Presentations

Immunology

V01

MPI cells as new in vitro model for macrophage research

M. D. Wegner^1,2, P. Engelhard^2,3, A. Prasse³, T. Jakob¹, S. Martin¹,

C. Galanos⁴, G. Fejer⁵, M. Freudenberg^3,6

¹Allergy Research Group, Department of Dermatology, Medical Center — University of Freiburg; ²Faculty of Biology , University of Freiburg; ³Department of Pneumology, Medical Center — University of Freiburg; ⁴Staatliches Weinbauinstitut Freiburg; ⁵School of Biomedical and Biological Sciences, University of Plymouth, Plymouth, United Kingdom; ⁶Centre for Biological Signalling Studies, University of Freiburg

Macrophages are diverse cell types in the first line of antimicrobial defense and according to subset they can promote or dampen inflammation. Only a limited number of primary models exist to study macrophage functions. Mouse bone marrow-derived, M-CSF-induced macrophages (BMM) with a limited lifespan are the most common in vitro model. We developed a simple method yielding self-renewing, non-transformed, GM-CSF/STAT5-dependent macrophages (MPI cells) from mouse fetal liver. Unlike other types of primary macrophages, MPI cells from various wild type, gene-deficient or transgenic mice strains can be propagated indefinitely in unlimited quantities and can be easily manipulated genetically.

MPI cells are highly sensitive to selected microbial agents, including LPS, lipopeptide, Mycobacterium tuberculosis, cord factor and adenovirus. In response to these stimuli they show a pattern of innate responses similar to alveolar macrophages (AM) but different from BMM. For example, MPI cells and AM induce a strong pro-inflammatory but no IL-10 response, whereas BMM mount a less pro-inflammatory but strong IL-10 response. Moreover, MPI cells show an as yet unknown regulation of IL-1α production upon LPS exposure, likely to play a key role in lung inflammation in vivo.

Due to their easy handling and characteristic alveolar macrophage-like properties, the newly established MPI cell lines may become a useful tool in biomedical research and especially for the study of macrophage responses to airborne pathogens.

V02

Comparison of the immune modulating properties of Monophosphoryl lipid A (MPL) and Lipopolysaccharide (LPS) in vitro

S. Schülke, L. Vogel, A. Flaczyk, S. Wolfheimer, B. Löschner, I. Spreitzer, S. Vieths, S. Scheurer

Abteilung Allergologie, Paul-Ehrlich-Institut, Langen

Monophosphoryl lipid A (MPL) is described as a non-toxic TLR4 ligand, derived from Salmonella minnesota R595 (Re) lipopolysaccharide (LPS) by chemical modification. MPL is clinically used as an adjuvant for cancer treatment (Ceravix®), hepatitis vaccination (Fendrix®), and allergen specific immunotherapy (Pollinex® Quattro). Nevertheless, reports on the mechanism of adjuvant activity are limited. The aim of this study was to compare the immune modulating capacities of MPL and LPS in vitro.

In both human and murine lung epithelial cell lines (LA-4, A549) LPS induced higher CCL2 secretion than MPL. In murine BM-derived myeloid dendritic cells (mDC), LPS and MPL stimulation resulted in the same cytokine secretion pattern (IL-1β, IL-6, IL-10 and TNF-α). At high concentrations of MPL, IL-1β secretion was 4-fold higher compared to LPS, whereas LPS stimulation resulted in higher secretion of IL-6, IL-10 and TNF-α, respectively. Moreover, stimulation with both adjuvants resulted in mDC activation characterized by CD40 and CD69 upregulation. Here LPS proved to be more potent than MPL (thresholds for mDC activation: MPL: 100 ng/ml, LPS: 1 ng/ml). Furthermore, compared to stimulation with Ova alone, the co-administration of MPL and Ova resulted in enhanced IL-5 secretion from DO11.10 CD4⁺ T cells co-cultured with mDC which was not observed for LPS. In line with this result, stimulation with MPL coupled to Ova (MPL: Ova) resulted in enhanced cytokine secretion from both mDC (IL-1β, IL-6, TNF-α, IL-10, IL-12) and CD4⁺ T cells (IL-5, IL-13, IL-2, IFN-γ, IL-17) compared to equimolar amounts of MPL and Ova provided as a mixture.

In summary, using in vitro assay systems we observed similar but attenuated immune responses induced by MPL and LPS. MPL applied together with an allergen on CD4⁺ T cells boosted allergen-specific Th1-, Th2-, and Th17-adaptive responses. Although considered safe in humans, further studies should critically assess the adjuvant capacity of MPL in order to evaluate potential non-desired immunological effects.

V03

Histamine 4 receptor stimulation modulates the differentiation process and chemokine release in human monocyte derived macrophages

L. Ratz, S. Mommert, R. Gutzmer, T. Werfel

Division of Immunodermatology and Allergy Research, Department of Dermatology and Allergy, Hannover Medical School

Histamine is an important mediator of biological functions and present in high amounts in inflammatory skin lesions. Such skin lesions evoke a migration of monocyte precursor cells, in part via histamine induced chemotaxis, into the inflamed tissue whereby they differentiate into macrophages. The expression and function of the histamine receptors, especially the histamine H4 receptor (H4R), has already been determined on monocytes and various subtypes of antigen presenting cells in our previous studies. However, the effects of histamine on macrophages seem rather unclear.

Therefore our aim was to assess a functional role of the histamine receptors, with focus on the histamine H4 receptor, on these professional phagocytes.

Here we could show that polarized M1 and M2 macrophages express the H1R, the H2R and the H4R but not the H3R on mRNA level. On M2 macrophages we observed an up-regulation of the H1R and H4R upon activation with IL-4. Interestingly we could show that the phenotype of M1 and M2 macrophages was significantly altered when H4R ligands were added continuously to the media during the period of differentiation. A significant up-regulation of the subtype specific surface marker CD68 and a down-regulation of CD163 were detected by flow cytometry in response to treatment with the H4R agonist.

Furthermore fully differentiated macrophages were stimulated and the cell free supernatants were analyzed by ELISA. When stimulated with IFN-γ and LPS in the presence of histamine or a H4R agonist, M1 macrophages produced substantially lower amounts of the chemokine CCL4. In conclusion we could show that the H4R is functionally expressed on activated macrophages. The down-regulation of CCL4 led to decreased migration of immune cells to the site of inflammation and might have implications for the treatment of allergic diseases since H4R agonists may attenuate the inflammatory response.

V04

The canonical wingless factor 1 affects the T cell stimulatory capacity of unstimulated dendritic cells, associated with impaired expression of MHCII and induction of immunomodulatory mediators

M. Bros¹, M. Kollek², S. Usanova¹, F. Bollmann³, V. Besche¹, E. Montermann¹, A. Cholaszczyńska1, H. Kleinert³, A. Reske-Kunz¹

¹Hautklinik, Universitätsmedizin Mainz; ²Kinder- und Jugendmedizin, Universitätsklinik Freiburg; ³Institut für Pharmakologie, Universitätsmedizin Mainz

Under basal conditions, β-catenin is complexed in the cytoplasm with a number of proteins to mediate its degradation or is engaged by E-cadherin and cytoskeletal proteins to serve as a signaling adaptor. Release of β-catenin from the degradation complex by binding of WNT factors to cellular frizzled (Fzd) receptors or from E-cadherin by mechanical stress results in its nuclear translocation, engagement of TCF/LEF proteins, and transcriptional regulation of target genes. More recently, by employment of transgenic mice and mechanical stress-induced activation, β-catenin was demonstrated to essentially contribute to the tolerogenic acitvity of dendritic cells (DCs).

Here we asked for effects of WNT-1 as a physiologically relevant stimulus to affect the phenotype and function of mouse bone marrow-derived DCs (BM-DCs). At unstimulated state, BM-DCs expressed all Fzd receptors assessed (Fzd-1, -3, -4, -7), which were markedly attenuated in response to stimulation with LPS. BM-DCs were treated with WNT-1 as a prototypical inducer of canonical WNT signaling to activate β-catenin (WNT-DCs). At unstimulated state, WNT-DCs displayed a somewhat attenuated expression of MHCII, while costimulatory markers were not affected. As assessed by total genomic expression analysis, WNT-DCs showed enhanced expression of several proinhibitory molecules not yet implicated in DC functions. In accordance, unstimulated WNT-DCs mediated lower proliferation of allogeneic T cells than control BM-DCs, which was not associated with alterations of regulatory T cell frequencies. Stimulation with LPS resulted in comparable activation of both WNT-DCs and contol BM-DCs.

Taken together, our findings suggest that canonical WNT factors may serve to enhance the tolerogenic function of immature DC under homeostatic conditions, overridden by pathogen-derived stimuli in order to ensure the acquisition of immunogenic function upon infection.

V05

The surface library of naive T cells - an advanced view on the early activation process

A. Gräßel¹, K. Dietz^1,2, S. Hauck³, C. von Törne³, E. Kloppmann⁴, C. Schmidt-Weber², K. Suttner²

¹ZAUM-Zentrum für Allergie und Umwelt, TU und Helmholtz Zentrum München; ²DZL — German Center for Lung Research, München; ³Research Unit Protein Science, Helmholtz Zentrum München; ⁴Department of Bio-informatics and Computational Biology, Technische Universität München

Background: It’s known that an imbalanced differentiation process of T cells into the Th cell subsets could be a reason for the development of allergies and asthma. Since this is an increasing problem, it is important to gain more knowledge about this central immunological process. We want to elucidate this process from a different point of view, because we think that the cell surface is the key access into the regulation. Monitoring the changes happening on the cell surface during the activation of naive T cells on transcriptional and translational level, may provide a deeper insight into this often misdirected differentiation process.

Aim: By combining RNA and corresponding surface protein expression data from naive and activated T cells, we will generate a more complete understanding of the T cell activation process.

Method: The LC-MS/MS based cell surface capturing (CSC) technology was used to enrich and identify cell surface glycoproteins and their quantitative changes during activation (αCD3/αCD28). The transcriptional state of naïve and activated T cells was investigated by microarray based gene expression profiling (8x60K) and analysis with GeneSpring Software (Agilent).

Results: In an early time window of T cell activation we detected fast occurring quantitative effects on RNA and protein level, which partly correspond. With the CSC technology it was possible to detect 140 cell surface proteins on naïve T cells. 16 % of these surface proteins could not be integrated into an immunological background and are novel proteins in this context. To investigate their role in T cell activation, functional and global localization studies are ongoing.

Conclusion: Matched datasets of RNA and surface protein expression of naïve and stimulated T cells created a surface library for T cell activation, where novel surface proteins on naive T cells were detected. The analysis of these proteins may result in the possibility to find new targets for drug and immunotherapy development in the future.

V06

Histamine regulates basophil degranulation and activation via the histamine H4 receptor

S. Mommert, S. Kleiner, R. Gutzmer, U. Raap, T. Werfel

Division of Immunodermatology and Allergy Research, Department of Dermatology and Allergy, Hannover Medical School

Basophils, first described by Paul Ehrlich in 1879, circulate at relatively low levels in the peripheral blood and migrate to sites of inflammation. Like mast cells basophils abundantly express the high-affinity IgE-receptor (FceRI) on their cell surface. Here we investigated the role of the histamine receptors, particularly of the histamine H4 receptor, in modulating basophil activation and degranulation. We could show that highly purified basophils express the H1R, the H2R and the H4R but not the H3R on mRNA level by means of real time PCR.

As a measure of degranulation sulfidoleukotrienes, produced by isolated IL-3 primed leukocytes, were determined by ELISA. The expression of basophil specific surface markers was evaluated by flow cytometry. Cross-linking of the FceRI upon stimulation with a specific antibody is a strong activation signal for basophils, resulting in the release of mediators stored in basophil granules such as histamines and proteases and de novo synthesis of mediators such as sulfidoleukotrienes which include leukotriene C4 (LTC4) and its derivates LTD4 and LTE4.

In this study we determined that simultaneous treatment with histamine or a specific H4R agonist significantly reduces the synthesis and release of sulfidoleukotrienes in these highly activated basophils. In patients suffering from bee or wasp venom allergy the allergen dependent degranulation and sulfidoleukotriene liberation were also decreased by treatment with histamine or the H4R agonist. Furthermore an up-regulation of the activation marker CD203c in response to H4R stimulation was detected.

These data imply that the H4R regulates IgE dependent processes and represent an interesting novel mechanism how histamine prevents an overwhelming reaction in a negative feed back loop. These findings will contribute to a better understanding of the complex interplay of inflammatory mediators such as histamine during allergen exposure at the site of allergic inflammation.

Skin

V07

10 years experience with omalizumab in chronic urticaria

P. Staubach, A. Groiffik, C. Stanger, A. Weber, O. Weirich, D. Mäck, J. Kunkel

Hautklinik, Universitätsmedizin Mainz

Background: Chronic Urticaria (CU) is a common illness. Some of the severe subgroups are often treated unsuccessfully. The International Urticaria Guidelines support the use of second generation antihistamines, if necessary up to 4-fold of the approved dosage. If there are still symptoms the additional use of leukotriene antagonists, omalizumab and/or cyclosporine are recommended. All those 3rd line treatment options are off-label. In the past, the International Urticaria Guidelines strongly recommended placebo controlled doppelblind studies for those off-label treatment options.

Methods: For the last 15 years the Department of Dermatology University Medical Center Mainz has had a well established specialized urticaria outclinic. Here especially severe cases of urticaria patients are diagnosed, treated with all possible treatment options and closely observed.. Extensive experiences have been collected in treating chronic urticaria patients with omalizumab over the last 10 years. Adverse events were observed. Standardized patient reported outcomes like quality of life questionnaires and daily diaries were filled out by the patients in the last years.

Results: Overall more than 150 patients were treated with omalizumab in different dose regimes (IgE- and body weight-related, individual dose regimes) and settings (off-label, Phase II and III studies). More than 1000 patient visits were observed, more than 2000 dosages were applied. All patients were closely monitored during and after therapy. The response to treatment was convincing in at least 9 of 10 patients. No severe side effects were seen.

Conclusion: Omalizumab is a safe, promising and convincing therapy for the treatment of severe chronic urticaria patients.

V08

Omalizumab in chronic inducible urticaria: what’s the method in the madness?

S. Müller, C. Schempp, T. Jakob

Klinik für Dermatologie und Venerologie, Universitätsklinikum Freiburg

Omalizumab has altered the outlook of otherwise treatment refractory chronic spontaneous urticaria. However, effective therapy of chronic inducible urticaria, in particular solar and heat contact urticaria still remains a challenge. We present 2 cases of inducible urticaria with divergent reponses to omalizumab.

A 64 year old caucasian woman had suffered from solar urticaria with systemic symptoms for 28 years. Urticae appeared within minutes of light exposure. Phototesting showed eliciting spectra within the UVA and visible light ranges. An autologous serum test with irradiated (but not with non-irradiated) serum was positive. Treatment with high dose antihistamines, light hardening, mycophenolate mofetil and plasmapharesis caused no significant improvement. In line with recent positive reports treatment with omalizumab was initiated. Unfortunately, however, her urticaria remained recalcitrant. A repeat phototest after 3 months of omalizumab resulted in a systemic reaction. Based on a perceived deterioration of her urticaria the patient refused further omalizumab treatment.

A 60 year old caucasian woman had suffered from heat contact urticaria for 9 years. Associated systemic symptoms occurred at work due to heat from the bakery ovens. Immersion of her forearm in water at 43 °C resulted in pruritus, erythema and brawny oedema which were sharply demarcated from normal, non-immersed skin. The heat urticaria proved refractory to combination therapy with high-dose H1 antihistamine, H2 antihistamine and leukotriene antagonist. Treatment with omalizumab resulted in complete remission of symptoms that was maintained despite dose reduction to 150 mg once monthly.

Previous reports show omalizumab to be effective in solar urticaria and less so in heat contact urticaria. In view of this the presented cases demonstrate the need for comprehensive characterisation of this patient group in order to define biomarkers that allow the identification of omalizumab responders.

V09

Bifunctional role of IL-31 in skin biology

K. Hänel¹, C. Cornelissen1, P. Amann², Y. Marquardt², K. Czaja², J. Lüscher-Firzlaff¹, B. Lüscher¹, J. Baron²

¹Institute of Biochemistry and Molecular Biology, Aachen; ²Department of Dermatology, Aachen

Atopic dermatitis (AD) is a prevalent chronic condition in children and adults resulting in skin barrier defects with increased infiltration of allergens and pathogens and unrelenting pruritus. The expression of IL-31 is increased in skin lesions of AD patients and correlates with disease severity. IL-31 interferes with the differentiation of keratinocytes, aggravating the AD phenotype. Aim of the study was to understand the molecular consequences of IL-31. Target genes were identified in organotypic 3-dimensional skin models using exon arrays. Detailed analysis showed that IL-31 affects various genes whose products are relevant for the mechanical barrier. Indeed IL-31 weakened this barrier, manifested by increased penetrability to an allergen and a small compound as well as enhanced transepidermal water loss. To follow up these results we used a Cell-sorted Skin Equivalent placed on the back of SCID mice. In this in vivo model we identified the influence of IL-31 on morphology, filaggrin expression and trans epidermal water loss. Surprisingly, we identified the IL-1 cytokine network as a key downstream effector of the IL-31/IL-31 receptor axis. Antagonizing the IL-1 receptor with Anakinra we could rescue the IL-31 effects on skin differentiation and barrier formation. Furthermore we identified this cytokine network to be responsible for an increased expression of a series of antimicrobial peptides (AMP), like members of the S100 family and human beta defensins. In additional experiments we could illustrate that at low doses of IL-31 treatment there is no disruption of the skin barrier but an increased AMP expression. Thus our study defines how IL-31 negatively affects the mechanical skin barrier and suggests a positive effect on the antimicrobial barrier by regulating the IL-1 network. The balance of these two activities will have to be considered during therapeutic targeting of IL-31.

V10

RNase7 regulates Th2 cytokine production by activated human T-cells

V. Kopfnagel¹, J. Harder², M. Kleine³, T. Werfel¹

¹Division of Immunodermatology and Allergy Research, Department of Dermatology and Allergy, Hannover Medical School; ²Department of Dermatology, University Hospital Schleswig-Holstein, Campus Kiel; ³Planton GmbH, Kiel

RNase7 is one of the major antimicrobial peptides (AMPs) secreted by keratinocytes. It is constitutively expressed in the epidermis of healthy human skin and has been found to be upregulated in chronic inflammatory skin diseases such as atopic dermatitis and psoriasis. In addition to their antimicrobial activity immunregulatory functions have been published for several AMPs. In lesional skin of patients with atopic dermatitis and psoriasis activated T-cells might be directly exposed to RNAse7. Here we investigated the influence of RNase7 on activated T-cells.

In the current study we demonstrate that treatment of activated human CD4⁺ T-cells and Th2 cells with RNase7 significantly reduced the production of the Th2 cytokines IL-4 and IL-13 by activated human T-cells while IFNγ and IL-2 were not regulated. To analyze if ribonuclease activity is required for the regulation of the Th2 cytokine release we repeated our experiments with a ribonuclease-inactive recombinant RNase7 mutant. Interestingly, our data show that RNase7 ribonuclease activity is dispensable for the observed regulatory effect. Furthermore, we were able to show that the inhibition of Th2 cytokine production by RNase7 is mediated by a down-regulation of GATA3 activity.

Our data indicate that RNase7 has immunmodulatory functions on Th2-cells and might foster a Th1 cytokine environment in the skin.

V11

Cytokine induction by the human autoallergen thioredoxin

S. Hradetzky¹, L. Rösner¹, A. Heratizadeh1, R. Crameri², A. Scheynius³, T. Werfel¹

¹Department of Dermatology and Allergy, Division of Immunodermatology and Allergy Research, Hannover Medical School; ²Swiss Institute of Allergy and Asthma Research (SIAF), University of Zürich, Davos, Switzerland; ³Department of Medicine Solna, Translational Immunology Unit, Karolinska Institutet, Stockholm, Sweden

Introduction: A subgroup of patients with atopic dermatitis (AD) exhibits specific IgE responses to autoantigens (autoallergens). Some of these autoallergens show high homology to exogenous allergens. One member of this group is the human thioredoxin (hTrx) with its corresponding homolog Mala s 13 from the skin-resident yeast Malassezia sympodialis. Aim of the study: Since only little is known about the immunomodulation by autoallergens, this study aimed to compare cytokine effects of hTrx and Mala s 13 in human immune cells.

Results: We could show induction of monocyte and T cell cytokines by hTrx and Mala s 13 in immune cells from patients with AD as well as from control donors. For some of these cytokines there were significant differences between the donor groups. Most striking was the reduced IL-10 release from hTrx- or Mala s 13-stimulated PBMCs of AD patients sensitized to Malassezia sympodialis, compared to the control donors. The main IL-10 producers were monocytes, not CD4⁺ T cells. Further data strengthen the activation of monocytes by hTrx, since it induced NLRP3 mRNA expression as well as the release of activated caspase-1 and IL-1β.

Conclusion: Sensitization to the skin-colonizing yeast Malassezia sympodialis, which can be found in about 50% of the AD patients, could contribute to disease exacerbation in several ways. One possibility is the secondary cross-sensitization to homologous human antigens. Cross-reactivity for Mala s 13 and hTrx has already been demonstrated at IgE and T cell level. Here we could show an activation of innate immune cells and subsequent induction of T cell cytokines. These findings could give further hints on the pathomechanisms of this cross-sensitization, whereas the reduced ability for IL-10 production in immune cells from sensitized AD patients may indicate their loss of tolerance towards these proteins.

V12

Increased frequencies of circulating differentiated autoreactive CD8⁺ T cells in patients suffering from atopic dermatitis

L. Rösner¹, A. Heratizadeh¹, S. Hradetzky¹, C. Hennig², G. Hansen², B. Eiz-Vesper³, I. Mittermann⁴, R. Valenta⁴, T. Werfel1

¹Division of Immunodermatology and Allergy Research, Department of Dermatology and Allergy, Hannover Medical School; ²Department of Pediatric Immunology, Allergology and Pneumology, Hannover Medical School; ³Department of Transfusion Medicine, Hannover Medical School; ⁴Division of Immunopathology, Department of Pathophysiology, Centre for Physiology and Pathophysiology, Medical University of Vienna, Austria

Autoreactivity in atopic dermatitis has been described by several independent studies within the last 20 years. While most data on “autoallergens” exist on autoreactive IgE, we and others identified and characterized also specific T cells by T cell cloning. More precisely, autoreactive T cell clones specific for human α-NAC, a subunit of the chaperone nascent polypeptide-associated complex, were not found to be restricted to the Th2-profile, but could also be assigned to Th1, Th17 or cytotoxic T cell subsets.

About 20% of skin infiltrating T cells are CD8⁺. This study aimed therefore to determine frequency as well as phenotype of circulating autoreactive cytotoxic T cells. To exclude cloning artefacts, MHC-tetramers are the method of choice.

Applying prediction algorithms, MHC affinity studies and proliferation analyses, immunodominant epitopes of α-NAC were identified. Generated MHC-tetramers bound specifically CD8⁺ T cells. The characterization of tetramer⁺ T cells of AD patients and control donors was carried out direct ex-vivo by surface marker staining of CD8, CD45R0, CD45RA, CD27, and a vital stain. While the frequency of α-NAC-specific CD8⁺ T cells was significantly elevated in AD patients, it could also be observed that these displayed significantly more often a T_EM or T_EMRA differentiated phenotype compared to the control group. These subtypes have been shown to be the main source of perforin, granzyme B, and interferon-γ within CD8⁺ T cells and argument for an involvement of autoreactive cytotoxic T lymphocytes in the inflammation of AD.

V13

Innate lymphoid cells (ILCs) in allergic contact dermatitis

S. van de Poel, S. Kunz, S. Martin, T. Jakob

Allergy Research Group, Department of Dermatology, Medical Center — University of Freiburg

Innate lymphoid cells (ILCs) are a recently identified family of heterogeneous innate immune cells belonging to the lymphoid lineage. In analogy to T helper cell subsets, ILCs can be classified into three groups based on the developmental dependency on transcription factors and expression of cytokines. Prototypical ILCs are NK cells (ILC1), natural helper cells (ILC2) and lymphoid tissue-inducer (ILC3) cells. ILCs are known to play a role in lymphoid organogenesis, tissue remodeling and inflammation including allergic asthma and reside especially in the intestine and the lung. Recent studies showed that ILCs are also present in skin and mediate pathology in a mouse model of atopic dermatitis as well as in psoriatic plaque formation.

Here we analyzed the involvement of ILCs in allergic contact dermatitis using the mouse model of TNCB-induced contact hypersensitivity. Using flow cytometry, we identified all three groups of ILCs in the skin of naïve BALB/c mice. Quantification of these cells using counting beads revealed a frequency of approximately 1150 ILC2, 350 ILC3, and 700 NK cells per 50 mg ear skin. During the elicitation phase of contact hypersensitivity we observed a distinct increase of NK cells in the inflamed skin, their number peaking at around 24 h after challenge. The frequency of ILC2 cells in the skin showed a slight increase 24 h after challenge, whereas no alterations in the frequency of ILC3 cells could be observed. Analysis of the activation profile using ICOS as an activation marker revealed a higher activation of ILC2 cells in mice with contact hypersensitivity compared to control groups. Upon primary contact with the allergen or an irritant, we observed no difference in the frequency of ILC2 cells, whereas ILC3 and NK cell increased at later time points.

In conclusion, our data indicate that numbers and activation profile of dermal ILCs are influenced by contact allergens and thus, ILCs might be functionally involved in allergic contact dermatitis.

V14

Improvement of the human T cell priming assay (hTCPA)

S. Straub, T. Jakob, S. Martin, P. Esser

Klinik für Dermatologie und Venerologie, Universitätsklinikum Freiburg

Background: The replacement of animal testing for the identification of chemical skin sensitizers by alternative in vitro methods is urgently needed both for ethical and legislatory reasons. One hallmark of contact sensitizers is the induction of T cell activation.

Aim: The goal is the improvement of our recently developed hTCPA to identify potential contact sensitizers and to assess their potency.

Method: Human dendritic cells (DCs) are pulsed with contact sensitizers. Autologous sorted naive human T cells are primed with these modified DCs. Sensitizer-specific re-stimulation is performed and cytokine production is used as readout to determine antigen-specific T cell responses.

To shorten the assay time we are testing FastDCs differentiated within 2 days with a cytokine cocktail instead of conventional monocyte-derived DCs generated within 5 days.

In addition, we investigate the potential de novo induction or expansion of regulatory T cells during the cultivation, both in assays with FastDCs and 5-days-DCs.

Results: In vitro priming of naive human T cells with chemical-pulsed DCs results in antigen-specific CD4⁺ and CD8⁺ T cell responses that allow in vitro identification of contact sensitizers as determined by intracellular cytokine staining.

Our recent results show that FastDCs work as well as the 5-days-DCs regarding the cytokine release; therefore we can shorten the assay time by 3 days. Moreover, the use of FastDCs seems to abrogate the de novo induction of regulatory T cells during the priming. This might allow for an enhanced sensitivity of the assay especially in the case of weak sensitizers.

Conclusion: The hTCPA is a highly specific and promising in vitro test for the identification of contact sensitizers. This assay may aid risk assessment and the replacement of animal testing.

V15

Comparative assessment of nickel sensitization based on medical history, patch test and lymphocyte transformation test

S. Ständer, P. Thomas, F. Rueff, B. Summer

Klinik und Poliklinik für Dermatologie und Allergologie, Ludwig-Maximilians-Universität, München

Introduction: Nickel (Ni) allergy is common. However it may not always be detected by patch test (PT) despite a history of cutaneous metal intolerance (CMI, e. g. eczema to jewelry or jeans button) — or PT cannot be performed because of actual eczema. The lymphocyte transformation test (LTT) might add to reveal Ni sensitization. Thus we assessed the correlation between history, PT and LTT.

Methods: In 30 patients (8 female, 22 male, 17-80yrs, mean 52,75) with PT in skin disease without history of CMI (“controls”) and 38 patients (34 female, 4 male, 44-75yrs, mean 61,28) with PT due to history of CMI we performed: Questionnaire-aided medical history, PT at least with the standard series and LTT. PBMC were obtained from heparinized blood at day 0 of PT, cultured at 1x106/ml for 5 days with control stimuli and serial dilutions of Ni, cobalt and chromium salts, followed by H3-thymidine addition. Stimulation index (SI) >3: +, SI 2–3: (+), < 2: -.

Results: 30 controls: PT no reaction to Ni; LTT 2 +, 4 (+); 24 -. 38 patients with CMI: PT 13 +, 25 -; LTT 19 +, 4 (+), 15 -. At the discrimination level of Ni-stimulation 1x10-5 M: LTT in the control group all -; in the 38 patients with CMI: 14 +, 3 (+), 21 -. Furthermore all PT + patients were also LTT +.

Conclusion: With regard to Ni sensitization, the LTT is a useful tool to detect the respective patients — if correctly performed. In particular, discrimination levels to distinguish false positive and negative reactivity have to be established.

V16

Effects of a GATA-3-specific DNAzyme in a subacute mouse model of oxazolone induced contact hypersensitivity

U. Purath¹, R. Ibrahim², A. Müller¹, H. Garn²

¹Sterna biologicals GmbH & Co. KG, Marburg; ²Institut f. Laboratoriumsmedizin und Pathobiochemie, Molekulare Diagnostik, Marburg

DNAzymes represent a particular class of antisense molecules combining the specificity of antisense molecules with an inherent catalytic activity which makes them an attractive tool for highly specific interference with disease-causing target molecules. In general, they are single-stranded DNA molecules with two sequence-specific RNA-binding domains flanking a central catalytic domain. We developed and patented a DNAzyme — named hgd40 — that targets GATA-3, the central transcription factor in T helper cell type 2 (Th2) differentiation and activation, as well as a specific formulation for topical dermal application.

Targeting GATA-3 can be a key for therapeutic interventions in predominantly Th2-driven diseases like atopic dermatitis. For this reason, we modified an oxazolone-induced contact hypersensitivity mouse model to establish elongated skin swelling reactions compared to the acute models, thereby enabling the analysis of treatment effects on T cell-mediated pathomechanisms. The therapeutic effects of hgd40 were analyzed in this model with regard to clinical symptoms and the expression pattern of relevant transcription factors and cytokines over time.

Prophylactic treatment with topically applied hgd40 in a water/oil/water emulsion, specifically developed for penetration enhancement and DNAzyme protection, significantly and dose dependently reduced oxazolone-induced reactions in the skin, e. g., skinfold thickness. Molecular analysis revealed reduced GATA-3 mRNA levels early after topical application of hgd40.

Based on these results, we think that the water/oil/water-formulated DNAzyme hgd40 may represent a new and promising therapeutic agent for the treatment of allergic skin diseases.

V17

The role of STAT1 signalling in the Th17 response in patients with chronic mucocutaneous candidiasis

J. Hiller¹, R. Effner², A. Puel³, S. Cypowyi⁴, M. Schaller⁵, B. Lögering⁶, J. Buters², J. Ring⁷, J. Laurent-Casanova⁴, C. Schmidt-Weber², C. Traidl-Hoffmann¹

¹Institute of environmental medicine, UNIKA-T, Technische Universität München; ²ZAUM —Center of Allergy and Environment, Technische Universität and Helmholtz Zentrum München; ³Laboratory of Human Genetics and Infectious Diseases, Neckar Branch, Neckar Medical School, Institut National de la Sante et de la Recherche Medicale U980 and University Paris Descartes, France; ⁴St. Giles Laboratory of Human Genetics of Infectious Diseases, Rockefeller Branch, The Rockefeller University, New York, USA; ⁵Department of Dermatology, Eberhard-Karls-Universität, Tübingen; ⁶Dermatologikum Hamburg; ⁷Department of Dermatology and Allergology, Technische Universität München;

Patients with chronic mucocutaneous candidiasis (CMC) carrying heterozygous gain-of-function (GOF) STAT1 mutations show a defect in IL-17 and IL-22 producing T cells pointing to an important role of STAT1 in the regulation of IL-17 and IL-22. However, mechanisms by which these STAT1 mutations impair IL-17/IL-22 immunity are not clarified so far. Aim of the study was to investigate the role of STAT1 in the regulation of IL-17 and IL-22 by analyzing T cell immunity in CMC patients with an overactive STAT1 protein on the one hand and STAT1 deficient mice on the other. Cytokine expression dependent on STAT1 in PBMCs of CMC patients was examined using the STAT1 inhibitor fludarabine. PBMCs of CMC patients (n = 2) and healthy controls (n = 3) were stimulated with Candida albicans or aCD3/aCD28 in the presence or absence of fludarabine. Cytokine production was characterized by ELISA and flow cytometry. Moreover IL-22 expression in activated splenocytes from STAT1-/- mice was analyzed by ELISA. Cytokine analysis revealed that STAT1 inhibition by fludarabine modulates the Th17 response in CMC patients after the stimulation with Candida albicans or aCD3/aCD28. Inversely to impaired IL-22 and IL-17 production in patients with STAT1 over-reactivity, STAT1-/- mice show a higher production of IL-22 and IL-17 in splenocytes compared to WT mice. The Th17 response in CMC patients after the inhibition of STAT1 together with the data of STAT1-/- mice support a role of STAT1 in the regulation of IL-17 and IL-22. In summary this work provides first evidence that a reduced over-reactivity of STAT1 in CMC patients restores Th17 immunity.

V18

Tumor bearing humanized mice: Identification of a novel feature of anti-EGFR antibodies in epidermoid cancer therapy

J. Kubach¹, M. Hubo¹, C. Amendt², C. Stroh², H. Jonuleit¹

¹Hautklinik, Universitätsmedizin Mainz; ²Merck Serono, Darmstadt

Numerous humanized monoclonal antibodies (mAb) have been generated for cancer therapy including melanoma and constitute a promising approach for treatment of patients with metastatic tumors. However, limitations in the predictive value of available animal models restricted the in vivo analyses of these antibody-mediated properties so far. Humanized mice hold promise to overcome these limitations, being able to concentrate on role of the human immune system in cancer defense. As a proof of concept we established a humanized mouse model to evaluate mAb in an epidermiod cancer setting using EGFR-expressing A431 tumor cells. In this context, Cetuximab has been designed to inhibit EGFR signaling and has been postulated as a potent mediator of antibody dependent cytotoxicity (ADCC). However, induction of ADCC in vivo is controversially discussed and could not be addressed in conventional animal models so far. Here, we established a human tumor-bearing mouse model in which human immune cells can engraft and mediate anti-tumor responses. Using immunodeficient NOD/Scid mice transgenic for human MHC class I molecule HLA-A2 and adoptively transferred human HLA-A2+ PBMC after engraftment of human epidermoid cell carci-noma A431 lead to a solid coexistence without evidence of rejection. Addition of high dose anti-EGFR mAb in absence of an immune system induced a strong tumor regression. However, tumor regression by low doses of anti-EGFR mAb treatment was immune cell-dependent and surprisingly not mediated by NK cells but tumor infiltrating CD8⁺ T effector cells. This CD8⁺ T cell-mediated ADCC was restricted to IgG1 anti-EGFR mAb. CD8⁺ T cell-mediated ADCC was also depending on binding to CD16 and could be inhibited after blockade. Furthermore, enhanced glycosylation of the Fc portion of anti-EGFR mAb and presence of IL-15 markedly improved ADCC activity of CD8⁺ T cells. Taken together, our results provide strong evidence of a novel mechanism of CD8⁺ T cell-mediated cytotoxicity induced by anti-EGFR mAb with an IgG1 isotype.

Respiratory tract

V19

Charakterisierung der endogenen pulmonalen Th17-Zellgenerierung im Kontext einer Th17-polarisierten Atemwegsentzündung

M. Albrecht¹, S. Lingner¹, M. Lochner², A. Dittrich¹

¹Pädiatrische Pneumologie, Allergologie und Neonatologie, Medizinische Hochschule Hannover; ²Infektionsimmunologie, TWINCORE, Zentrum für Experimentelle und Klinische Infektionsforschung, Hannover

Unsere Vorarbeiten zu pulmonalen Sensibilisierungsprozessen haben gezeigt, dass eine akute, durch Th17 Zellen ausgelöste Entzündung in der Lunge ein Risikofaktor für Neusensibilisierungen ist. Im Zuge dieser Neusensibilisierung konnte die Generierung von endogenen antigenspezifischen T-Zellpopulationen beobachtet werden, die Interleukin(IL)-17 oder Interferon-gamma (IFNγ) sezernieren. Ort und Zeitpunkt der endogenen T-Zellgenerierung, sowie deren Phänotyp sollten genauer bestimmt werden.

In Modell der Th17-abhängigen Atemwegsinflammation durch adoptiven T-Zelltransfer und zwei Provokationsphasen wurden transgene Reportertiere als Rezipienten verwendet, in denen die Induktion des Th17-spezifischen Transkriptionsfaktors (RORgamma-t) durch Ko-expression von GFP nachgewiesen werden kann. Analysen mittels Durchflusszytometrie zeigten, dass sowohl nach der ersten, als auch nach der zweiten Provokationsphase der Anteil an IL-17- oder IFNγ-produzierenden Zellen in der Lunge deutlich höher war als in den drainierenden Lymphknoten (LN). Das Verhältnis zwischen IL-17- und IFNγ-produzierenden Lymphozyten verschiebt sich zwischen den beiden Inflammationsphasen. Im Vergleich zu IFNγ-sekretierenden T-Zellen zeigen endogene Th17 Zellen (IL-17+GFP+CD4+) in der Lunge eine erhöhte Expression der “Memory”-Marker CD62L und CCR7. Die Bildung von endogenen Th17 (IL-17+GFP+ CD4+CCR6+) Zellen konnte bereits 48h nach Induktion der Th17-polarisierten Atemwegsinflammation nachgewiesen werden. Zu diesem Zeitpunkt waren auch IFNγ-produzierende T-Zellen (IFNγ+CD4+CXCR3+) detektierbar.

Diese Daten legen die Vermutung nahe, dass die Induktion der IL-17+ Zellen in der Lunge stattfinden könnte und möglicherweise der Induktion der IFNγ+ Zellen vorangeht. Weitere Untersuchungen in diesem Modell werden zur Aufklärung der Mechanismen der pulmonalen Sensibilisierung durch Th17 Zellen beitragen.

V20

Investigation of the influence of an allergen specific Th17 response on remodeling of the airways

M. Peters, S. Köhler-Bachmann, T. Lenz-Habijan, A. Bufe

Experimentelle Pneumologie, Ruhr-Universität Bochum

We have previously shown that allergen pulsed DCs that were stimulated with a low dose of LPS tend to induce a Th2 response whereas high dose activated cells induce a mixed Th2/Th17 response. The aim of this study was to investigate whether these mixed Th2/Th17 response to allergens has an influence on airway remodeling in a mouse model of asthma.

Therefore we have generated dendritic cells (BMDCs) from mice and pulsed them with LPS free ovalbumin (OVA). Subsequently these cells were activated with different doses of LPS with or without ATP as an additional inflammasome activating stimulus. The activated cells were then used to sensitize mice via the airways followed by three consecutive challenges with OVA aerosol. Moreover we have investigated the direct effect of IL-17A on lung fibroblasts in vitro and in vivo.

The activation of allergen pulsed BMDCs with as few as 0.1 ng/ml LPS is sufficient to induce a Th2 response with airway eosinophilia. Increasing doses of LPS+/-ATP led to induction of a mixed Th2/Th17 response. Interestingly, the increasing Th17 response correlates with an increased influx of neutrophilic granulocytes into the airways. Even more interesting we found a correlation between neutrophil counts in the airways and remodeling detected by increased numbers of alpha-smooth muscle actin (alpha-SMA) positive cells in the airway walls and goblet cell metaplasia. Furthermore we were able to show in vitro that direct stimulation of mouse lung fibroblasts with purified IL-17A protein results in alpha-SMA transcription in these cells. This holds also true for the situation in vivo where application of IL-17A via the airways led to enhanced transcription of alpha-SMA in the lung tissue.

We conclude that an increased allergen specific Th17 response in the airway goes along with increased airway remodeling. Our in vitro and in vivo studies with purified IL-17A suggest that increased remodeling is not only based on neutrophilic inflammation but also on the direct action of IL-17A on airway structural cells.

V21

Wnt signaling in allergic asthma

K. Dietz¹, K. Suttner¹, M. Königshoff², C. Schmidt-Weber¹, U. Zissler¹

¹ ZAUM — Center of Allergy and Environment, Technische Universität München, Helmholtz Center Munich and member of German Center for Lung Research, München; ²CPC-Comprehensive Pneumology Center, Ludwig-Maximilians-Universität München, Helmholtz Center Munich and member of German Center for Lung Research, München

Background: The pathological hallmarks of asthma are a Th2 driven chronic inflammation of the airways, including airway hyperresponsiveness, mucus hyperproduction and airway remodeling. This remodeling process is known to be influenced by several signaling pathways. Wnt signaling is differentially regulated in asthma patients compared to healthy subjects and may therefore be one of these pathways. Since a strong interaction between the epithelium and effector immune cells was previously described, we hypothesize that the Th2 driven asthmatic immune response is influencing the Wnt signaling in the airway epithelium.

Aim: To identify the role of the Wnt signaling in bronchial epithelial cells which are influenced by a Th2 immune response.

Method: Primary bronchial epithelial cells were cultured in the presence of Th-subtype specific cytokines and RNA was harvested after 6h and subjected to Agilent single color microarray gene expression profiling (44K). Existing datasets were analyzed under constant significance restrictions (p < 0.05) using Agilent Genespring Software. The expression of regulated Wnt genes was validated using SYBR Green based quantitative PCR.

Results: Significant gene expression changes of Wnt5a and FZD10 were observed in Th-subtype specific cytokine induced bronchial epithelial cells. The mRNA levels of both genes were significantly increased upon stimulation with IL-4 in comparison to a medium control.

Conclusion: The results show that a Th2 driven immune response induces changes in the Wnt signaling of bronchial epithelial cells via autocrine effects of secreted Wnt ligands like Wnt5a or by inducing Wnt receptors like FZD10. The current results form the basis for characterization of Wnt-mediated airway remodeling, biomarker definition and therapeutic considerations.

V22

Analysis of the Wnt5a/cGMP/Calcium Signaling Axis in Airway Remodeling and Fibrosis S. Köhler-Bachmann¹, E. Mergia², D. Koesling², A. Bufe¹

¹Abteilung für Experimentelle Pneumologie, Ruhr-Universität Bochum; ²Institut für Pharmakologie und Toxikologie, Ruhr-Universität Bochum

Background: There is currently little known about the molecular mechanisms that lead to airway remodeling in asthma. One of the key pathological features of airway remodeling is fibrosis, which is characterized by an increase of ECM (Extracellular Matrix) deposition by myofibroblasts organized in fibrotic foci. One profibrotic key factor is TGFβ (Transforming Growth Factor beta) which either initiates EMT (Epithelial Mesenchymal Transition) or the conversion of fibroblasts to myofibroblasts. This conversion is partially blocked by NO/sGC/cGMP signaling (NO: Nitric Oxide; sGC: soluble Guanylyl Cyclase; cGMP: cyclic Guanosine Monophosphate) in vitro. Interestingly, an inhibitory interplay of Wnt5a/Ca²⁺ and NO/sGC/cGMP signaling was shown in murine F9 terratocarcinoma cells. We hypothesize that a Wnt5a/cGMP/Ca ²⁺ signaling axis supports and even worsens airway remodeling and fibrosis in chronic asthma.

Methods and Results: We analyzed the expression of Wnt5a, TGFβ1 and collagen in primary murine lung fibroblasts. These cells exhibit a moderate activity of sGC. As expected, stimulation with recombinant TGFβ1 activated the fibroblasts by inducing the expression of collagen. Additionally we found increased expression of Wnt5a and TGFβ1 itself. This effect is remarkably potentiated by recombinant Wnt5a. Addition of 8-Br-cGMP, a cellpermeable cGMP analog, to TGFβ1 stimulated cells did not affect the collagen and TGFβ1 expression significantly, whereas 8-Br-cGMP considerably counteracts the activation of fibroblasts by Wnt5a /TGFβ1 costimulation.

Discussion: Our results show that Wnt5a acts as a multiplier in TGFβ1 triggered transition of lung fibroblast to myofibroblasts. Furthermore, our results indicate that a Wnt5a/cGMP/Ca2⁺ signaling axis exists in lung fibroblasts and that this axis affects their activation. At present the investigation of α1sGC-knock-out mice should illuminate the impact of NO/sGC/cGMP signaling on airway remodeling in chronic asthma.

V23

Chronic airway inflammation and viral influenza infections as a model of virus-host immune interaction

C. Hudemann¹, C. Skevaki¹, M. Matrosovich², H. Renz¹

¹Institute of Laboratory Medicine and Pathobiochemistry, Philipps University, Marburg; ²Institute of Virology, Philipps University, Marburg

Allergic asthma and other chronic airway inflammatory diseases are heavily affected by viral infections. Epidemiological and clinical data reveal contradictory effects in terms of asthma prevention and asthma exacerbation in already diseased patients. The molecular nature of these phenotype-modifying virus-host interactions, however, still remains largely unknown. This leads to the hypothesis that the impact of viral infections on the development of chronic inflammation depends on (i) virus origin and frequency of viral infection as well as (ii) on host immune status and localization of infection. Here, we describe a novel prevention model of influenza airway infection followed by succeeding OVA-sensitization and -challenge in Balb/c mice. While a single infection itself causes a quick influx and activation of CD4 and CD8 T-cell subpopulations peaking at day 8-12 combined with a pronounced TNF-α, IFN-γ and IL-6 secretion pattern, a subsequent OVA-sensitization and challenge displays a decreased severity of a wide range of allergic phenotypic parameters (e. g. eosinophilia p < 0.05, goblet cell hyperplasia p < 0,05) compared to only OVA-treated group. In-vitro and in-vivo analysis of CD8 memory cell subpopulations suggest a cross reactive critical role in the virus induced dampening of a subsequent allergic phenotype.

The delineation of the underlying molecular and cellular rearrangements will lead to a better understanding of the regulations of chronic inflammation and should identify novel targets for virus induced protection and exacerbation.

V24

Arabinogalactan binds to DC-SIGN and MMR-1 modulating the activation of human T-cell by dendritic cells

P. Guidato¹, K. Peters¹, D. Megger², B. Sitek², A. Bufe¹, M. Peters¹

¹Abteilung für experimentelle Pneumologie; Ruhr Universität Bochum; ²Medizinisches Proteomcenter, Ruhr Universität Bochum

Recent studies showed that Arabinogalactan (AG) isolated from Cowshed Dust Extract or extracts of Alopecurus pratensis prevents allergic airway inflammation and sensitization in murine models of asthma. One common problem of substances isolated from environmental sources is the contamination with pathogen associated molecular patterns (PAMPs).

The aim of this study was to produce PAMP-free AG from sterile cultures of callus tissue (CT) to investigate its mechanisms of allergy protection.

AG was prepared from cell culture supernatants of Phleum pratense CT. To investigate the allergy protective activity of AG mice were sensitized in an OVA-Alum model and treated with AG during the sensitization phase. To investigate effects in a human cell culture model we used AG in a mixed lymphocyte reaction (MLR) of human monocyte derived dendritic cells (moDC) and allogeneic human naïve CD4⁺ T-cells.

We can show that i. n. treatment of mice with callus-AG during the sensitization phase significantly reduces airway inflammation via reducing numbers of eosinophils and lymphocytes in the bronchoalveolar lavage. Moreover we found that callus-AG significantly reduces IL-5 in OVA restimulated splenocytes. Stimulating moDCs with callus-AG resulted in IL-10 secretion and a reduced expression of costimulatory CD83 molecules, when moDCs were afterwards treated with LPS. Furthermore, in a human in vitro model of allogeneic T cell activation by moDCs (MLR) callus-AG significantly reduced the T-cell proliferation and IFNγ secretion. Furthermore, we show that AG binds to DC-SIGN and MMR-1 in vitro.

We were able to show that PAMP-free AG from plant CT is able to protect mice from allergic airway inflammation and to reduce T-cell proliferation in a human in vitro model of T cell activation. Since ligation of DC-SIGN and MMR-1 was described in the literature to reduce the immune regulatory capacity of DCs we hypothesize that callus-AG may prevent allergic sensitization and inflammation via the activation of these receptors.

V25

Interleukin 10 signaling in T cells, B cells or neutrophils/monocytes is not involved in allergen specific tolerance induction

A. Preuhsler1, S. Kunz¹, W. Müller², R. Jack³, S. Martin¹, A. Roers⁴, T. Jakob¹

¹Allergy Research Group, Department of Dermatology, University Medical Center, Freiburg; ²Faculty of Life Sciences, University of Manchester, UK; ³Department of Immunology, Institute of Immunology and Transfusion Medicine, University Hospital Greifswald; ⁴Institute of Immunology, Medical Faculty, Carl Gustav Carus University of Technology, Dresden

Human studies suggest that allergen immunotherapy leads to regulatory immune responses that suppress the development of allergic inflammation. Interleukin (IL)-10 production by allergen-specific T cells has been suggested as one of the regulatory mechanisms. This was supported by in vivo studies in mice in which treatment with IL-10 receptor (IL-10R) blocking antibody abrogated the beneficial effects of immunotherapy. In the present study we address the cellular targets of IL-10 in the process of tolerance induction by using mice with a cell type specific inactivation of the IL-10R gene generated by Cre/loxP-mediated recombination.

Ovalbumin (ova) sensitized mice were treated with three s.c. ova injections on alternate days for tolerance induction. After challenge by ova inhalation allergen specific antibody and cytokine responses as well as allergen induced airway inflammation were analyzed. Tolerance induction was effective in the suppression of airway inflammation in wildtype mice but not in IL-10R null mutants (IL-10R^FL/FL Cre deleter⁺), confirming the involvement of IL-10R in tolerance induction. In contrast, in mice that lack IL-10 signaling specifically in T cells (IL-10^RFL/FL CD4-Cre⁺) the degree of tolerance induction was comparable to that of Cre negative littermate controls both displaying strongly reduced eosinophilic infiltration into the bronchoalveolar space and reduced Th2 responses to allergen specific restimulation. Effective tolerance induction was also observed in mice with a B cell specific (IL-10R^FL/FL CD19-Cre⁺) as well as in mice with a neutrophil/monocyte specific (IL-10R^FL/FL LysM-Cre⁺) deletion of the IL-10R.

In summary, our results show that in the murine model of allergen induced airway inflammation direct effects of IL-10 on T cells, B cells or neutrophils/monocytes are not critical for tolerance induction. Thus different cellular targets of IL-10 are likely to be involved in the benefical effects of allergen specific tolerance induction.

V26

Einfluss von 25-Hydroxyvitamin D auf die Typ-I-Sensibilisierung und die spezifische Immuntherapie im Mausmodell

G. Heine¹, C. Tabeling², B. Hartmann³, C. Gonzälez-Calera², A. Kühl⁴, J. Lindner¹, A. Radbruch⁵, M. Worm¹

¹Klinik für Dermatologie, Venerologie und Allergologie, Allergie-Centrum-Charité, CCM, Charité — Universitätsmedizin Berlin; 2Department of Infectious Diseases and Pulmonary Medicine, Charité — Universitätsmedizin Berlin; ³Division of Immunology & Rheumatology, Stanford University School of Medicine, CA, USA; ⁴Medizinische Klinik m. S. Rheumatologie und Klinische Immunologie/Research Center Immuno Sciences, CBF, Charité — Universitätsmedizin Berlin, ⁵Deutsches Rheuma-Forschungszentrum Berlin

Calcitriol (1α,25-dihydroxyvitamin D3) ist der biologisch wirksame Vitamin D Metabolit und vermittelt immunologische Wirkungen, die während allergischer Immunreaktionen relevant sind. Eigene Daten zeigen, dass Calcitriol den IgE-Isotypenklassenwechsel direkt in B-Lymphozyten hemmt. Aufgrund der hyperkalzämischen Toxizität steht Calcitriol jedoch therapeutisch nicht zur Verfügung. In Vorarbeiten zeigten wir, dass Antigen-abhängig aktivierte B-Zellen aus der Calcitriolvorstufe 25-Hydroxyvitamin D3 (25(OH)D) biologisch wirksames Calcitriol synthetisieren können. In dieser Untersuchung wurde der Einfluss von 25(OH)D auf die Typ-I-Sensibilisierung und die Funktion während der allergenspezifischen Immuntherapie untersucht. BALB/c Mäuse wurden gegen Ovalbumin (OVA) sensibilisiert; in 25(OH)D-Defizienz oder -Suffizienz. Die Kinetik der humoralen Immunantwort wurde mittels ELISA bestimmt. Ein OVA-spezifisches Immuntherapieprotokoll wurde etabliert und im Mausmodell der allergischen Atemwegsentzündung mittels Histologie, pulmonaler Zytokinexpressionsanalyse, und Funktionsanalyse in isolierten und perfundierten Lungen untersucht. Die OVA-spezifischen IgE und IgG1 Serumkonzentrationen waren in 25(OH)D-defizienten Mäusen höher als in Kontrolltieren. Durch die OVA-spezifische Immuntherapie wurde dosisabhängig die humorale Immunreaktion nach OVA-Exposition vermindert. Die gleichzeitige Anwendung von 25(OH)D mit der OVA-spezifischen Immuntherapie reduzierte die allergische Atemwegsentzündung und Hyperreagibilität nach OVA-Provokation. Entsprechend war die Th2-Zellinfiltration und Th2-Zytokinexpression in der Lunge vermindert. Zusammenfassend deuten diese Daten an, dass 25(OH)D an der Regulation der Typ-I-Sensibilisierung sowie der spezifischen Immuntherapie beteiligt ist.

V27

Petasites hybridus (butterbur) extract exerts anti-inflammatory effects on primary human nasal epithelial cells in vitro

S. Steiert¹, U. Zissler¹, A. Chaker², H. Bier², J. Drewe³, C. Zahner³, C. Schmidt-Weber¹, C. Traidl-Hoffmann⁴, S. Gilles⁴

¹ZAUM — Center of Allergy & EnvironmentTechnische Universität and Helmholtz Center Munich; ²HNO-Klinik, Klinikum rechts der Isar, Technische Universität München; ³Max Zeller Söhne AG, Romanshorn, Schweiz; ⁴Institut für Umweltmedizin am Klinikum rechts der Isar, Technische Universität München / UNIKA-T, Augsburg

Background: As integral part of the innate immune system, nasal epithelial cells are involved in early steps of allergic inflammation and defence against respiratory viruses.

Aim of study: To evaluate the anti-inflammatory effect of a butterbur leaf extract (Tesalin®) for potential topical application in allergic and non-allergic rhinitis and to elucidate its mechanism of action in nasal epithelial cells.

Methods: Human primary nasal epithelial cells were obtained from turbinoplastic surgeries. Cells were cultivated to 80 % confluency and stimulated with the viral double-stranded RNA mimick Poly IC either alone or in combination with Tesalin® or different petasin isoforms (petasin, isopetasin, neopetasin) contained in Tesalin®. Readouts were production of IL-8, neutrophil chemotaxis and whole transcriptome analysis.

Results: Stimulation of human primary nasal epithelial cells with Poly IC leads to the release of high amounts of IL-8 (range 13–130ng/ml) and induces neutrophil chemotaxis towards cell supernatants. Tesalin®, as well as the petasin isoforms, block the Poly IC-induced IL-8 production, with a combination of all three isoforms being more efficient than any of the isoforms alone. Inhibition of IL-8 release was paralleled by significantly decreased neutrophil chemotaxis towards supernatants of Poly IC/Tesalin®-treated cells.

Conclusion: Functionality of the human primary nasal epithelial cells was shown by inflammatory mediator release upon stimulation with a viral mimic and by neutrophil chemotaxis. Tesalin® inhibited, through the combined action of petasin isoforms, Poly IC induced IL-8 release and neutrophil chemotaxis.

Allergens

V28

Influence of natural and anthropogenic pollen exposition on pollen allergenicity and allergic sensitization

I. Beck¹, S. Gilles-Stein¹, S. Breitner², A. Schneider², K. Wolf², C. Biernath⁴, J. Cyrys², A. Peters², C. Traidl-Hoffmann¹

¹Institute for environmental medicine, UNIKA-T, Augsburg / Technische Universität München; ²Institute of Epidemiology II, German Research Center for Environmental Health, Helmholtz Zentrum München; ⁴Institute of Soil Ecology, Helmholtz Zentrum München

Evidence is compelling for a positive correlation between urbanisation and increment of allergic sensitisation and diseases. The reason for this association is not clear to date. Some data point to a pro-allergic effect of anthropogenic factors on susceptible individuals. Data analysing the impact of environmental — natural and anthropogenic — factors on the allergenicity of allergen carriers such as pollen grains are scarce. Within an interdisciplinary research team (REKLIM Initiative) this study evaluates the effect of natural (e.g. climate) and anthropogenic (e. g. traffic pollutants) factors on birch pollen in a holistic approach. Moreover allergenicity of pollen will be placed in context to allergic sensitisation and symptoms of subjects (KORA study) living in the surrounding of the respective tree. The results of this study will significantly add to our understanding how urbanisation and climate change influence the allergenicity of birch pollen and how this will impact on allergic sensitisations and symptom severity. Therefore birch trees from urban and rural sites in the surrounding of KORA subjects in Augsburg were selected and sites were characterised for NO₂ and O₃ by passive samplers (n = 40). Temperature, NO_x, PM2.5, PM10 and black smoke exposition of the trees will be determined by temperature- and pollution models. Pollen was categorised according to maturation state and allergenicity was analysed by ELISA for Bet v 1, LTB4 and PGE₂. First results show that urban trees are exposed to significant higher NO₂- and lower ozone concentrations, while Bet v 1 concentrations of urban and rural trees do not significantly differ. The results of this study will significantly add to our understanding how urbanisation and climate change influence the allergenicity of birch pollen and how this will impact on allergic sensitisations and symptom severity.

V29

A non-allergenic fraction of aqueous pollen extracts mediates aggravation of the allergic immune response to pollen allergen in humans

S. Gilles¹, I. Beck¹, A. Chaker², M. Bas², M. McIntyre³, L. Cifuentes³, A. Petersen⁴, H. Behrendt⁵, J. Ring³, C. Schmidt-Weber⁶, C. Traidl-Hoffmann¹

¹Institute of Environmental Medicine, UNIKA-T, Augsburg / Technische Universität München; ²ENT Department, Klinikum rechts der Isar, Technische Universität München; ³Department of Dermatology and Allergy, Technische Universität München; ⁴Division of Clinical and Molecular Allergology, Research Center Borstel, Airway Research Center North (ARCN), Member of the German Center for Lung Research, Borstel; ⁵Christine-Kühne-Center for Allergy Research and Education (CK Care), Munich; ⁶ZAUM — Center for Allergy & Environment, Technische Universität & Helmholtz Center München

Objective: This study aimed at elucidating the effect of a non-allergenic, low molecular weight fraction from pollen (APE < 3 kDa) on the human allergic immune response in vivo.

Methods: Birch and grass pollen-allergic individuals were skin-pricked with allergen alone, allergen plus APE < 3 kDa, or allergen plus candidate substances, and wheal size was recorded. In a nasal provocation study, healthy and pollen allergic individuals were subjected to repeated intranasal challenges with either allergen alone or with allergen plus APE < 3 kDa. Local cytokine and IgE production was measured, and whole transcriptome was assessed in nasal specimens of a patient subgroup. Clinical end-points were nasal secretion weights, obstruction, and symptom scores.

Results: Patients pricked with pollen allergens developed larger wheals if allergens were diluted in the low molecular weight fraction. Candidate mediators are linoleic acid derivatives and serotonin. In nasal secretions of allergic patients challenged with allergen plus APE < 3 kDa, IL-8 and IgE were significantly increased. These patients showed increased nasal secretion and reported more severe rhinorrhea than the allergen-only group. Serotonin receptors were differentially regulated in nasal specimens after challenge with allergen plus APE < 3 kDa.

Conclusions: APE < 3 kDa exacerbates the allergic immune response in the skin and nose. In the skin, pollen-associated lipid mediators boost the wheal-and-flare reaction. In the nose, pollen-derived serotonin might play a role in differential aggravation of symptoms.

V30

Rolle von Zeckenstichen bei der Entstehung einer Typ-I-Sensibilisierung auf das Oligosaccharid Galaktose-alpha-1,3-Galaktose (alpha-GAL) bei Forstmitarbeitern und Jägern im Naturpark Schönbuch und den angrenzenden Landkreisen

J. Fischer¹, E. Lupberger¹, J. Hebsaker¹, E. Aichinger², R. Oehme², D. Reick², T. Biedermann¹

¹Universitäts-Hautklinik Tübingen; ²Landesgesundheitsamt Baden-Württemberg, Stuttgart

Hintergrund: Das Oligosaccharid Galaktose-alpha-1,3-Galaktose (α-GAL) wurden als Panallergen für Fleisch von Säugetieren identifiziert. In den Südstaaten der USA fand sich eine Korrelation mit Zeckenstichen der Gattung Amblyomma americanum. Zeckenstiche werden in dieser Region als wichtigste Quelle ein Sensibilisierung gesehen. Ziel dieser Studie war es zu untersuchen, ob auch Zeckenstiche in Süddeutschland heimische Zeckenarten der Gattung Ixodes und Dermatocentor eine Rolle bei der Entstehung einer Typ-I-Sensibilisierung auf α-GAL spielen können.

Methoden: Im Rahmen einer Querschnittstudie wurde Forstmitarbeiter und Freizeitjäger mit Tätigkeit im Naturpark Schönbuch und den angrenzenden Landkreisen Tübingen, Reutlingen, Esslingen, Stuttgart und Böblingen serologisch mittels Phadia-CAP auf eine Typ-I-Sensibilisierung auf α-GAL untersucht. Begleitend wurde das Gesamt-IgE und die Blutgruppeneigenschaften im Serum bestimmt. Fragenbogenbasiert wurde der zeitliche Umfang einer Tätigkeit im Wald, die Anzahl von Zeckenstichen und mögliche allergische Symptome erhoben.

Ergebnisse: Die Studie wurde am 28.11.2013 geschlossen (last patient in). Die Phase der Auswertung hat gerade begonnen. Konkrete Angabe zu Ergebnissen können daher zum derzeitigen Zeitpunkt noch nicht gemacht werden.

Schlussfolgerung: Typ-I-Sensibilisierungen auf α-GAL sind häufiger in Süddeutschland als bislang bekannt.

V31

Allergen levels in hair of different cattle breeds — do breed-specific differences really exist?

E. Zahradnik, I. Sander, T. Brüning, M. Raulf

Institut für Prävention und Arbeitsmedizin der Deutschen Gesetzlichen Unfallversicherung, Ruhr-Universität Bochum

Bovine allergens are important inducers of occupational allergic airway diseases in agricultural workers. The lipocalin, Bos d 2, is a major allergen in cow hair and dander extracts. Breed-specific differences in Bos d 2 content have been suggested in the literature. The aim of the study was to compare protein and total and major allergen content of hair samples collected from different cattle breeds.

In total, 80 hair samples from 17 different cattle breeds were analyzed (Simmental, n = 31; German Holstein, n = 15; Brown cattle, n = 11; “Others”, n = 23). Protein concentration was determined by the Bradford assay. The allergen content was measured using a sandwich ELISA based on polyclonal antibodies against bovine hair protein extract and a commercial immunoassay based on monoclonal antibodies against the major allergen Bos d 2 (Indoor Biotechnologies). Results were given in μg/g of hair. Statistical analyzes were performed using the Kruskal-Wallis test and Spearman’s rank correlation.

In all three tested parameters, a wide variability was observed between individual samples. The protein content differed about 40-fold (0.3–1.2 mg/g), the cow hair allergen content about 500-fold (37–18553 μg/g) and Bos d 2 content about 1200-fold (5-6323 μg/g). Protein, cow hair allergen and Bod d 2 values correlated strongly and significantly with each other (rS between 0.85 and 0.92; p < 0.0001). Cow hair allergen concentrations were on average 3.5-fold higher than Bos d 2 quantities. No significant differences between different breeds were found. The median of Bos d 2 content was 770.4 μg/g for Simmental, 693.5 μg/g for German Holstein, 600.7 μg/g for Brown cattle and 645.3 μg/g for the “Others”.

Our data concerning protein, cattle hair allergen and Bos d 2 concentrations show high variability between individual animals without any association to a particular breed.

V32

A novel protein tool for the experimental analysis of functional epitopes of Bet v 1-related allergens

D. Schiller¹, H. Berkner², M. Hartl², C. Seutter von Loetzen², S. Randow¹, J. Nürnberg¹, M. Gubesch¹, L. Vogel¹, F. Husslik¹, A. Reuter¹, J. Lidholm³, B. Ballmer-Weber⁴, S. Vieths¹, P. Rösch²

¹Paul-Ehrlich-Institut, Langen; ²Department of Biopolymers, Bayreuth; ³Thermo Fisher Scientific, ImmunoDiagnostics Division, Uppsala, Sweden; ⁴Allergy Unit, University Hospital Zürich, Switzerland

Background: Birch pollen-allergic subjects often develop Bet v 1-specific IgE that cross-reacts with related food allergens. Bet v 1 and its homologs in pollen and food display exclusively conformational epitopes. We established a system to specifically analyze functional epitopes and epitope cross-reactivity of Bet v 1-related allergens. The enzyme norcoclaurine synthase (NCS) from Thalictrum flavum is structurally homologous to Bet v 1 but does not bind IgE reacting with Bet v 1-like allergens. By substituting amino acids in variants of NCS, the impact of individual residues of Bet v 1-like allergens in IgE binding can be studied.

Methods: Putative functional IgE epitopes of Bet v 1 were grafted onto NCS. NCS variants were purified and protein structures were evaluated by CD and NMR spectroscopy. Sera of birch pollen allergic patients were analyzed for IgE binding to NCS variants. Cross-reactivity of patients’ IgE was assessed by competitive binding assays using variants of NCS, Bet v 1 and Bet v 1-homologous allergens. The capability of NCS variants harboring functional IgE epitopes to induce mediator release in humanized rat basophil leukemia (RBL) cells was tested.

Results: CD and ¹H¹⁵N-HSQC NMR analyses indicated Bet v 1-like structure of NCS variants. sIgE binding increased significantly with the number of amino acids substituted in NCS. IgE binding to NCS variants could be competitively inhibited with Bet v 1 and Bet v 1-like allergens. Structural analysis of IgE binding sites of NCS variants showed high conformational similarities among cross-reactive allergens. NCS variants carrying more than one functional IgE epitope were able to induce mediator release in RBL cells.

Conclusions: Amino acids critical for IgE binding of Bet v 1 and cross-reactivity in Bet v 1-like allergens were identified. The induction of Ig binding to the non-allergenic NCS with its Bet v 1-like structure allows the unambiguous identification and analysis of critical amino acids in epitopes of Bet1-related allergens.

V33

Recombinant Polistes dominula protease allergen Pol d 4 as candidate for dissection of sensitisation to vespid venoms

S. Wolf¹, S. Blank², F. Bantleon³, T. Jakob⁴, E. Spillner¹

¹Aarhus University, Denmark; ²ZAUM — Center of Allergy and Environment, Technical University and Helmholtz Center Munich; ³Institute of Biochemistry and Molecular Biology, University Hamburg; ⁴Allergy Research Group, Department of Dermatology, University Medical Center Freiburg

Background: Sensitization to cross-reactive allergens poses significant problems in Hymenoptera venom allergy. Molecular and structural similarity of vespid venom allergens from Vespula and the increasingly spreading Polistes species makes reliable identification of culprit venom and therapeutic decision highly difficult. Species-specific and CCD-free allergens could provide opportunities to dissect sensitisation of venom allergic patients.

Objective: Recombinant production of the heavily glycosylated putative protease allergen Pol d 4, analysis of cross-reactivity with other venom proteases and analysis of sensitization of venom allergic patients.

Methods: Recombinant Pol d 4 was cloned and expressed via baculovirus-mediated infection of insect cells. The purified protease was assessed for the presence of CCDs and specific IgE reactivity of venom-sensitized patients as compared to Pol d 5, Ves v 1 and Ves v 5. Diagnosis of venom allergy was based on history, skin test and sIgE to venom. Moreover, we built homology models of Pol d 4 and other putative venom proteases.

Results: Recombinant Pol d 4 could be produced in high yields as secreted protein in Sf9 insect cells. The purified, highly glycosylated protein exhibited no detectable CCD reactivity. Molecular modeling of Pol d 4 revealed a typical fold of serine proteases and emphasized the reduced molecular similarity with other putative venom proteases. Using the recombinant Pol d 4 sIgE reactivities of venom allergic patients were assessed.

Conclusion: Recombinant and CCD-free Pol d 4 might provide a valuable tool for differentiating Polistes and Vespula venom sensitization.

V34

Analysis of IgE and IgG4 profiles to a panel of CCD free bee venom allergens in bee venom allergic patients treated with venom immunotherapy

M. Frick¹, F. Bantleon², S. Blank³, J. Huss-Marp¹, F. Ruëff⁴, A. Helbling⁵, E. Spillner², T. Jakob¹

¹Allergy Research Group, Department of Dermatology, University Medical Center Freiburg; ²Department of Engineering, Aarhus University, Denmark; ³ZAUM — Center of Allergy and Environment, TU and Helmholtz Center, Munich; ⁴Department of Dermatology and Allergology, Ludwig-Maximilians-Universität, München; ⁵Allergy Unit Zieglerspital, Department of Internal Medicine, Spital Netz Bern, Switzerland

Background: Detection of IgE to recombinant (r) Hymenoptera venom allergens has been suggested to improve the diagnostic precision in Hymenoptera venom allergy. Using a panel of CCD free honeybee venom(HBV) allergens (rApi m 1, rApi m 2, rApi m 3, nApi m 4, rApi m 5 and rApi m 10) we recently demonstrated improved diagnostic sensitivity as compared to use of rApi m 1 alone.- We identified Api m 3, Api m 5 and Api m 10 as additional major allergens and revealed distinct sensitisation profiles some of which are dominated by IgE to allergens that have been reported to be absent or underrepresented in therapeutic HBV preparations. Here we now report the IgE and IgG4 profiles of HBV allergic patients before and under treatment with venom immunotherapy (VIT).

Methods: Diagnosis of HBV allergy (n = 56) was based on history, skin test and sIgE to HBV extract (i1). IgE reactivity was analyzed to the entire panel of HBV allergens, IgG4 reactivity was analysed to rApi m 1 (i208), rApi m 3, nApi m 4, and rApi m 10 using ImmunoCAP research prototypes (Thermo Fisher Scientific, Uppsala, Sweden).

Results: The IgE sensitisation frequency to rApi m 1, rApi m 2, rApi m 3, nApi m 4, rApi m 5 and rApi m 10 was 80.3 %, 64.3 %, 46.4 %, 32.1 %, 67.9 % and 77.4 % respectively and confirmed previously reported frequencies. The analysis before and after at least 6 months of VIT demonstrated a more than 2 fold increase in IgE reactivity to rApi m 1, rApi m 2, rApi m 3, nApi m 4 and rApi m 10 in 17.9 %, 21.4 %, 7.1 %, 14.3 %, and 8.9 %, respectively. VIT induced IgE levels above the cutoff (> 0,35 kU/L) in previously negative patients in 4/11 for Api m 1, 5/20 for Api m 2, 2/30 for Api m 3, 5/38 for Api m 4 and 1/16 for Api m 10. Analysis of sIgG4 demonstrated that VIT induced a pronounced IgG4 response to HBV (9.4 ± 15.2 fold), Api m 1 (22.8 ± 59.2 fold) and Api m 4 (11.9 ± 26.7 fold), while only little induction of IgG4 to Api m 3 (1.6 ± 1,4 fold) and Api m 10 (2.6 ± 3,6 fold) was observed.

Conclusion: Our data confirms the presence of a broad spectrum of different sensitisation profiles to a panel of HBV allergens. In addition, it demonstrates a certain degree of new sensitisations to single allergens during VIT, a robust induction of allergen specific IgG4 responses to high abundance allergens and a relative lack to allergens that have been reported to be absent or underrepresented in therapeutic HBV preparations. Future analysis will have to address if these parameters are of relevance for the outcome of VIT.

V35

Detection of blocking antibody responses in patients with hymenoptera venom allergy by the ELIFAB assay

J. Müller¹, J. Pickert¹, A. Rudzio¹, S. Blank², E. Spillner³, C. Möbs¹, W. Pfützner¹

¹Department of Dermatology and Allergology, Philipps University Marburg; ²Institute of Allergy Research, Helmholtz Zentrum, Munich; ³Department of Engineering — BCE Protein Engineering, Aarhus University, Denmark

Allergen-specific immunotherapy (ASIT) results in synthesis of allergen-specific IgG antibodies, especially of the IgG4 isotype, which have been shown to inhibit allergen binding by IgE in patients suffering from pollen- or food-induced immediate type allergy. So far, assessment of the allergen blocking capacity was based on complex and cumbersome cellular assays, e. g. on detecting FceRII/CD23-bound allergen-IgE complexes on the surface of immortalized EBV-transformed B cells. Recently, a cell-free ELIFAB (enzyme-linked immunosorbent facilitated antigen binding) assay was described, which allows to determine the functional activity of allergen-specific serum antibodies by measuring their influence on the binding of allergen to IgE captured by immobilized CD23 monomers. To test the applicability of this novel assay in determining blocking antibody responses in patients suffering from HV allergy, serum samples were collected at different time-points during HV-ASIT, i.e. before initiation and during the build-up phase, directly before and after sting challenge, and after cessation of ASIT. The ELIFAB assay was modified by utilizing either the major honey bee (Api m 1) or common wasp (Ves v 5) venom allergen. To correlate ELIFAB data with ASIT-induced humoral changes, Api-m-1- and Ves-v-5-specific IgE, IgG and IgG4 antibodies were measured by ImmunoCap. Results showed that ASIT led to increased concentrations of HV-specific IgG and IgG4 antibodies. Furthermore, depletion of HV-specific IgG resulted in an abrogation of the allergen-blocking capacity confirming that these antibodies were able to inhibit HV-allergen binding to IgE. Our data suggests that the ELIFAB assay is a prom-ising tool to characterize potentially protective IgG antibody responses induced by ASIT with HV. Further studies might shed light upon whether functional or rather quantitative levels of HV-specific IgG antibodies are a surrogate marker for ASIT-induced allergen tolerance in patients suffering form HV allergy.

V36

Acid-labile poly(ethylene glycol) nanocarriers for allergen encapsulation and controlled release for specific immunotherapy

H. Köhring¹, I. Bellinghausen², H. Frey¹, J. Saloga²

¹Institute of Organic Chemistry, Graduate School Materials Science Mainz, Johannes-Gutenberg-Universität, Mainz; ²Department of Dermatology, Johannes-Gutenberg-Universität Mainz

Polymer nanoparticles are used to protect therapeutic proteins or drugs from degradation, obtaining higher local concentrations and enabling targeted transport. Encapsulation of allergens inside polymeric nanoparticles could aid to avoid severe side effects occurring sometimes during allergen-specific immunotherapy. We have synthesized a novel type of difunctional, water soluble poly(ethylene glycol) dimethacrylate macromonomer with acetal-sites that degrade at acidic pH. The allergen and the macromonomer were entrapped inside liposomes as templates that were produced by dual asymmetric centrifugation. Radical polymerization of the methacrylate groups inside the liposomes generated PEG-nanocarriers. The allergen-loaded nanocarriers were approximately 150–200 nm in size and showed low polydispersity indices. It could be demonstrated in in-vitro studies that immature dendritic cells (DC) internalize these protein-loaded, non-toxic PEG-nanocarriers. Viability and surface marker expression of DC was not affected by allergen-loaded nanoparticles. Furthermore, nanoparticles did not lead to DC maturation. To investigate the targeted delivery of allergen into the antigen-presenting pathway, immature monocyte-derived DC from allergic donors were pulsed with Phleum pretense allergens (free or loaded into nanoparticles) and simultaneously matured with pro-inflammatory cytokines. Then, DC were co-cultured with autologous CD4+ T cells to induce proliferative responses. DC treated with allergen-loaded nanoparticles induced an allergen-specific proliferation reaching about 50% of the proliferation intensity observed for T cells stimulated with allergen-treated DC alone. These data indicate that allergen-loaded nanoparticles are capable of inducing specific immune responses necessary for specific immunotherapy. Furthermore, this finding encourages functionalization of nanoparticles with targeting molecules like mannose for increased allergen uptake and presentation by DC.

Food/Gastro-intestinal tract

V37

Folate status as a modifier of epigenetic profile in human neonatal CD4⁺ T cells

H. Harb¹, M. Amarasekera², S. Ashley³, M. Tulic⁴, P. Pfefferle¹, D. Potaczek¹, H. Renz¹, D. Martino³, D. Kesper¹, S. Prescott²

¹Institute for Laboratory Medicine and Pathobiochemistry, Molecular Diagnostics, University Marburg; ²School of Paediatrics and Child Health, University of Western Australia, Perth, Australia; ³Murdoch Childrens Research Institute, Melbourne, Australia; ⁴Tolerance Immunitaire, Hôpital de l’Archet, Université de Nice Sophia-Antipolis, France

Introduction: Normal immune development in early life is important for the protection against inflammatory diseases such as allergies. Changes in the dynamics of gene-environment interactions in early life are implicated in the rising of disease predisposition. Exposure to different environmental factors during pregnancy can play a very important role in the development of immune response. In the present study we sought to examine the effects of high in utero exposure to folate on neonatal epigenetic profiles of genes associated with allergic inflammation.

Methods: 23 neonates were selected from a large prospective cohort including the two extremes of maternal folate levels. Different epigenetic modifications, including DNA methylation and histone acetylation were analyzed at allergy-associated genes. Furthermore, cytokine levels were measured following both polyclonal and allergen specific stimulation of cord blood mononuclear cells.

Results: There was a bias toward a Th2 phenotype by looking at the whole cohort epigenetic profile. Histone acetylation on both H3 and H4 histones at the GATA3 locus and H4 acetylation at the IL9 locus was increased in children born to mothers with high folate levels together with decreased acetylation at the IFNG locus in the same group. Cytokine levels were not significantly differed between the two groups. Nevertheless, cytokine ratios of Th2/Th1 showed an increase in the high folate group.

Conclusion: Maternal folate as a known methyl donor is likely influencing the development of the fetal immune system by modifying epigenetic marks like histone acetylation. High folate levels render allergy-associated genes more permissive for transcription and might thus affect disease development later in life.

V38

Immune modulatory properties of non-digestible oligosaccharides mimicking the functionality of human breast milk oligosaccharides ; — potential for allergy prevention in bottle fed infants

S. Lehmann¹, J. Hiller¹, W. Back², J. van Bergenhenegouwen³, J. Ring⁴, C. Schmidt-Weber⁵, L. Knippels³, J. Garssen⁶, C. Traidl-Hoffmann¹

¹Institute of Environmental Medicine, UNIKA-T, Augsburg/Technische Universität München; ²Brewing and Beverage Technology, Technische Universität München; ³Nutricia Research, Department of Immunology Utrecht, Netherlands; ⁴Department of Dermatology and Allergology, Technische Universität München; 5ZAUM ; — Center of Allergy and Environment, Technische Universität & Helmholtz Zentrum München; ⁶Division of Pharmacology, Utrecht Institute for Pharmaceutical Science, Faculty of Science, Utrecht University, Netherlands

As combinations of lactic acid bacteria (LAB) and non-digestible oligosaccharides have been shown to harbor preventive effects towards immune-regulatory disorders like allergies it is current practice for bottle fed infants to apply oligosaccharides derived from bovine milk (galactooligosaccharides) and plants (fructooligosaccharides) mimicking the functionality and molecular size of human milk oligosaccharides as a supplement in infant formulas.

The aim of this study was to reveal underlying mechanisms and direct effects of non-digestible oligosaccharides in combination with certain LAB on human monocyte-derived dendritic cells (MoDCs). Immature human MoDCs prepared from peripheral blood of healthy non-atopic volunteers were screened in vitro after stimulation with different LAB strains in the presence of specific neutral and acidic galacto- and fructooligosaccharide mixtures. Cytokine, chemokine and galectin release by MoDCs was analyzed after 24h in cell-free supernatants by luminex-based assay and ELISA.

Neutral and acidic oligosaccharide mixtures exert a significant additive effect on bacteria induced anti-inflammatory IL-10 secretion by MoDC, while no ability to increase pro-inflammatory IL-12p70 production was observed. T cells induced by these MoDC show a regulatory T cell phenotype characterized by Foxp3 expression and an enhanced IL-10/IFNγ ratio of secreted cytokines.

These results indicate anti-inflammatory and immune-modulatory properties of the investigated LAB in the presence of neutral and acidic non-digestible oligosaccharide mixtures in vitro. The tested combinations might represent a useful therapeutic strategy for immune regulatory disorders, and could be considered as allergy preventing ingredients in food.

V39

Characterization of food allergens using mass spectrometry: Is our picture of plant allergen isoforms complete?

J. Brockmeyer, T. Wigger, M. Hummel

Institut für Lebensmittelchemie, Westfälische Wilhelms-Universität, Münster

Allergen diagnosis and research projects dealing with plant food allergens often take advantage of the use of recombinant allergens that can be produced in high amounts with excellent purity. However, the initial identification of allergens in food is in many cases based on the use of cDNA libraries and cloning techniques using degenerated primers that tend to be incomplete on allergen isoform level. In addition, posttranslational processing might be missed when the natural allergen is not analyzed thoroughly. We therefore characterized the 2S albumins from mustard (Sin a 1) and sesame (Ses i 1) as model food allergens by mass spectrometry to investigate whether data on isoforms of these allergens are comprehensive.

2S albumins are members of the prolamin superfamily and are widely distributed in seeds and tree nuts. They are synthesized as a single approx. 13 kDa precursor protein that is subjected to extensive posttranslational proteolytic processing. The mature 2S albumins are composed of two subunits of approximately 9;–10 kDa and 3–4 kDa size, which are linked by two disulfide bridges. Using mass spectrometry and purification of the 2s albumin fraction from sesame and mustard seeds, we were able to detect sequence polymorphisms of the 2S albumins from mustard and sesame on peptide level and confirmed the occurrence of novel isoforms by direct mass spectrometrical analysis of subunits and intact proteins. For Sin a 1, we identified 6 novel isoforms, while for Ses i 1 pronounced differences in proteolytic processing of the single allergen precursors was observed (“C-terminal clipping”) leading to sequence identity of clipping variants of Ses I 1 down to only 86%.

Altogether, we present here an overview of the possibilities and pitfalls of protein mass spectrometry in the field of allergen research and discuss the complexity of food allergen isoforms in natural sources.

V40

Analysis of the IgE epitope profile of soybean allergen Gly m 4

F. Husslik¹, C. Seutter von Loetzen², M. Hartl², L. Vogel¹, B. Ballmer-Weber³, J. Kleine-Tebbe⁴, R. Treudler⁵, J. Simon⁵, K. Hanschmann⁶, A. Reuter¹, M. Gubesch¹, P. Rösch², S. Vieths¹, T. Holzhauser¹, D. Schiller¹

¹Division of Allergology, Paul-Ehrlich-Institut, Langen; ²Department of Biopolymers, University of Bayreuth; ³Allergy Unit, Department of Dermatology, University Hospital Zurich, Switzerland; ⁴Allergy and Asthma Center Westend, Berlin; ⁵Department of Dermatology, Venereology and Allergology, University of Leipzig; ⁶Department of Biostatistics, Paul-Ehrlich-Institut, Langen

Background: Individuals with birch pollinosis may show allergic reactions after consumption of soybean-containing food. This is caused by cross-reaction of IgE directed against the major birch pollen allergen Bet v 1 with its homologue Gly m 4 from soybean.

Objective: To identify IgE epitopes of Gly m 4 in birch pollen-allergic subjects with and without allergy to soybean.

Methods: Sera from 33 birch pollen-allergic patients with and without clinical reactivity to soy were included in the study. Specific IgE against Bet v 1 and Gly m 4 was determined by ImmunoCAP and IgE binding to rGly m 4 and soy extract was tested in western blot. To predict putative IgE epitopes of Gly m 4, a bioinformatical approach was used. Epitopes were analyzed with Gly m 4 variants with substituted epitope residues that were purified from E. coli. Secondary and tertiary structures were assessed by CD and NMR spectroscopy. IgE binding of variants was analyzed by competitive immunoblotting and inhibition ELISA. Residues critical for IgE binding were analyzed with model protein norcoclaurine synthase (NCS).

Results: Gly m 4- and Bet v 1-specific IgE levels ranged from 0 to > 100 kUA/L in sera of both patient groups. Intensity of IgE binding in western blot corresponded to ImmunoCAP analysis. Surface mapping resulted in four distinct epitopes of Gly m 4. Several substitutional rGly m 4 variants were generated and showed a typical Bet v 1-like structure according to CD spectra and 1D-1H-NMR spectroscopy. IgE binding was reduced with several variants, indicating a multiple number of epitopes on Gly m 4 surface. Selected residues were grafted onto NCS to induce IgE binding.

Conclusion: No significant differences in Gly m 4-specific IgE binding could be observed between birch pollen-allergic patients with and without clinical soy allergy. However residual IgE reactivity persists in substitutional rGly m 4 variants, indicating the presence of further IgE epitopes not yet identified.

V41

LTP cross-reactivity — primary sensitization to mugwort pollen LTP Art v 3 facilitates subsequent sensitization to peach LTP Pru p 3 in mice

A. Wangorsch¹, S. Schülke¹, G. Gadermaier², M. Albrecht¹, M. Wallner², S. Randow¹, S. Wolfheimer¹, J. Lidholm³, I. Lauer¹, F. Ferreira², M. Toda¹, S. Vieths¹, G. Reese¹, S. Scheurer¹

¹Paul-Ehrlich-Institut, Langen; ²Universität Salzburg, Österreich; ³Thermo Scientific, Uppsala, Schweden

Lipid transfer proteins (LTPs) are important pollen and food allergens in the Mediterranean area. Peach LTP Pru p 3 is considered as primary sensitizer contributing to clinical cross-reactivity. Despite the evidence on the importance of Pru p 3, it is unclear whether reactivity to multiple LTPs is due to antibody cross-reactivity or to independent sensitization events.

Aim of the study was to compare antigenicity of LTPs from pollen and food and to investigate whether primary sensitization to LTPs promote the secondary sensitization to LTPs from unrelated sources.

IgE sensitization to peach LTP was compared in different mouse strains. IgE high responder mice (CBA/J) were used to compare antigenicity and IgE cross-reactivity of different LTPs. To investigate whether an established Th2 immune response to LTPs facilitates a secondary immune response and cross-reactivity to unrelated LTPs, CBA/J mice were immunized with Pru p 3 followed by Art v 3, Art v 3 followed by Pru p 3, and a mixture of both. Induction of LTP specific Ig-titers and cross-reactivity were determined by ELISA.

In contrast to BALB/c mice, CBA and C3H mice showed IgE-responses to Pru p 3. A species-specific immune response lacking any IgE and IgG cross-reactivity was observed for most of the tested LTPs. Although Pru p 3 and Art v 3 show high structural similarity, Art v 3 displayed stronger antigenicity. In line with this, IgE response to Pru p 3 was promoted in mice previously sensitized to Art v 3, which was even the case upon concurrent administration of both LTPs. Primary sensitization with Pru p 3 did not show similar effects to the Art v 3 IgE response.

In summary, the immune response to LTPs is mice strain dependent. LTPs displayed different immunogenic properties and did not induce cross-reactive IgE antibodies. Primary sensitization to Art v 3 conditioned the animals for subsequent sensitization to Pru p 3. The extent to which these findings are applicable to the manifestation of clinical cross-reactivity in humans needs to be investigated.

V42

Potential allergens in the lipophilic matrix of peanut: Isolation and characterization of oil body proteins (oleosins)

C. Schwager¹, A. Petersen¹, S. Kull¹, W. Becker¹, U. Jappe²

¹Division of Clinical and Molecular Allergology, Research Center Borstel / Airway Research Center North, German Center for Lung Research, Gießen; ²Division of Clinical and Molecular Allergology, Research Center Borstel / Department of Dermatology and Allergology, University of Lübeck

Background: Peanut (Arachis hypogaea) allergy is one of the most frequent class I food allergies worldwide. Ingestion of peanut-containing products can cause severe allergic reactions. So far, little is known about the clinical relevance of a group of lipophilic oil body proteins, termed oleosins. Two oleosin have been described in peanut so far, Ara h 10 and Ara h 11. Allergenic properties of oleosins have been published for sesame, hazelnut and buckwheat, but little is known on the impact of peanut oleosins. Due to the fact that lipophilic allergens are underrepresented in aqueous extracts, generally used for diagnostic tests, the isolation and characterization of peanut oleosins may add to the efforts to complete component-resolved analysis.

Methods: In order to isolate oleosins, different methods for the enrichment of these components were applied. Lipophilic protein fractions obtained from the extraction method of Tzen et al. (1997), a chloroform/methanol extraction and a centrifugation-flotation method were analyzed using 2D-PAGE electrophoresis, SDS-PAGE and immunoblotting techniques. Further identification of oleosins was achieved performing N-terminal sequencing, homology search and mass spectrometry.

Results: The method of Tzen et al. (1997) and the chloroform/methanol extraction were not suitable for the isolation of oleosins due to low amounts and impurities. In contrast, the centrifugation-flotation method provides already highly purified oil body proteins. SDS-PAGE analysis revealed oleosin-characteristic bands of 14 –18 kDa. Subsequent N-terminal sequencing confirmed the presence of the oleosin Ara h 11. MALDI-TOF-MS analysis identified additional oleosins e. g. Ara h 10 and Oleosin 3.

Conclusion: After evaluation of three different methods, the isolation of the known peanut oleosins was achieved using the centrifugation-flotation method. Further investigation will focus on the preparation of the single oleosins.

V43

Allergen-induced inflammation of the intestine in a humanized mouse model of allergy is inhibited by activated regulatory T cells

M. Eschborn¹, B. Weigmann², S. Reissig³, A. Waisman³, J. Saloga¹, I. Bellinghausen¹

¹Department of Dermatology, University Medical Center of the Johannes Gutenberg-University Mainz; ²Department of Internal Medicine I, University Hospital Erlangen, University Erlangen-Nürnberg; ³Institute for Molecular Medicine, University Medical Center of the Johannes Gutenberg-University Mainz

Recently, we have developed a humanized mouse model of allergen-induced IgE-dependent gut inflammation in PBMC-engrafted immunodeficient mice. In the present study we investigated the role of regulatory T cells (Treg) in this model. Therefore, NOD-scid-γc-/- mice were injected intraperitoneally with human PBMC from allergic donors together with the respective allergen or with NaCl as control in the presence or absence of different concentrations of CD4⁺CD25⁺ Treg of the same donor. After an additional allergen boost one week later, mice were challenged with the allergen rectally on day 21 and gut inflammation was monitored by a high resolution video mini-endoscopic system. Allergen-specific human IgE in mouse sera, which was only detectable in PBMC plus allergen-treated mice, was strongly inhibited by co-injection of Treg at a ratio of at least 1 : 10. The presence of Treg also reduced allergen-specific proliferation and cytokine production of human CD4⁺ T cells recovered from spleens at the end of the experiment. Furthermore, the allergen-induced endoscopic score evaluating translucency, granularity, fibrin production, vascularity, and stool after rectal allergen challenge was significantly decreased by Treg. Activation of Treg prior to injection further increased all inhibitory effects which could be prevented by blockade of the Treg activation marker GARP. These results demonstrate that allergen-specific gut inflammation in human PBMC-engrafted mice can be avoided by enhancing the numbers of autologous Treg in these mice which is of great interest for therapeutic intervention of allergic diseases in the intestine.

Posters

P01

Establishment and validation of chromatin immunoprecipitation (ChIP) method for cohort studies

H. Harb, D. Kesper, H. Renz, P. Pfefferle

Institute for Laboratory Medicine and Pathobiochemistry, Molecular Diagnostics, University Marburg

Introduction: Over the past few decades, the incidence of allergic diseases such as asthma has been increasing especially in westernized countries. This increase has been suggested to be of an environmental basis and might be triggered by epigenetic modifications. The aim of the work is to establish and validate a ChIP method suitable for cohort studies. To analyse epigenetic modifications like histone acetylation or histone methylation in samples e. g. cord blood cells, a redundant and validated method should be implemented.

Methods: Chromatin Immunoprecipitation (ChIP) method was established for the measurments of different histone modifications primairly in CD4⁺ cells which are the main vessel for immunological changes in asthma and allergy. Furthermore, different validation steps and techniques were implemented starting by measuring the lower limit of detection, lower limit of quantification and the reference range. Additionally, the effect of freezing and thawing the samples, long time storage and the temperature sensitivity of the samples were analysed.

Results: Several Th1/Th2/Treg and Th17 loci were analysed and validated and measured. Moreover, it seemed that freezing and thawing does not have a significant effect on the enrichment values of different genes as well as long time storage by -80 °C. On the other hand working on room temperature (RT) and leaving the samples on RT can have a destructive effect on enrichment values.

Conclusion: a validated method to measure different histone modifications for more Th1/Th2/Treg and Th17 loci is established, covering a wide range of genes, which might help to improve our understanding of epigenetic changes by environmental factors and their influence on disease susceptibility. This method can be implemented in different cohorts with different questions covering a whole range of immune diseases and can be spread to other cell types if needed.

P02

A novel tool for monitoring free serum IgE in patients treated with omalizumab

B. Stöcker, R. Brehler

Klinik und Poliklinik für Hautkrankheiten, Universitätsklinikum Münster

The humanized monoclonal anti-IgE antibody omalizumab generate its mode of action by reducing free IgE serum concentrations. This has a therapeutic effect in many IgE-mediated disorders like asthma and chronic urticaria. It is recommended that free IgE serum concentrations should be reduced to 20.8 IU/ml or less, in order to benefit from omalizumab treatment. Monitoring free IgE levels is useful to ensure a sufficient reduction and to evaluate cases without therapeutic benefit. We are testing a lateral flow immunoassay for the quantitative measurement of non complexed IgE in serum, which is quick and easy to perform. The detection limit of the assay is 30 IU/l. Free IgE serum levels of patients with severe allergic asthma are analysed before and during a therapy with omalizumab. Additionally, correlation analysis concerning changes in asthma control scores and free IgE concentrations are made.

P03

Pattern recognition pathways leading to a Th2 cytokine bias in ABPA patients

K. L. Becker, M. Gresnigt, S. Smeekens, C. Jacobs, C. Magis-Escurra, L. Joosten, M. Netea, M. Reijers, F. van de Veerdonk

Radboud University Medical Center Nijmegen, Niederlande

Background: Allergic bronchopulmonary aspergillosis (ABPA) is characterized by an exaggerated Th2 response to Aspergillus fumigatus, but the immunological pathways responsible for this effect are unknown.

Objective: The aim of this study was to decipher the pattern recognition receptors (PRRs) and cytokines involved in the Aspergillus-specific Th2 response, and to study Aspergillus-induced responses in healthy controls and ABPA patients.

Methods: Peripheral blood mononuclear cells (PBMCs) were stimulated with heat-killed Aspergillus conidia, various other pathogens, or PRR-ligands. PRRs and cytokine pathways were blocked with PRR-blocking reagents, anti-TNF (Etanercept or Adalimumab), IL-1Ra (Anakinra), or IFNγ (IFN-gamma). ELISA and FACS were used to analyze cytokine responses.

Results: Aspergillus was the only pathogen that stimulated the Th2 cytokines IL-5 and IL-13, while Gram-negative bacteria, Gram-positive bacteria, Candida albicans, chitin, β-glucan, or Toll like receptors (TLR) ligands did not. Depletion of CD4⁺ cells abolished IL-13 production. Blocking complement receptor 3 (CR3) significantly reduced IL-5 and IL-13, while blocking TLR2, TLR4 or dectin-1 had no effect. ABPA patients displayed increased Aspergillus-induced IL-5 and IL-13, and decreased IFNγ production compared to healthy controls. All biological agents tested showed the capability to inhibit Th2 responses, but also decreased Aspergillus-induced IFNγ.

Conclusions: Aspergillus conidia are unique in triggering Th2 responses in human PBMCs, through a CR3-dependent pathway. ABPA patients display a significantly increased Aspergillus-induced Th2/Th1-ratio that can be modulated by biologicals. These data provide a rationale to explore IFNγ therapy in ABPA as a corticosteroid-sparing treatment option, by dampening Th2 responses on the one hand and supplementing the IFNγ deficiency on the other hand.

P04

Anaphylaxie nach Verzehr von Maronenpilzen

I. L. Suarez, H. Prucha, C. Kugler, K. Brockow, J. Ring, U. Darsow

Klinik und Poliklinik für Dermatologie und Allergologie am Biederstein, Technische Universität München

Eine 25-jährige Patientin erlitt an einem Abend im September eine anaphylaktische Reaktion mit generalisierter Urtikaria, Juckreiz der Nasen- und Mundschleimhaut, Schluckbeschwerden, Kloßgefühl im Hals und Dyspnoe. Sie wurde notfallmäßig behandelt. Das Abendessen der Patientin bestand aus einem Maronenpilz-Omelett mit Salz, Pfeffer, Eiern und selbst gesammelten Pilzen sowie Kaffee. Weitere Nahrungsmittel, die am selben Tag von der Patientin konsumiert wurden, habe sie seitdem wieder gegessen und vertragen.

Kofaktoren wie Infektionen, Alkohol oder körperliche Anstrengung waren nicht involviert.

Weiterhin sind bei der Patientin eine Ciprofloxacin-Unverträglichkeit, eine Rhinoconjunktivitis allergica saisonalis auf Gräser mit Zustand nach Hyposensibilisierung, ein atopisches Ekzem, eine Tierhaarallergie und ein orales Allergiesyndrom auf Nüsse bekannt.

Native Pricktestungen zeigten positive Ergebnisse u. a. auf das selbstgemachte Maronenpilz-Omelett, Maronenpilz, Shiitakepilz, Austernpilz und Champignon. Im Blut zeigten sich spezifische IgE-Antikörper gegen Gräser- und Birkenpollen, Katzenepithel und Haselnuss. In der oralen Provokationstestung wurden das selbstgemachte Omelett ohne Maronenpilze und die im Pricktest negativen Pilzsorten Egerlinge, Pfifferlinge und Steinpilze vertragen. Auf eine Provokation mit dem angeschuldigten sowie mit den Prick-positiven Pilzen wurde aufgrund der schweren Reaktion verzichtet. Eine Allergen-Charakterisierung im Maronenpilz wird derzeit versucht.

Aufgrund der Anamnese und des positiven Hauttests stellten wir eine Maronenpilz-Allergie fest.

Maronenpilze (Boletus badius) sind eine Pilzart aus der Familie der Dickröhrlinge. Sie sind beliebte und häufige Speisepilze in Deutschland. Eine Allergie ist nach unserer Kenntnis bisher nicht publiziert.

In den letzten Jahren wurden einige Berichte über Pilzallergien, insbesondere über Boletus edulis (Steinpilz) publiziert. In unserem Fall wurden Steinpilze gut vertragen. Eine mögliche Kreuzallergie konnte hier nicht gezeigt werden.

P05

Typ-I-Allergie auf Heparine

A. Kolbinger, R. Franz, J. Ring, U. Darsow

Klinik und Poliklinik für Dermatologie und Allergologie am Biederstein, Technische Universität München

Hintergrund: Unverträglichkeiten gegenüber Heparin sind oftmals lokale Typ-IV-Allergien. Generalisierte Typ-I-Reaktionen wurden bisher sehr selten beschrieben.

Methoden: Wir untersuchten zwei Patienten mit Soforttypreaktionen auf Mono-Embolex® (Certoparin). Es erfolgten Prick- und Intrakutantestungen mit Sofort- und Spätablesung sowie eine subkutane Provokationstestung.

Fall 1: 54-jähriger Patient mit urtikariellem Exanthem und Dyspnoe 3 Tage nach Gabe von Certoparin. Hier blieb die Pricktestung mit niedermolekularen Heparinen negativ, die Intrakutantestung auf Certoparin (1 : 10) dagegen war in der Sofortablesung positiv (Hirudin und Fondaparinux negativ). Die subkutane Provokationstestung mit einer Kumulativdosis von 0,5ml Certoparin führte nach 6h zu Urtikaria.

Fall 2: Bei der 61-jährigen Patientin traten anamnestisch wiederholt innerhalb von 30-60 min nach s.c. Gabe von Certoparin, bzw. Nadroparin (Fraxiparin®) generalisierter Juckreiz mit Dyspnoe, Quincke-Ödem und katecholaminpflichtiger Blutdruckabfall auf. In diesem Fall erwies sich bereits die Pricktestung mit Certoparin, Nadroparin und Heparin-Natrium als positiv, ebenso die Intrakutantestung (1 : 10) auf Fondaparinux und Enoxaparin. Auf die Provokation mit Certoparin wurde aufgrund der Schwere der Reaktion verzichtet.

Beide Patienten zeigten negative s.c.-Provokationstests auf Hirudin und Fondaparinux, so dass Ausweichmöglichkeiten zur Antikoagulation gefunden werden konnten; entsprechende Allergiepässe wurden ausgestellt.

Schlussfolgerungen: Selten basiert eine Heparin-Allergie auf einer Typ-I-Reaktion. Obwohl bis dato nur wenige Berichte über eine mögliche Kreuzreaktivität zu Hirudinen oder Faktor-Xa-Inhibitoren existieren, sollten diesbezüglich stets Haut- und Provokationstests erfolgen.