Background: Atopic disease is an increasing problem. Eczema affects 10-20% of young children, and 33-37% of children with eczema are food allergic. Among other factors, nitric oxide (NO) is thought to play a role in eczema and food allergy. Following the atopic march, pproximately 80% of children with atopic eczema will become sensitized to aeroallergens and develop asthma and/or allergic rhinitis. Skin prick test is used for investigating sensitization and is considered a safe method. However, systemic allergic reactions may appear when the test is performed. In diagnosing food allergy and for evaluating achievement of tolerance, the oral food challenge is the method of choice, and the double-blind placebocontrolled fashion is 'the gold standard'. Skin prick test: We examined six cases of generalized allergic reactions in connection with skin prick testing in order to identify risk factors, and thereby increase safety, and we investigated the necessity of performing skin prick tests in duplicate. We found that all six children with generalized reactions were <6 months of age. When analyzing skin prick tests in duplicate, we found only 1.3% that showed diverging results, and in infants <6 months even fewer, 0.9%. Food challenge: We developed recipes and a protocol for low-dose oral food challenge to milk and egg to be used in young children outgrowing their food allergy so as to facilitate early re-/introduction of small amounts of milk and egg. We performed 52 challenges, both open and double-blind placebo controlled. The recipes were validated for blinding. The lowdose challenge was tolerated well by the children and was easy to perform. Four children had a positive challenge outcome, all reacting to very small amounts of milk. All but two of the non-reacting children were able to introduce milk and egg into their diet. Nitric oxide and eczema: We investigated the effect of eczema treatment on the NO levels in urine. The sum of nitrite and nitrate was measured in urinary samples from 94 infants at two visits, with an interval of 6 weeks, and the results were compared with clinical data. The levels of NO products increased significantly when the eczema improved. The atopic march: The aim was to evaluate the atopic march in children with eczema, from referral at <2 years until 4½ years of age. We followed 123 children with eczema, 78 sensitized and 45 not sensitized to milk and/or egg, with respect to eczema severity, other allergic manifestations, development of airway sensitization, and achievement of food tolerance. The difference in severity of eczema at referral was significant when comparing food-sensitized with non-sensitized children. At follow-up, 62% were still affected by eczema, although 56% only mildly so. Tolerance was achieved in 81% of the children allergic to milk and 68% of those allergic to egg. Fifty-eight percent of the food-sensitized children and 26% of the non-sensitized children had become sensitized to aeroallergens, a significant difference. The difference in airway symptoms was not significant. Very few children were exposed to tobacco smoke in their homes. Conclusions: Increased precautions should be considered when performing skin prick tests in infants <6 months of age. The use of a single prick, to avoid the risk of summation of reactions, is justified when performing skin prick tests. We report recipes and a protocol for standardized open and double-blind placebo-controlled low-dose food challenge in young children, enabling the introduction of small amounts of egg and milk into the diet during tolerance development. NO products in urine increases when eczema improves. This might be due to a Th2/Th1 shift induced by the eczema treatment and skin healing, and the variation in NO response may be due to individual variations in NO-induced feedback downregulation of Th1 and Th2 proliferation. The prognosis for achieving clinical tolerance is very good in children early sensitized and allergic to milk and egg, but they will become significantly more often sensitized to aeroallergens.

Chemokines and their receptors are involved in many aspects of immunity. Chemokine CX3CL1, acting via its receptor CX3CR1, regulates monocyte migration and macrophage differentiation as well as T cell-dependent inflammation. Two common, nonsynonymous polymorphisms in CX3CR1 have previously been shown to alter the function of the CX3CL1/CX3CR1 pathway and were suggested to modify the risk for asthma. Using matrix-assisted laser desorption/ionization time-of-flight technology, we genotyped polymorphisms Val249Ile and Thr280Met in a cross-sectional population of German children from Munich (n = 1,159) and Dresden (n = 1,940). For 249Ile an odds ratio of 0.77 (95% confidence interval 0.63–0.96; p = 0.017) and for 280Met an odds ratio of 0.71 (95% confidence interval 0.56–0.89; p = 0.004) were found with atopy in Dresden but not in Munich. Neither polymorphism was associated with asthma. Thus, amino acid changes in CX3CR1 may influence the development of atopy but not asthma in German children. Potentially, other factors such as environmental effects may modify the role of CX3CR1 polymorphisms. / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

No description available.

Atopische Dermatitis (Atopisches Ekzem) ist die häufigste Erkrankung der Haut, und sie ist mit der Entwicklung anderer schwerwiegenden atopischen Erkrankungen wie Lebensmittelallergien, allergischer Rhinitis, Heuschnupfen und Asthma vergesellschaftet. Inaktivierende Mutationen im Filaggrin (FLG) Gen zeigten die stärkste Assoziation mit dem Krankheitsbild der atopischen Dermatitis. FLG wird fast ausschließlich in der Epidermis der Haut ausgeprägt und trägt maßgeblich zur normalen Differenzierung von Keratinozyten und zur Integrität der Hautbarriere bei. Bi-allelische inaktivierende Mutationen in dem Gen verursachen die Hauterkrankung Ichthyosis vulgaris, bei es zu Ausbildung einer übermäßig trockenen und schuppigen Hautoberfläche kommt. Etwa die Hälfte aller Ichthyosis vulgaris Patienten entwickeln ebenfalls atopische Dermatitis im Laufe Ihres Lebens. Zusätzlich treten in Ichthyosis vulgaris Patienten häufiger atopische Krankheitsbilder auf als in Gesunden. Als Modell zur Erforschung molekularer Grundlagen der Pahtologie des atopischen Ekzemes und von Pathomechanismen der Atopie eignet sich die „flaky tail“ Mauslinie. Diese Mäuse entwickeln typische Merkmale einer systemischen Atopie wie z.B. einen Barriere Defekt der Haut, ein Hautekzem, eine Entzündung der Lunge, sowie erhöhte Mengen an Immunglobulin E im Serum. In diesem Modell wird die Krankheitsentwicklung durch zwei natürlich entstandene Mutationen in den Genen Flg und Tmem79 verursacht. Die das Gen Flg betreffende Mutation bewirkt eine reduzierte Expression, welche einen Ichthyosis vulgairs ähnlichen Phänotyp auslöst, der durch Schuppenartige („flaky“) Haut am Schwanz („tail“) gekennzeichnet ist (Flgft). Die Mutation im Tmem79 Gen bewirkt eine matte Fellfarbe und wird daher als „matted“ Mutation (Tmem79ma) bezeichnet. Obwohl die Mutationen in benachbarten Regionen des Maus Chromosoms 3 zu finden sind, konnten diese genetisch getrennt werden. Dadurch gelang es zu zeigen, dass allein die Tmem79ma Mutation ausreicht, um Ekzeme und systemische Atopie in Mäusen auszulösen. Nach Rückkreuzung der Flgft Mutation auf den pro-allergischen BALB/c genetischen Hintergrund entwickelten die Flgft/ft BALB/c Mäuse eine - Erkrankung ähnlich der atopischen Dermatitis, die ebenfalls von spontanem Asthma und hohen IgE Konzentrationen im Serum gekennzeichnet war. Beides sind Hauptmerkmale des sogenannten atopischen Marsches. Diese Versuche implizierten, dass Filaggrin eine wichtige Funktion im Schutz vor der Entwicklung von Atopie in Mäusen ausübt. Im Gegensatz zu diesen Ergebnissen wurden nach dem Rückkreuzen von Flg knockout (Flg-/-) Mäusen auf den BALB/c Hintergrund nur ein transienter neonataler Ichthyose Phänotyp, aber keine Hautentzündung oder Atopie beobachtet. Um diese Diskrepanz besser zu verstehen, generierten wir Flg knockout Mäuse direkt im BALB/c Hintergrund. Diese genetisch reinen Flg-/-/BALB/c Mäuse rekapitulierten den neonatalen Ichthyose Phänotyp, und sie zeigten einen Barriere Defekt, der die perkutane Sensibilisierung förderte. Spontane Entzündungen der Haut oder systemische Atopie wurden allerdings nicht beobachtet. Um die genetische Ursache der Atopie in Flgft/ft BALB/c Mäusen zu verstehen, sequenzierten wir das Genom mittels PacBio long read sequencing und verglichen es mit dem BALB/c Referenzgenom. Überraschenderweise zeigte die Analyse, dass die kongenen Flgft/ft BALB/c Mäuse ebenfalls die Atopie-verursachende Tmem79ma Mutation im homozygoten Zustand trugen. Zuvor wurde berichtet, dass die Flgft und Tmem79ma Mutationen vor dem Rückkreuzen des Flgft Allels auf den BALB/c Hintergrund voneinander getrennt werden konnten. Unsere Beobachtungen erklären nun die phänotypische Diskrepanz zwischen den Flgft/ft BALB/c und den Flg-/-/BALB/c Mäusen. Die Daten implizieren, dass der alleinige Funktionsverlust von FLG keine Atopie auslöst und werden durch Beobachtungen in einem Teil der FLG-defizienten Ichthyosis vulgaris Patienten gestützt, die ebenfalls keine Atopie zeigen. Es ist trotzdem wahrscheinlich, dass der Verlust von Filaggrin im Zusammenspiel mit weiteren genetischen Barriere Defekten, wie z.B. Mutationen im Tmem79 Gen das Krankheitsbild der Atopie qualitativ beeinflusst.:Abbreviations 6 Summary 9 Zusammenfassung 11 Introduction 13 Skin 13 Epidermis 13 The immune system 15 Adaptive immune response effector mechanisms 17 Allergic diseases 19 Atopic march 19 Atopic dermatitis 19 Metabolics of AD skin 23 Filaggrin and AD 25 Current mouse models of barrier defect and AD 29 Induced AD mouse models 29 Transgenic and knock out (KO) AD mouse models 30 Inbred mouse strains spontaneously developing AD-like disease 31 Mouse models of FLG-deficiency 32 AD and microbiome 34 Aim 37 Material and methods 38 Generation of Flg-/- mice 38 Crispr targeting strategy 38 α-FLG Western Blot 39 Protein extract preparation 39 SDS Electrophoresis 39 α-FLG Immunofluorescence staining 40 PCR Typing 40 Isolation of genomic DNA from mouse tail tips 40 PCR typing strategy 41 H&E staining of neonatal mouse ear skin 42 Ear thickness measurement 44 Quantification of transepidermal water loss (TEWL) 44 Quantification of total and antigen-specific IgE 44 Whole back skin RNA Sequencing (RNA-Seq) 44 Sorting of basal ear skin keratinocytes for RNA extraction, by fluorescence-activated cell sorting (FACS) 45 Quantitative real time PCR (qRT-PCR) 47 Flow cytometric analysis of ear skin 48 Analysis of DO11.10+/4get transgenic T cell response to epicutaneous OVA immunization 49 Skin microbiome analysis 50 S.aureus colonization of Flg mutant mice 51 Long-term epicutaneous OVA treatment 52 Mapping of reads, assembly, annotation and variant calling 53 Statistical analysis 55 Results 56 Generation and validation of Flg-/-/ BALB/c mice 56 Large sequence deletions detected in Flg-mutant mice by PCR 56 Complete loss of FLG protein expression in Flg-/- mice 57 Flg-/-/BALB/c mice show a barrier-defective skin 59 FLG-deficient skin is devoid of inflammation 61 FLG-deficient skin shows age-related inside-out barrier defect 62 Mild T cell expansion and no other changes in the epidermal compartment of the Flg-/-/BALB/c mice 62 γδ T cell expansion in the whole ear skin suspensions of FLG-deficient mice 64 Lack of systemic atopy in FLG-deficient mice, as indicated by IgE quantification 67 S. aureus colonization does not trigger eczema or atopy in FLG-deficient mice 68 Sensitization with a protein Ag does not trigger inflammation in FLG-deficient mice 69 FLG-deficient skin shows age-related outside-in barrier defect 69 Mild inflammatory signature detected in Flg-/-/BALB/c back skin by RNA sequencing 71 Type 2 immune response signalling detected in Flg-/-/BALB/c ear skin keratinocytes by RNA sequencing 72 FLG-deficient ear skin shows elevated IL-1b expression 74 Reduced cutaneous commensal microbial diversity in FLG-deficient mice 75 Presence of atopy-causing Tmem79ma mutation in Flgft/ft BALB/c mouse genome account for phenotypic differences with our Flg-/-/BALB/c mice 76 Discussion 81 FLG-deficiency in mice does not trigger spontaneous atopic disease 81 FLG-deficiency in mice causes mild immunological perturbation 82 Atopic march, observed in Flgft/ft BALB/c congenic strain, can be explained by the presence of the matted mutation 88 Relevance of filaggrin deficiency for the pathogenesis of atopy in mouse and man 89 References 92 Acknowledgments 136 Erklärung zur Eröffnung des Promotionsverfahrens 137 Erklärung über die Einhaltung der gesetzlichen Vorgaben 138

No description available.

Untersuchungen der letzten Jahre deuten darauf hin, daß Virusinfektionen ein potentieller Modulator der menschlichen Allergieentstehung sind. Die folgende Studie untersucht, ob frühkindliche Infektionen mit CMV, EBV und VZV einen protektiven Effekt auf die Entstehung atopischer Erkrankungen im späteren Leben haben. Die Studie berücksichtigt sowohl die humorale, als auch die zelluläre Immunantwort auf Virusinfektion. Die humorale Immunantwort wurde serologisch im Verlauf bei 672 Kindern von Geburt bis zum 7. Lebensjahr untersucht (ELISA). Die Antikörper-Titer für CMV, EBV und VZV im Alter von 1 Jahr und 3 Jahren wurden mit den atopischen Manifestationen (atopische Dermatitis, Asthma bronchiale, Rhinokonjunktivitis, Gesamt-Serum-IgE und atopische Sensibilisierung) verglichen. Die TH1 Immunantwort (intrazelluläre IFN gamma Produktion) wurde bei 100 Kindern mit und ohne atopische Manifestationen im Alter von 1 Jahr bis 16 Jahren nach CMV Stimulation untersucht (Durchflußzytometrie). Signifikante Unterschiede in den Häufigkeiten atopischer Symptome zwischen Kindern mit positivem und negativem Antikörper-Titer gegen CMV, EBV und VZV konnten nicht gezeigt werden. Auf zellulärer Ebene zeigte sich, daß Kinder und Jugendliche ohne atopische Symptome auf Virus-Stimulation (CMV-Antigen bzw. Peptid) statistisch ebenso häufig mit einer IFN-gamma-Produktion (TH1-Immunantwort) reagieren wie Kinder und Jugendliche mit atopischen Krankheitszeichen. Die Ergebnisse der vorliegenden Studie können die Hypothese eines protektiven Effekts viraler Infektion, zumindest der Herpesgruppe, in den ersten Lebensjahren hinsichtlich einer späteren Allergieentstehung nicht bestätigen. Die Zusammenhänge der Entwicklung atopischer Erkrankungen sind bisher noch nicht vollständig geklärt. Es gilt weiterhin zu erforschen, in welchem Maße frühkindliche Infekte, neben Allergenexposition, Ernährung, Luftschadstoffen und familiärer Prädisposition die Allergogenese beeinflussen. / Studies of the last years indicated that virus infections are a potential modulator of the human allergogenesis. The following study analysed if infections with CMV, EBV and VZV in early childhood have a protective effect on the development of atopic disorders in later life. The study considered the humoral and cellular immuneresponse to virus infection. The humoral immuneresponse of 672 children were serologically followed up from birth to the age of seven years (ELISA). The antibody titres of CMV, EBV and VZV at the age of 1 year and 3 years were compared with the atopic manifestations (atopic dermatitis, asthma bronchiale, rhinoconjunctivitis, total serum IgE levels and atopic sensitiziation) at the age of seven years. The TH1 immuneresponse (intracellular IFN gamma production) of 100 atopic and nonatopic children at the age of 1 year to 16 years were analysed after CMV stimulation (flowcytometry). There was no significant difference in atopic manifestations between seven-year-old children with seropositivity and seronegativity (CMV, EBV, VZV) in early childhood. Nonatopic children showed the same T cell reactivity after CMV stimulation like atopic children. The study could not show a protective effect of herpesvirus infections in early childhood on the development of atopic disorders in later life. Further studies will help to understand the influence of virus infections on the human allergogenesis.

Vaccinia virus (VACV) is an enveloped DNA virus, member of the Orthopoxviridae genus. VACV genome size is about 200 kbp. This huge genome capacity allows VACV to encode a set of factors that are non-essential for virus replication and spread in vitro. While these factors are needed for interfering with host immune responses, VACV remains strongly immunogenic. Cell-mediated and humoral immune responses in atopic disorders are deregulated to a certain extent, leading to complications in case of infection or vaccination with vaccines based on replicating viruses, such as eczema vaccinatum caused by VACV. VACV effects on immune responses consist among others in the inhibition of expression of type I interferon (IFN) at various levels - for example in a specific inhibition of phosphorylation of the interferon regulatory factor-3 (IRF-3) via inhibition of the activity of TANK-binding kinase 1 (TBK 1) that normally phosphorylates IRF-3. Phosphorylation allows IRF-3 to translocate into the nucleus where it initiates transcription of IFNβ followed by induction of expression of IFN and interferon stimulated genes. Expression of these genes is shut down when IRF-3 activity is inhibited. To overcome this block, a recombinant VACV expressing murine IRF-3 under VACV p7.5 promotor (WR-IRF3) was generated....

<p>Otitis media with effusion (OME) is the major form of chronic relapsing inflammatory disease of the middle ear, constitutes the most common diagnosis for children under 15 years old and is the major cause of auditory dysfunction in pre-school children. OME is a disease more commonly found in allergic children. These studies sought to investigate the inflammatory response in the middle ear of patients and test the hypothesis that an allergic-like response might occur in the ear. Atopy was diagnosed by standard in vitro tests. Immunochemical techniques used to study classic allergic rhinitis and asthma were extrapolated to the evaluation of OME children whose effusion persisted beyond 2 months. Not only eosinophil cationic protein (ECP), tryptase, CD3-positive and IL-5 producing cells, but also myeloperoxidase (MPO) was found in middle ear fluid and/or mucosa in the majority of patients with OME and atopy. </p><p>Initially, levels of ECP, MPO, and tryptase were measured in effusions from 97 random OME patients whose atopic status was determined by in vitro testing to 12 inhalants and 5 foods. The response of eosinophils, neutrophils and mast cells in the middle ear was distinctly different between atopic and non-atopic patients (p<0.001) with higher levels of the cell markers in the atopic group of patients. This suggested that 1) perhaps OME was predominantly a disease of atopics and that 2) they differed in their response from non-atopics.</p><p>Tryptase was measured in middle ear effusions from 38 patients with OME, 94.7% of whom were atopic by in vitro testing. Tryptase was elevated only in the effusion of atopic patients as compared to 5 controls (p<0.01). Biopsies stained histochemically for tryptase showed evidence of mast cells in the mucosa and submucosa from 6 of 8 OME ears but absent in 4 normals.</p><p>Middle ear biopsies, embedded in a plastic resin to improve the structural preservation, from 5 patients with OME and 5 normals were evaluated for the presence of eosinophils and neutrophils with monoclonal antibodies against 4 specific granule proteins. Eosinophils and neutrophils were present in the mucosa and mucus in significantly higher numbers than in the control group.</p><p>In an effort to determine whether the middle ear itself might be involved in allergic disease, evidence that some of the cells, mediators and cytokines associated specifically with a Th-2 response were sought for in the middle ear mucosa of these children. Middle ear biopsies from 7 atopic patients with OME and 4 controls demonstrated the presence of activated eosinophils, CD-3+ T cells and IL-5 mRNA cells only in the mucosa from atopic OME children. </p><p>Conclusion: Effusion and mucosal biopsies containing ECP, tryptase, and/or IL-5 mRNA cells, CD3+ T cells, eosinophils, and mast cells indicate that many of the mediators and cells essential to the production of a Th-2 immune mediated response are present in ears with chronic effusion. The increased levels of MPO in atopic patients further suggest that the general inflammatory response to putative inciting agents such as bacterial and viral products may be altered in atopy. These studies support the hypothesis that the exaggerated inflammation within the middle ear associated with most cases of OME is possibly the result of an atopic response within the middle ear itself.</p>

Otitis media with effusion (OME) is the major form of chronic relapsing inflammatory disease of the middle ear, constitutes the most common diagnosis for children under 15 years old and is the major cause of auditory dysfunction in pre-school children. OME is a disease more commonly found in allergic children. These studies sought to investigate the inflammatory response in the middle ear of patients and test the hypothesis that an allergic-like response might occur in the ear. Atopy was diagnosed by standard in vitro tests. Immunochemical techniques used to study classic allergic rhinitis and asthma were extrapolated to the evaluation of OME children whose effusion persisted beyond 2 months. Not only eosinophil cationic protein (ECP), tryptase, CD3-positive and IL-5 producing cells, but also myeloperoxidase (MPO) was found in middle ear fluid and/or mucosa in the majority of patients with OME and atopy. Initially, levels of ECP, MPO, and tryptase were measured in effusions from 97 random OME patients whose atopic status was determined by in vitro testing to 12 inhalants and 5 foods. The response of eosinophils, neutrophils and mast cells in the middle ear was distinctly different between atopic and non-atopic patients (p&lt;0.001) with higher levels of the cell markers in the atopic group of patients. This suggested that 1) perhaps OME was predominantly a disease of atopics and that 2) they differed in their response from non-atopics. Tryptase was measured in middle ear effusions from 38 patients with OME, 94.7% of whom were atopic by in vitro testing. Tryptase was elevated only in the effusion of atopic patients as compared to 5 controls (p&lt;0.01). Biopsies stained histochemically for tryptase showed evidence of mast cells in the mucosa and submucosa from 6 of 8 OME ears but absent in 4 normals. Middle ear biopsies, embedded in a plastic resin to improve the structural preservation, from 5 patients with OME and 5 normals were evaluated for the presence of eosinophils and neutrophils with monoclonal antibodies against 4 specific granule proteins. Eosinophils and neutrophils were present in the mucosa and mucus in significantly higher numbers than in the control group. In an effort to determine whether the middle ear itself might be involved in allergic disease, evidence that some of the cells, mediators and cytokines associated specifically with a Th-2 response were sought for in the middle ear mucosa of these children. Middle ear biopsies from 7 atopic patients with OME and 4 controls demonstrated the presence of activated eosinophils, CD-3+ T cells and IL-5 mRNA cells only in the mucosa from atopic OME children. Conclusion: Effusion and mucosal biopsies containing ECP, tryptase, and/or IL-5 mRNA cells, CD3+ T cells, eosinophils, and mast cells indicate that many of the mediators and cells essential to the production of a Th-2 immune mediated response are present in ears with chronic effusion. The increased levels of MPO in atopic patients further suggest that the general inflammatory response to putative inciting agents such as bacterial and viral products may be altered in atopy. These studies support the hypothesis that the exaggerated inflammation within the middle ear associated with most cases of OME is possibly the result of an atopic response within the middle ear itself.

The aim of this thesis was to identify risk factors for the onset of adult asthma. Other objectives were to study determinants of smoking habits and the association between sensitization and outcome of asthma. In 1990, a questionnaire was distributed to 12,732 individuals from three age groups (16, 30-39 and 60-69 years) in two counties of Sweden. In a second phase, 2538 subjects who had reported respiratory symptoms and 600 controls were invited to clinical investigations, 81% participated. At the follow-up in 2003 subjects of the remaining cohort (11,282) were re-invited. Analyses are based on the 67% (n=7563) who responded to both questionnaires 1990 and 2003. In 2003, 17.2% of the young adults, 11.4% of the middle-aged and 10.3% of the elderly reported having, or having had, asthma. A total of 791 subjects reported onset of asthma during the 13-year study period. Lifestyle factors such as smoking, obesity, hard physical training and a low consumption of fruit and fish were constant risk factors for onset of asthma after adjusting for socioeconomic group. A smoker’s risk of asthma onset was increased by 37%. The impact of risk factors differed between the age-groups. BMI had a significantly higher impact in the middle-aged and elderly. In subjects participating in the clinical investigations in 1990, sensitization to pets, were determinants of both persistent asthma and onset of asthma in 2003. The risk for persistent asthma was threefold. The risk for onset of asthma was more than doubled. Smoking at baseline in 1990 was the strongest determinant of being a smoker in 2003. Allergic sensitization and clinically verified asthma were not associated with smoking habits in 2003. No differences in changing smoking habits could be identified between smokers with or without asthma. In conclusion, modifiable lifestyle factors are important risk factors for adult onset asthma. The co-occurrence and interplay between asthma and cigarette smoking is still puzzling.