was first focused on AhR, a ligand-dependent transcription factor that have been suggested to modulate IDO levels in mouse DCs. It was shown for the first time that TLR4 induction of IDO was dependent on AhR in human DCs. In addition, AhR gene expression as well as AhR activity were reduced in DCs stimulated with mannan and LPS compared with LPS only controls. This data suggest that MR can regulate IDO levels by interfering with AhR expression. Furthermore, it was shown that the NF-κB pathway is key in regulating IDO levels through CLRs and TLRs in human DCs. Particularly, RelB, a member of the non-canonical pathway, expression correlates with the levels of AhR, suggesting that a functional and/or physical association between them might be involved in regulating IDO levels in human DCs. Future studies should aim at elucidating such interactions. To sum up, this project has shown the pivotal role of CLRs and TLRs in modulating the IDO pathway in human DCs, which can affect DC phenotype and function and impact immune responses. In addition, we have gained some understanding into the molecular mechanisms of IDO modulation which might involve cooperation between different transcription factors such as AhR and RelB. These data give new insights into how glycosylated allergens can induce Th2 immune responses, which can pave the way to develop better therapeutic strategies to fight back allergy related diseases.

Background: Allergic diseases are characterised by dysregulated immune responses. The first manifestation of the atopic phenotype is often food allergy, with symptoms like eczema. Food allergy in children is generally outgrown before 3 years of age, but a temporary food elimination diet is often advocated. The prevalence of allergic diseases has increased in affluent countries during the last decades, possibly as a consequence of a changed lifestyle leading to decreased microbial load. Aim: To investigate humoral, mucosal and cell-mediated immunity in association to allergy and allergy development in young children and relate this to environmental factors. Subjects: Two cohorts of children were investigated; 1) Children from countries with high (Sweden) and low (Estonia) prevalence of allergy that were followed prospectively from birth to 5 years of age. 2) Infants with eczema and suspected food allergy that were followed prospectively to 4 ½ years of age. Methods: Endotoxin levels were analysed in house dust samples. Antibodies were measured in serum and saliva samples with ELISA. Food allergen induced cytokine responses were analysed in mononuclear cells. Results: The microbial load, delineated as endotoxin levels, was higher in house dust from Estonia than Sweden and was, in Swedish children, inversely associated with sensitisation and clinical symptoms of allergy. The decreased microbial load in Sweden may have an impact on mucosal immune responses as different IgA antibody patterns were observed in Sweden and Estonian children with much lower secretory (S)IgA antibody levels and high proportion of non-SIgA, i.e. IgA antibodies lacking the secretory component, in the Swedish children. Moreover, low levels of SIgA were associated with clinical symptoms in sensitised children. High IgG4 antibody levels to food allergens during infancy were associated with faster tolerance development in food allergic children. Cytokine responses by mononuclear cells after allergen stimulation was upregulated with age in children with prolonged food allergy, but not in children who develop tolerance before 4 ½ years of age, possibly because of the prolonged elimination diet in the former group. Summary: Reduced microbial exposure in affluent countries may affect the mucosal immune responses during infancy, possibly resulting in an increased risk of developing allergic disease. High levels of IgG4 antibodies during infancy are associated with faster achievement of tolerance in food allergic children. Allergen elimination during infancy may result in a dysfunctional cytokine response.

Allergies are emerging as a major public health concern in the westernized world as they are increasing for reasons that remain poorly understood. Allergies involving polysensitization and multiple organ involvement result in decreased quality of life, increased morbidity and mortality. Allergic subjects can be poly-sensitized to different allergens due to phylogenetic relatedness; several species contain shared allergenic epitopes. This phenomenon occurs both at the IgE as the T cell level. While IgE cross-reactivity has been well documented, T-cell cross-reactivity has not been thoroughly studied. Well-designed studies that underlie the T-cell mechanisms that accompany allergic inflammation are highly useful in future design for successful immunotherapy as T-cells play a crucial role in the perpetuating cycle of allergic response. Cross-reactivity at the T-cell level was studied with the use of HLA class II Tetramers in two distinct models: grass-pollen and tree nut allergy. In the first study, grass pollen from the Pooideae subfamily was studied. We identified that Timothy grass derived epitopes did not cross-reacted with all homologous epitopes from distinct grass-pollen species indicating that timothy grass alone does not cover the whole set of epitopes. Moreover, these minimally cross-reactive epitopes were detected with comparable frequency, phenotype and functionality to Phl p-specific T-cells, suggest that a multiple allergen system should be considered for immunotherapy instead of a mono allergen system. In the tree nut allergy model, we used cashew as a model to study tree-nut cross-reactivity against hazelnut, pistachio and walnut at the T-cell level. T-cell-clones specific to 7 out of 12 were generated to assess cross-reactivity by tetramer co-staining and proliferation experiments. TCC specific to cashew derived epitopes could readily proliferate with hazelnut and pistachio but not walnut peptides. This study leads to the conclusion that exposure to other tree nut allergens can trigger cashew-specific T-cell responses in these cashew allergic subjects. Strict avoidance of other tree nuts should be recommended in cashew allergic subjects. Food allergies have become a major public health concern as they can cause severe and sometimes fatal reactions. Nonetheless, food allergy-specific T-cell epitopes remain unidentified and uncharacterized. In the food allergy characterization study, we examined walnut allergen-specific T-cells. T-cell reactivity towards Jug r 1 and Jug r 2, as their corresponding allergens in peanut, 2S albumin (Ara h 2) and 7S vicilin-like seed storage protein (Ara h 1) respectively, are highly immunogenic in peanut allergic subjects. Jug r 3 was also studied since we have a small cohort of samples from Spain, where LTP is the major plant food allergen. We initially investigated Jug r 1 Jug r 2 and Jug r 3-specific T-cell responses using CD154 activation assay. Jug r 2, but neither Jug r 1 nor Jug r 3, elicited dominant T-cell responses in allergic subjects. Several Jug r 2 derived epitopes were then identified by using tetramer-guided epitope mapping (TGEM). The magnitude and phenotype of the response of Jug r 2-specific CD4+ T-cells in allergic and non-allergic subjects were determined directly ex-vivo. The predominant phenotype for Jug r 2 reactive T-cells is central memory phenotype. Results show that allergic subjects have a predominant TH2 phenotype, however, TH17 responses in some individuals were also observed. T-cells with CCR4+CD27+, CCR4+CD27-, CCR4+CCR6+ and CCR4+CCR6- surface phenotypes were detected in allergic subjects. T-cells from non-allergic subjects have a TH1 and TH1/TR1 phenotypes characterized by surface expression of CXCR3. Similar observations were observed in the cashew model. Both Cashew and Walnut-specific T-cells with TH2, TH17 and TH2/TH17 phenotypes could be detected in non-asthmatic and asthmatic walnut allergic subjects. Understanding this T-cell heterogeneity may improve our understanding of disease manifestation.
Las alergias están aumentando por razones que aun son desconocidas. Los alérgenos, pueden actuar al nivel de IgE como al nivel de células T. Generalmente, los pacientes alérgicos están polysensitizados a distintas especies que contienen alérgenos debido a su relación filogenética. Aunque la reactividad cruzada ha sido estudiada a nivel de IgE, la misma ha sido poco estudiada a nivel de células T. En este estudio utilizamos tetrámeros de clase II para estudiar este fenómeno en dos modelos: uno de gramíneas y otro de frutos secos. Para el primero encontramos que un sistema de especies múltiples debería de ser utilizado, debido a que células que mínimamente reactivas están presentes con fenotipos, frecuencias y funcionalidad a la de la gramínea Timothy. Para los frutos secos encontramos que los clones de células T derivados de anacardos reconocen y tienen capacidades funcionales hacia pistacho y almendra pero no a la nuez. Evitar estos alimentos debería de ser estrictamente sugerido. Finalmente, nuestro estudio que se baso en la caracterización de células T especificas contra alimentos fue enfocado en la alergia a la nuez. Encontramos que los pacientes alérgicos contienen una variedad heterogenia en la respuesta celular T, caracterizada por respuestas TH2, TH2/TH17 y TH17, mientras que los pacientes no alérgicos tenían una frecuencia de células T baja o ausente y un fenotipo TH1. Además, la mayoría de las células tienen un fenotipo de memoria central. El entendimiento de esta heterogeneidad debería de ser aplicado para la mejora de tratamientos.

Allergic airway diseases such as rhinitis, asthma, and chronic rhinosinusitis are responsible for causing a huge economic burden on patients and society. Patients suffering from asthma often have allergies to pollen, dust mite, and mold. Interestingly, studies have shown that there is a correlation between severe asthma and sensitization to fungi including Aspergillus, Alternaria, Cladosporium, and Penicillium. This project has been focused on studying the innate immunomodulatory activities of the major allergen Alt a 1, from the ubiquitous airborne fungus, Alternaria alternata. In several studies, 90-100% of allergic patients who are sensitized to Alternaria, have Alt a 1 specific IgE antibodies indicating that it is a major and clinically relevant allergen. Although progress has been made over the past few decades regarding elucidating the mechanistic underpinnings of allergic inflammation, more research needs to be done, especially in regards to innate immunity and its role in the sensitization and exacerbation aspects of allergic diseases. Published studies have increasingly made it clear that Toll-like receptors (TLRs) are key players in innate immunity to several allergens. For example, the dust mite allergen, Der p 2, has been shown to mimic the activity of human and mouse MD2 in the presence of LPS to trigger a response through TLR4. Bet v 1, an allergen from Birch tree, has been shown to enter and be transported through lung epithelium in patient cells. It is hypothesized that transcytosis of allergens like Bet v 1 may contribute to sensitization and exacerbation in atopic individuals. This project was focused on two primary aims; (1) Characterize the innate immune response of Alt a 1 in human airway epithelial cells, and (2) Identify if and how Alt a 1 can enter human airway cells. We found that Alt a 1 was able to stimulate innate immune responses in bronchial epithelial cells and this was dependent upon TLR2, TLR4 and the downstream adaptor proteins MyD88 and TIRAP. We also found in our studies that Alt a 1 rapidly enters bronchial epithelial cells. Furthermore, our data suggests that endocytosis of Alt a 1 may be partially dependent upon interaction with phosphatidyl-inositol-3-phosphate (PI-3-P).
Ph. D.

Der globale Prävalenzanstieg allergischer Erkrankungen wird mit der westlichen Ernährung und einem sich ändernden Fettsäurespektrum assoziiert. Die omega-3 Fettsäure Docosahexaensäure (DHA) wurde bereits bei verschiedenen chronisch-entzündlichen Erkrankungen erfolgreich therapeutisch eingesetzt. Die dabei zugrunde liegenden Wirkmechanismen sind jedoch nicht vollständig geklärt. Deshalb wurde hier der molekulare Mechanismus der DHA-vermittelten Hemmung der IgE-Produktion in humanen B-Zellen sowie der verminderten Differenzierung IgE-produzierender Plasmazellen in vitro untersucht. Analysen der beteiligten Signaltransduktionswege offenbarten eine Reduktion der IL-4-abhängigen STAT6-Phosphorylierung und der CD40-vermittelten NFkappaB-Translokation, was zu einer Inhibition des IgE-Klassenwechsels auf dem Niveau des epsilon-Keimbahntranskriptes sowie der Aktivierungsinduzierten Desaminase führte. Weiterhin wurde in einer randomisierten, kontrollierten Doppelblindstudie die Supplementierung mit hochdosierter DHA bei Patienten mit atopischem Ekzem hinsichtlich klinischer und immunologischer Parameter geprüft. Dabei führte DHA zu einer Reduktion des Schweregrades der Erkrankung und zu einer verminderten IgE-Produktion anti-CD40/IL 4-stimulierter Blutzellen ohne Beeinflussung des Serum-IgE-Spiegels. Schließlich wurden die lokalen Prozesse nach DHA-Verabreichung in einem Mausmodell für proteininduzierte Dermatitis analysiert. Dabei war die Reduktion der klinischen Ekzemausprägung mit der verminderten Zahl dermaler CD8+ T-Zellen verbunden. Andere histologische Parameter und das Serum-IgE blieben jedoch unbeeinflusst. Durch die Fähigkeit von DHA, in den IgE-Klassenwechsel in B-Zellen einzugreifen, stellt die Supplementierung mit DHA somit eine mögliche präventive Maßnahme gegen allergische Erkrankungen dar. Ebenso ist DHA in der Lage, den Schweregrad des atopischen Ekzems durch die positive Beeinflussung lokaler inflammatorischer Prozesse signifikant zu verbessern.
The prevalence of allergic diseases has increased worldwide. Westernised diet with its changing polyunsaturated fatty acid (PUFA) proportions is considered to contribute to this development. The omega-3 PUFA Docosahexaenoic acid (DHA) has been reported to be antiinflammatory, but its way of action is not completely understood. Initially, the molecular mechanisms of DHA impact on IgE production in human B cells were examined in vitro. Thereby, DHA inhibited IgE production and the differentiation of IgE secreting cells. This was mediated through direct inhibition of the immunoglobulin isotype switching process by decreasing epsilon germline transcript and activation induced desaminase transcription. Analysis of involved signalling pathways revealed an inhibition of IL-4 driven STAT6 phosphorylation and a reduced NFkappaB translocation into nucleus upon CD40 ligation. Next, in a randomised, double bind, controlled clinical study the efficacy of high-dose DHA supplementation in atopic eczema was determined by investigating the impact on clinical and immunological parameters. In the DHA, but not in control group a clinical improvement of atopic eczema and a reduction of CD40/IL-4 mediated IgE synthesis of peripheral blood cells were observed whereas serum IgE levels remained unchanged. Finally, in a mouse model the impact of oral DHA application on allergen induced dermatitis as well as the underlying local mechanisms were investigated. Thereby, the DHA mediated reduction of clinical skin score was associated with decreased dermal CD8+ T cell numbers, whereas other histological parameters or serum IgE values were not affected. In summary the results indicate that dietary DHA may be effective in prevention of allergic diseases by interference with the IgE switching process and improve the clinical outcome of atopic eczema by its positive impact on local inflammatory processes.

Background: The incidence of allergic diseases in industrialized countries has increased, and a relation between allergy and dietary fatty acids has been proposed. Modulation of the maternal immune function during pregnancy may have an impact on future clinical outcome in the child. Aim: The aim of this thesis was to add knowledge on the relationship between long chain polyunsaturated fatty acids, sensitization and allergic disease and possible immunological events regulating this. Subjects: The thesis is based on results obtained from two cohorts. The first, including 300 cord blood samples collected from 1985-2005. The second, a double-blind placebo controlled multi-centre study comprising 145 families with allergic disease. Methods: Phospholipid fatty acids and total IgE antibodies were analyzed in cord blood samples with gas chromatography and Uni-CAP™, respectively. The families participating in the double-blind placebo controlled multi-centre study were recruited at antenatal units in Linköping and Jönköping and the mothers were supplemented with 2.6 g ω-3 long-chain polyunsaturated fatty acids (LCPUFA) or placebo daily from gestational week 25 until 3 months of breast feeding. Phospholipid fatty acids in maternal serum were analysed before and during the intervention to assess compliance. Prostaglandin E2, leukotrienes B4 and cytokines were analyzed with ELISA technique in supernatants from maternal LPS-stimulated whole blood cultures. Clinical outcome was allergic disease with positive skin prick test and/or specific circulating IgE to food allergens at one year of age. Cytokines, chemokines, SIgA antibodies and prostaglandin E2 were analyzed in breast milk with Luminex and ELISA techniques. Results: The proportions of cord serum linoleic acid (LA, C18:2 ω-6) and α-linolenic acid (LNA, C18:3 ω-3) decreased significantly from 1985 to 2005. However, the LA/LNA ratio did increase, revealing a relatively larger decrease in LNA than in LA. The proportions of both arachidonic acid (AA; C20:4 ω-6) and docosahexaenoic acid (DHA, C22:6 ω-3) as well as other ω-6 and ω-3 fatty acids increased significantly during the same time period. No correlations were found between ω-6 and ω-3 fatty acids and total IgE antibodies. Proportions of ω-3 LCPUFA increased in the ω-3 supplemented group of mothers. Lipopolysaccharide-induced prostaglandin E2 secretion in whole blood culture decreased in a majority of ω-3 PUFA supplemented mothers (18 of 28, p < 0.002).The decreased prostaglandin E2 production was more pronounced among non-atopic than atopic mothers. Lipopolysaccharide induced cytokine and chemokine secretion was not affected. The period prevalence of food allergy was lower in the ω-3 group (1⁄52, 2%) compared to the placebo group (10⁄65, 15%, p <0.05) as well as the incidence of IgE-associated eczema (ω-3 group: 4 ⁄ 52, 8%; placebo group: 15 ⁄ 63, 24%, p < 0.05) at one of year. There were no differences in breast milk cytokine, SIgA and PGE2 levels between the two intervention groups. However, the levels of several cytokines tended to be higher in colostrum from non-atopic ω-3 supplemented mothers as compared to non-atopic placebo supplemented mothers. Higher levels of TGFß2 and SIgA in 3 months milk were associated with allergic disease at one year of age both with and without detectable IgE. Conclusions: Cord blood LA proportions decreased and LA/LNA ratio increased over the 20 year period between 1985 and 2005 this was not related to total IgE. ω-3 fatty acid supplementation of pregnant and lactating mothers resulted in a lower period prevalence of IgE associated eczema and food allergy in the children at one year of age. This was most pronounced in children of non-allergic mothers. The underlying mechanism requires further clarification.

Thèse (Ph. D.)--Université de Montréal, 2002.
"NQ-73468." "Thèse présentée à la faculté des études supérieures en vue de l'obtention du grade de philosophiae doctor (Ph. D.) en sciences biomédicales." Version électronique également disponible sur Internet.

[ES] Antecedentes: Los patrones de colonización microbiana alterados durante la infancia pueden ser en parte responsables del aumento de enfermedades alérgicas en los países desarrollados. La microbiota intestinal difiere en composición y diversidad durante los primeros meses de vida en niños que luego desarrollan o no una enfermedad alérgica. Sin embargo, poco se sabe sobre la importancia de las respuestas inmunitarias tempranas de la mucosa a la microbiota intestinal en el desarrollo de alergias infantiles. Además, los estudios con respecto al efecto protector de la microbiota de la leche materna en el riesgo de desarrollar alergias no han sido concluyentes. Aunque la cavidad bucal es el primer lugar de encuentro entre la mayoría de los antígenos exógenos y el sistema inmunológico, no existen datos sobre la influencia de las bacterias orales en el desarrollo de alergias durante la infancia. Objetivos: El objetivo general de esta tesis fue evaluar la composición y diversidad microbiana en muestras orales, intestinales y de leche materna, junto con su interacción con IgA, para estudiar el papel de la colonización microbiana durante edades tempranas de la vida en condiciones de salud y de enfermedad alérgica. Sujetos: Los bebés y las madres incluidas en este estudio forman parte del ensayo aleatorio doble ciego más grande de Suecia, entre 2001 y 2003, donde se evaluaron los posibles efectos preventivos sobre la alergia de Lactobacillus reuteri ATCC 55730 hasta los 2 y 7 años. En esta tesis, utilizamos muestras de heces recogidas a los 1 y 12 meses, y muestras orales de bebés, obtenidas longitudinalmente a los 3, 6, 12, 24 meses y 7 años. Además, analizamos muestras de leche materna, recogidas a un mes después del parto de las madres correspondientes. Métodos: Se utilizaron tecnologías de secuenciación de segunda generación dirigidas al gen 16S rARN, en combinación con citometría de células marcadas por fluorescencia, para abordar las respuestas de IgA de la mucosa hacia las bacterias intestinales y de la leche materna. Además, se utilizó la secuenciación del gen 16S para describir la colonización oral de la microbiota, en muestras de saliva, de niños que desarrollaron alergias o de aquellos que se mantuvieron sanos. Los niveles de carga bacteriana en diferentes hábitats microbianos se obtuvieron mediante la metodología de qPCR y los niveles totales de IgA de las muestras de heces se determinaron mediante inmuno-ensayo ELISA. Resultados y conclusión: La colonización de la cavidad bucal durante la infancia temprana es progresiva, aumenta en complejidad con el tiempo, y varios factores externos parecen influir en gran medida en la maduración de la microbiota oral, ya sea con un impacto a corto o largo plazo. Los cambios tempranos en la composición microbiana oral parecen influir en la maduración inmune y el desarrollo de alergias en la infancia, y la presencia de especies bacterianas específicas puede ser importante para este proceso. Además, las respuestas de IgA alteradas hacia la microbiota intestinal durante la infancia precedieron a las manifestaciones de asma y alergia durante los primeros 7 años de vida, y el consumo de leche materna con una riqueza microbiana reducida en el primer mes de vida puede aumentar el riesgo de desarrollar alergia durante la infancia. Los hallazgos observados en la presente tesis deben confirmarse en cohortes más grandes y la importancia de los factores ambientales postnatales para el desarrollo temprano de la microbiota debe abordarse más a fondo. Las investigaciones futuras deben ir más allá de la caracterización de la composición de la comunidad bacteriana e investigar los mecanismos funcionales entre los microorganismos colonizadores tempranos, la maduración inmunitaria y la alergia, así como el desarrollo del asma durante la infancia.
[CAT] Antecedents: S'ha proposat que els patrons de colonització microbiana alterats durant la infància podrien ser en part els responsables de l'augment de malalties al·lèrgiques als països desenvolupats. La microbiota intestinal difereix en composició i diversitat durant els primers mesos de vida en els nens que després van desenvolupar una malaltia al·lèrgica. No obstant això, poc es sap sobre la importància de les respostes immunes de la mucosa a la microbiota intestinal en el desenvolupament d'al·lèrgies infantils. A més, les investigacions amb relació a l'efecte protector de la microbiota de la llet materna en el risc de desenvolupar al·lèrgies no han sigut concloents. Encara que la cavitat bucal és el primer lloc de trobada entre la majoria dels gèneres externs i el sistema immunològic, encara no s'ha descobert la influència dels bacteris en el desenvolupament d'una al·lèrgia durant la infància. Objectius: L'objectiu general d'aquesta tesi va ser avaluar la composició microbiana i la diversitat de mostres orals, fecals i llet materns, juntament amb la seva interacció amb IgA, per estudiar el paper del desenvolupament microbià durant el període de la infància primerenca a la salut i la malaltia al·lèrgica. Subjectes: Les mares i xiquets inclosos en aquest estudi formen part d'un estudi aleatori doble-cec a Suècia, entre el 2001 i el 2003, on es van avaluar els possibles efectes preventius de la suplementació amb Lactobacillus ATCC 55730 fins als 2 i 7 anys. En aquesta tesi, s'utilitzaren mostres de bebès arreplegades longitudinalment, obtinguts a 1 i 12 mesos, 3, 6, 12, 24 mesos i 7 anys, respectivament. A més, s'analitzaren les mostres de llet materna, arreplegades a un mes postpart de les corresponents mares. Mètodes: S'han utilitzat tecnologies de seqüenciació de nova generació dirigides al ARNr 16S, en combinació amb la classificació de les cèl·lules activades, per abordar les respostes de la mucosa cap als bacteris intestinals i de la llet materna. A més, s'utilitzà la seqüenciació d'Illumina MiSeq del gen 16S per descriure la colonització microbiana oral, i es van obtenir mostres longitudinals de saliva de menuts que varen desenvolupar al·lèrgies i d'alguns que es van mantenir saludables. Els nivells de càrrega bacteriana en diferents nínxols microbians s'han obtingut mitjançant la metodologia de qPCR i els nivells totals d'IgA de les mostres fecals es determinaren mitjançant l'immunoassaig ELISA. Resultats i conclusions: La colonització de la cavitat bucal durant la primera infància és transitòria, augmenta la seva complexitat amb el temps, i diversos factors externs influeixen en gran mesura el procés de maduració de la microbiota oral, amb un impacte a curt i llarg termini. Els canvis primerencs en la composició microbiana oral pareixen influir en la maduració del sistema immunològic i el desenvolupament d'al·lèrgies a la infància, així com la presència d'espècies bacterianes específiques pot ser important per a aquest progrés. A més, les respostes d'IgA alterades cap a la microbiota intestinal durant la infància precedeixen a les manifestacions relatives a la malaltia asmàtica i al·lèrgiques durant els primers 7 anys de vida. Per altra banda, el consum de llet materna amb una microbiota de riquesa reduïda al primer mes de vida podria augmentar el risc de desenvolupar al·lèrgia durant la infància. Els resultats observats en aquest estudi haurien de confirmar-se en cohorts humanes més grans i la importància dels factors ambientals post natals que influeixen en el desenvolupament de la microbiota primerenca han de ser més estudiats. Les investigacions futures deuen anar més enllà de la caracterització de la composició de la comunitat bacteriana i investigar els mecanismes funcionals entre els microorganismes colonitzadors primerencs, la maduració del sistema immunològic i el desenvolupament de l'al·lèrgia i l'asma durant la in
[EN] Background: It has been proposed that altered microbial colonization patterns during infancy may be partly responsible for the increase of allergic diseases in developed countries. The gut microbiota differs in composition and diversity during the first months of life in children who later do or do not develop allergic disease. However, little is known about the significance of early mucosal immune responses to the gut microbiota in childhood allergy development, and the findings regarding the protective effect of breastmilk microbiota in the risk of allergy development have been inconclusive. Furthermore, even though the oral cavity is the first site of encounter between a majority of foreign antigens and the immune system, the influence of oral bacteria on allergy development during childhood has not yet been reported. Objectives: The general aim of this thesis was to assess the microbial composition and diversity of oral, fecal and breastmilk samples, together with its interaction with IgA, in order to study the role of microbial development during early childhood in health and allergic disease. Subjects: The infants and mothers included in this study were part of a larger randomized double-blind trial in Sweden, between 2001 and 2003, where potential allergy preventive effects of Lactobacillus reuteri ATCC 55730 were evaluated until 2 and 7 years of age. In this thesis, we used longitudinally collected stool and oral samples from infants, obtained at 1 and 12 months and 3, 6, 12, 24 months and 7 years of age, respectively. Furthermore, we analyzed breastmilk samples, collected at one month post partum, from the corresponding mothers. Methods: Next-generation sequencing technologies targeting the 16S rRNA gene, in combination with cell activated cell sorting, were used in order to address mucosal IgA responses towards gut and breastmilk bacteria. Furthermore, sequencing of the 16S rRNA gene was used in order to describe oral microbiota colonization, in longitudinally obtained saliva samples, from children developing allergy or staying healthy. Bacterial load levels in different microbial habitats were obtained by qPCR methodology and total IgA levels of stool samples were determined by ELISA immunoassays. Results and conclusion: Colonization of the oral cavity during early childhood is transitional, increasing in complexity with time, and several external factors appear to greatly influence oral microbiota maturation, having either a short or a long-term impact. Early changes in oral microbial composition seem to influence immune maturation and allergy development in childhood, and the presence of specific bacterial species may be important for this progress. Furthermore, altered IgA responses towards the gut microbiota during infancy preceded asthma and allergy manifestations during the first 7 years of life, and consumption of breastmilk with a reduced microbial richness in the first month of life may increase the risk for allergy development during childhood. Findings observed here need to be confirmed in larger cohorts and the importance of postnatal environmental factors for early microbiota development should be addressed further. Future research should go beyond characterization of bacterial community composition and investigate the functional mechanisms between early colonizing microorganisms, immune maturation and allergy and asthma development during childhood.
Dzidic, M. (2019). Microbiota development and mucosal IgA responses during childhood in health and allergic disease [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/125479
TESIS

The mechanistic factors responsible for the increase in allergic diseases are still not fully understood, but a reduced microbial stimulation seems to be one of the key issues. Research is now aiming at investigating the relationship between the innate immune system, involving the toll-like receptors, and allergy development. Further, the maternal influence on the child, possibly through in utero effects, but also through the breast milk, has shown to be of great importance. This thesis aimed at understanding how the maternal immune system is influenced by early exposures and allergic disease, but also to investigate the consequences of the maternal phenotype on the innate immune system of the developing child. The Th1/Th2 cytokine pattern in allergic diseases has been extensively studied. Here we were interested in comparing the innate cytokines in allergic and non-allergic women, and to see if the allergic status was influencing the effect of pregnancy differently. We demonstrate that IL-1β, IL-6, IL-10 and IL-12 production in cells from adult women are not influenced by allergic status, neither during pregnancy nor 2 years after. However, pregnancy had an apparent effect on cytokine levels, regardless of allergic status. Also, total IgE levels in allergic women were significantly lower 2 years after pregnancy in comparison with the levels during pregnancy, pointing to the fact that pregnancy indeed has an immunomodulatory role. We further wanted to investigate the immune system of mothers who had migrated to Sweden in comparison with indigenous mothers. The reason for our interest here was that children born from immigrated mothers have shown to have an increased risk of developing diseases such as allergy and Crohn’s disease. The results showed that immigrants from a developing country had significantly higher levels of breast milk IL-6, IL-8 and TGF-β1. Further, regardless of maternal country of birth, a larger number of previous pregnancies was associated with down-regulation of several substances, statistically significant for soluble CD14 and IL-8. The results suggest that maternal country of birth may indeed influence adult immune characteristics, potentially relevant to disease risk in offspring. The influence of allergic status of the mother on the expression of CD14, TLR2 and TLR4 was further investigated in monocytes from mothers and their newborn babies upon microbial stimulation. We could not find any differences in monocytic TLR levels between the groups. No significant differences regarding cytokine levels between allergic and non-allergic mothers in response to stimuli were found either. However, the cytokine and chemokine release triggered by TLR2 stimulation in CB revealed that CBMC from children with maternal allergic disease released significantly less IL-6, and a trend towards less IL-8. As we could not find differences in TLR levels attributed to maternal allergy, but an impaired IL-6 response, we turned our focus on an intracellular event taking place after TLR ligation. The results confirmed our results of decreased IL-6 levels in CB from children to allergic mothers. At 2 years of age, the children of allergic mothers still displayed a diminished IL-6 response. Additionally, they also had a decreased activity of p38 MAPK. p38 has an important role in driving Th1 responses, suggesting that the p38 pathway could be one of the responsible mechanisms behind the impaired responses correlated to allergic heredity found in CB as well as at 2 years of age. Infancy is a crucial time period for the developing immune system. Further, the relative composition of the two major monocytic subsets CD14++CD16- and CD14+CD16+ is altered in some allergic diseases. TLR levels are different in the two subsets, proposing a possible link to the reduced responding capacity of monocytes from children with allergic heredity. We followed up our earlier studies of children at birth and at 2 years of age by looking at 5 year old children. There were no differences regarding monocytic subsets, nor in TLR levels in unstimulated cells. However, when stimulating the cells with PGN, both monocytic subsets in allergic subjects were less capable of upregulating TLR2 compared to the age-matched controls. Taken together, the work in this thesis suggests that the maternal immune system is affected by the process of pregnancy and childhood exposures. It further suggests that maternal allergy affects the young child, in terms of impaired responses to microbial stimuli, which later in infancy correlates with allergic disease in the child. These impaired innate responses could lead to a diminished Th1 response, or alternatively to a deficiency in regulatory mechanisms, and thereby cause allergic disease.