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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. Methods: Monocyte-derived DC and monocyte-derived macrophages were exposed to cat allergen extract or Escherichia coli. Purified monocytes were stimulated with allergen extracts from cat or house dust mite (HDM) or the major allergenic protein Fel d 1 and induction of proinflammatory cytokines by monocytes was analyzed before and after blocking CD14. Results: We show that cat allergen extract induced tumor necrosis factor (TNF) and interleukin (IL)-6 production by CD14-positive macrophages but not by CD14-negative DC. Moreover, monocytes produced significantly higher levels of TNF in response to cat allergens than in response to HDM allergens. We observed no differences in levels of TNF and IL-6 from either macrophages or monocytes after exposure to cat allergen when comparing healthy and cat-allergic individuals. Finally, the proinflammatory cytokine production from monocytes in response to cat allergen extract but not to HDM allergen was significantly reduced by blocking CD14. Conclusion: These results indicate that closely related innate immune cells from the myeloid lineage respond differentially to cat allergen extract and that the pattern-recognition receptor CD14 might be one of the mediators involved in the inflammatory responses to inhalant allergens.

Summary 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. Objective To characterize the human T cell response to antigen 5 from Vespula vulgaris, Ves v 5. Methods Recombinant Ves v 5 was used to establish allergen-specific T cell lines (TCL) and T cell clones (TCC) from the peripheral blood of vespid-allergic and non-allergic individuals. Ves v 5-specific TCL were mapped for T cell epitopes using overlapping synthetic peptides representing the complete amino acid sequence of Ves v 5. Ves v 5-specific TCC were analysed for antigen-induced secretion of IL-4, IFN-γ and IL-10. Results Seventeen distinct T cell epitopes were recognized by allergic individuals among which Ves v 5181–192 was identified as a dominant T cell epitope. Partially different epitopes were observed in TCL from non-allergic subjects and the dominant epitope Ves v 5181–192 was not prevalent in these cultures. Ves v 5-specific TCC isolated from allergic individuals did not show the typical T helper type 2 (Th2)-like cytokine profile in response to specific stimulation, i.e. high amounts of IL-4 and low IFN-γ. TCC from non-allergic individuals showed a Th1-like cytokine pattern. Conclusions Our findings provide evidence that the allergic T cell response to Ves v 5 is not Th2-dominated and that different immunogenic sites on this major wasp venom allergen are recognized by allergic and non-allergic individuals.

The clinical significance and molecular specificity of hypersensitivity reactions to raw and cooked potatoes remain ambiguous. We therefore investigated the clinical hypersensitivity to raw and cooked potato in infants suspected to have potato allergy and compared the findings with the occurrence of immunoglobulin E (IgE) antibodies to patatin (Sol t 1), characterized as the primary allergen of potato. Twelve infants (10 to 24 months of age) suffering from atopic dermatitis (AD) and suspected to have adverse reactions to potato, were examined. As a skin exposure test we used rubbing with both raw and cooked potato, and used open oral challenge with cooked potato for 7 days. A special eczema scoring system (SCORAD) was used to assess the severity of symptoms and signs of AD. Skin-prick tests (SPTs) were performed with raw potato and natural Sol t 1, and serological studies included measurement of total serum IgE and IgE antibodies to Sol t 1, and potato radioallergosorbent testing (RAST). The skin-rubbing test with raw potato was positive in seven (58%) and the oral challenge positive in eight (67%) infants. One infant presented with an immediate reaction and seven with a delayed reaction, i.e. exacerbation of AD, after oral challenge responses to cooked potato. Nine (75%) infants had IgE antibodies to Sol t 1 in enzyme-linked immunosorbent assay (ELISA), and SPT to natural Sol t 1 was positive in six (50%) potato-allergic infants. In conclusion, we observed positive challenge responses to both raw and cooked potato in food-allergic atopic infants. The presence of IgE antibodies and concomitant positive SPTs to the heat-stable potato allergen, Sol t 1, suggest that cooked potato can be an allergenic food for infants suffering from AD.

Background: We have previously identified patatin (Sol t 1) of potato tubers as a major food allergen among atopic children. In addition to Sol t 1, concomitant IgE binding to other, then unidentified, potato proteins was observed. Methods: Purification and identification of the putative allergens were done by both standard and advanced methods of protein chemistry. The patient series comprised 39 children with positive skin prick test (SPT) to raw potato. Immunoblotting and ELISA were used to examine IgE-binding ability and skin prick testing to assess in vivo reactivity of the purified potato proteins. Results: Four IgE-binding potato proteins with molecular masses ranging from 16 to 20 kDa were purified and identified as cathepsin D-, cysteine-, and aspartic protease inhibitors belonging to the family of soybean trypsin inhibitors (Kunitz type). The proteins were designated Sol t 2, Sol t 3.0101, Sol t 3.0102, and Sol t 4. In ELISA, 51% of the sera of the 39 atopic children showed specific IgE to Sol t 2, 43% to Sol t 3.0101, 58% to Sol t 3.0102, and 67% to Sol t 4, respectively. All these four allergens were able to produce positive wheal-and-flare responses in SPT. Conclusions: In addition to Sol t 1, potato tubers contain several proteins belonging to the family of soybean trypsin inhibitors against which atopic children with positive SPT responses to raw potato have in vitro and in vivo reactive IgE antibodies.

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-gamma 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.

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-beta 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.

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 ofatopic 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.(J Allergy Clin Immunol 1999;103:165-71.)

Rationale Exposure and sensitization to domestic cat (Felis domesticus) is a significant risk factor for developing allergic rhinitis and asthma. Earlier studies have shown that majority of the cat allergic patients develop allergic responses against major cat allergen Fel d 1. Several attempts have been made to produce of recombinant (r) Fel d 1 as heterodimer. We have recently shown that baculovirus expression system is able to produce rFel d 1 that has compatible characteristics to nFel d 1. Methods In the present study an immunological characterization of rFel d 1 in comparison to the purified nFel d 1 and cat allergen extract (IMP Fel d, ALK-Abello') was performed by use of IgE inhibition, lymphocyte proliferation and basophile histamine release assay(s). Results In inhibition assay against cat allergen extract, natural and rFel d 1 were able to inhibit 87% of the binding of cat allergen specific serum IgE antibodies. The basophile histamine release responses with rFel d 1 and nFel d 1 showed to be in line with the responses given by the cat allergen extract. Comparable T-cell responses towards natural and rFel d 1 was obtained in lymphocyte proliferation assay and when the response of Fel d 1-specific T-cell lines was investigated even though nFel d 1 seemed to be recognized more frequently. Conclusions These results show that heterodimeric rFel d 1 elicits immunological responses that are comparable to nFel d 1. The present study demonstrates that recombinant allergen(s) with well defined biochemical and immunological characteristics are feasible for development of therapeutic vaccines.

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.