Ulla Seppälä

Identification of patatin as a novel allergen for children with positive skin prick test responses to raw potato

BACKGROUND Peeling of raw potatoes may cause allergic symptoms, such as sneezing, wheezing, and contact urticaria, for adults. For children, potatoes as food may cause various allergic reactions. However, the identity and molecular structure of the putative allergens in potato have remained unknown. OBJECTIVE The aim of our study was to identify possible major allergens in potato by using sera of atopic children suspected of having food allergy and having positive skin prick test responses to raw potato. METHODS Potato proteins were purified by standard methods of protein chemistry and characterized with amino-terminal sequencing and mass spectrometry. The IgE-binding ability of the purified proteins was verified by immunoblotting and ELISA with IgE antibodies from the sera of atopic children having positive skin prick test responses to raw potato. RESULTS Immunoblotting showed strong IgE binding to a 43-kd protein, identified as patatin, the main storage protein of potato tubers. In ELISA, 20 of 27 (74%) children with positive skin prick test responses to potato and none of the control subjects showed specific binding of IgE antibodies to purified patatin. A positive wheal-and-flare reaction was seen in 8 of 14 children prick tested with purified patatin. CONCLUSION These results show that patatin in potatoes is a significant, previously unrecognized,IgE-binding protein for children with a positive skin prick test response to raw potato. Further studies are needed to confirm the clinical importance of potatoes, and especially patatin, as a food allergen.

Structure of recombinant Ves v 2 at 2.0 Å resolution: structural analysis of an allergenic hyaluronidase from wasp venom

Wasp venom from Vespula vulgaris contains three major allergens: Ves v 1, Ves v 2 and Ves v 5. Here, the cloning, expression, biochemical characterization and crystal structure determination of the hyaluronidase Ves v 2 from family 56 of the glycoside hydrolases are reported. The allergen was expressed in Escherichia coli as an insoluble protein and refolded and purified to obtain full enzymatic activity. Three N-glycosylation sites at Asn79, Asn99 and Asn127 were identified in Ves v 2 from a natural source by enzymatic digestions combined with MALDI-TOF mass spectrometry. The crystal structure of recombinant Ves v 2 was determined at 2.0 A resolution and reveals a central (beta/alpha)(7) core that is further stabilized by two disulfide bonds (Cys19-Cys308 and Cys185-Cys197). Based on sequence alignments and structural comparison with the honeybee allergen Api m 2, it is proposed that a conserved cavity near the active site is involved in binding of the substrate. Surface epitopes and putative glycosylation sites have been compared with those of two other major group 2 allergens from Apis mellifera (honeybee) and Dolichovespula maculata (white-faced hornet). The analysis suggests that the harboured allergic IgE-mediated cross-reactivity between Ves v 2 and the allergen from D. maculata is much higher than that between Ves v 2 and the allergen from A. mellifera.

IgE reactivity to patatin-like latex allergen, Hev b 7, and to patatin of potato tuber, Sol t 1, in adults and children allergic to natural rubber latex.

Backround: Patients allergic to natural rubber latex (NRL) frequently show positive skin prick tests (SPT) and hypersensitivity reactions to various fruits, such as avocado, banana, and kiwi, as well as to vegetables such as potato.

Allergen-specific immunosuppression by mucosal treatment with recombinant Ves v 5, a major allergen of Vespula vulgaris venom, in a murine model of wasp venom allergy.

Up to 5% of the population suffer from systemic, 19% from local allergic hypersensitivity reactions to stinging insects. Even though specific immunotherapy is very effective in treating allergy to insect venom, new concepts of treatment strategies with only the disease eliciting allergen in recombinant form, along with antigen application via a less invasive route might be suggested for enhanced treatment efficacy and compliance. In the present study we aimed (i) to establish a mouse model of wasp venom allergy, mimicking the natural mode of sensitization, and (ii) to develop a prophylactic treatment strategy based on mucosal tolerance induction, using one major wasp venom allergen in recombinant form, i.e. recombinant (r)Ves v 5. Immunization with wasp venom – with or without the use of the adjuvant aluminium hydroxide – led to comparable T helper 2‐like immune responses in vivo and in vitro. Intranasal administration of rVes v 5 prior to sensitization with wasp venom resulted in a significant reduction of wasp venom‐specific antibody levels (immunoglobulin E (IgE)/IgG2a), type I hypersensitivity reactions in vivo and cytokine production in vitro. Pretreatment with the whole venom was less effective and caused toxic side reactions in higher concentrations, suggesting a favourable use of the recombinant venom allergen for mucosal application. Increased mRNA levels of transforming growth factor‐β and interleukin‐10, along with adoptive cell transfer experiments indicated that the immunosuppression after intranasal rVes v 5‐application has been mediated by regulatory mechanisms. This is further supported by the fact that the immunosuppression to rVes v 5 was associated with a bystander suppression to the unrelated aero‐allergen Bet v 1. In conclusion, we demonstrated that the intranasal application of recombinant Ves v 5 prevented subsequent allergic sensitization to all components of the whole wasp venom. As allergy to insect venom develops in dependence of the frequency of insect stings, a prophylactic treatment based on mucosal tolerance induction with recombinant allergens might be of interest for people at high risk to frequent exposure to the stinging insects.

Structural and immunological characterization of the N-glycans from the major yellow jacket allergen Ves v 2: the N-glycan structures are needed for the human antibody recognition.

Yellow jacket (Vespula vulgaris) hyaluronidase (Ves v 2) is a glycoprotein and a mixture of two isoallergens, Ves v 2.01 and Ves v 2.02. Wasp and bee sensitized individuals frequently show IgE antibodies that in vitro recognize common carbohydrate structures across the hymenoptera species. The aim of the study was to characterize the glycosylation patterns in Ves v 2 isoallergens and to assess their immunological properties regarding antibody binding and T cell activation. The glycosylation sites and the carbohydrate structures were verified by use of tandem mass spectrometry (MS/MS). The immunological characterization of the N-glycan structures was assessed by antibody binding, T cell proliferation and T cell epitope assays comparing native (n) and non-glycosylated recombinant (r) Ves v 2. Analyses of the Ves v 2 glycopeptides revealed that glycan attachments were found for residues 79, 99 and 127 of Ves v 2.01, and residues 66 and 81 of Ves v 2.02. Structural analysis of the glycopeptides showed that the majority of the N-glycans contained at least one alpha1,3-fucose and/or alpha1,6-fucose residues in a structure. Interestingly, serum IgE antibodies from vespid allergic patients recognized nVes v 2 but not rVes v 2. Non-glycosylated rVes v 2, however, induced T cell and cytokine responses comparable to glycosylated nVes v 2. The present study shows that N-glycan structures are needed for the antibody recognition but not for the T cell reactivity of Ves v 2 in vitro. The occurrences of carbohydrate-specific antibodies against nVes v 2, however, suggest that non-mammalian glycan structures as in nVes v 2 may provide a link between T cells and other effector cells in allergic responses.

Characterization of the human T cell response to antigen 5 from Vespula vulgaris (Ves v 5)

Background The T cell reactivity to the major allergen of bee venom, phospholipase A2, has been thoroughly characterized. In contrast, only little is known about the human cellular response to major allergens from wasp venom.

Clinical characterization and IgE profiling of birch (Betula verrucosa)--allergic individuals suffering from allergic reactions to raw fruits and vegetables.

BACKGROUND Hypersensitivity to raw fruits and vegetables is often associated with respiratory allergy to birch (Betula verrucosa) pollen and is considered to be the most prevalent form of food allergy in adults sensitized to birch pollen. OBJECTIVE The aim of the study was to investigate the association of clinical allergy and IgE profiles in individuals with birch pollen allergy and hypersensitivity to raw fruits and vegetables. METHODS A total of 59 adults with clinical and skin prick test confirmed birch pollen allergy were included in the study. All the subjects were interviewed by using a structured questionnaire and were examined in vivo by the open test, with the appropriate fruits and vegetables. ImmunoCAP and ImmunoCAP ISAC were used as in vitro diagnostics to assess sensitization profiles for each individual, and principal components analysis was used to analyze the IgE data sets. RESULTS Of 59 individuals, 54 (92%) had positive prick-prick test with raw potato, carrot, apple, and/or hazelnut, and the skin prick test was always positive when the corresponding skin challenge was defined as positive. Specific IgE in the ImmunoCAP and inhibition assays with rMal d 1 and rBet v 1 demonstrated that Bet v 1 is driving the sensitization against pathogenesis related-10 proteins. However, positive IgE in vitro results could not be used to predict clinical reactivity to raw fruits and vegetables. CONCLUSIONS The present study showed that component-based IgE profiling does not enhance the diagnostic potential in case of pollen-food syndrome, which may be associated with other as yet unidentified components.

Activation of human neonatal monocyte-derived dendritic cells by lipopolysaccharide down-regulates birch allergen-induced Th2 differentiation

Epidemiological studies describe an inverse association between the level of environmental endotoxin exposure during infancy and the prevalence of allergic disease in children. To study the effect of lipopolysaccharide (LPS) and lipopeptide Pam3Cys signaling via Toll‐like receptor (TLR)4 and TLR2 on dendritic cells (DC), respectively, on birch allergen‐induced T cell differentiation, cord blood monocyte‐derived DC were exposed to birch allergen extract alone or in combination with LPS or Pam3Cys and thereafter co‐cultured with naive autologous T cells. We demonstrate that birch allergen alone induced high levels of IL‐13 from neonatal T cells, whereas the production of IL‐5 and IFN‐γ was modest. Stimulation of DC with birch allergen together with LPS but not Pam3Cys resulted in a decreased IL‐13 production by T cells compared to birch allergen alone. Furthermore, birch allergen together with LPS induced increased up‐regulation of activation markers expressed on the surface and production of cytokines from DC relative to stimulation with birch allergen alone. Finally, birch allergen partially suppressed both LPS‐ and Pam3Cys‐induced DC maturation. Our results indicate that concomitant TLR4 stimulation during the initial phase of immune activation to birch allergen in infants may inhibit the development of a T helper 2‐type response.

In situ imaging of honeybee (Apis mellifera) venom components from aqueous and aluminum hydroxide-adsorbed venom immunotherapy preparations

BACKGROUND Treatment with aqueous and aluminum hydroxide (Al[OH](3))-adsorbed purified honeybee (Apis mellifera) venom (HBV) preparations can reduce the incidence of side effects associated with venom immunotherapy. OBJECTIVE The aim of the present study was to assess these purified HBV immunotherapy preparations in situ. METHODS Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) was used to visualize the distribution of HBV components. The preparations were administered on the back legs of naive Wistar rats. The rats were killed, and cryosectioned tissue sections were subjected to hematoxylin and eosin staining and MALDI-MSI analyses. RESULTS Low-density maps of tissue distribution of HBV peptides, such as secapin, mast cell degranulating peptide, and melittin (Api m 4) were detected in the tissue after administration of HBV immunotherapy preparations. In addition, release of biogenic amines, cytokines, and leukotrienes was observed, and the distribution of HBV allergens, such as Api m 1 and Api m 2, was shown. At the 24-hour time point, the major HBV allergen Api m 1 was still detected at the site of Al(OH)(3)-adsorbed HVB injection, whereas in the case of aqueous HBV preparation, all the allergens, as well as most of the biogenic amines, were cleared at the 24-hour time point. CONCLUSION The present study shows that the majority of low-molecular-weight HBV components are rapidly removed from the site of venom immunotherapy administration. Furthermore, Al(OH)(3)-adsorbed HBV preparation demonstrated a depot effect, prolonging the availability of bee venom allergens at the site of administration.

Cat allergen induces proinflammatory responses by human monocyte-derived macrophages but not by dendritic cells.

Background:  The upper airway mucosa of healthy humans contains a dense network of cells with dendritic morphology of which the majority express a macrophage‐like phenotype (CD14+CD64+CD68+), whereas the smaller population are immature dendritic cells (DC; CD11c+CD14−). Our aim was to study the proinflammatory response of human monocytes and in vitro‐generated macrophages and DC after contact with cat allergens.