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The Journal of allergy and clinical immunology 03/2013; · 9.17 Impact Factor
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ABSTRACT: Although rice (Oryza sativa) is one of the most common cereals produced and consumed around the world, there have been only a few reports on immediate hypersensitivity reactions after ingestion of rice. Few clinical studies on rice allergy in Asia have been reported concerning rhinitis, asthma and atopic dermatitis. In this case study, we identify allergens presumably responsible for anaphylaxis after ingestion of rice in a German patient.
Prick-to-prick tests, determination of specific IgE and the basophil activation test (BAT) were performed to confirm IgE-mediated allergy. IgE reactivity was further analyzed by immunoblotting of protein extracts from cooked commercial rice products. Rice allergens were purified, subjected to N-terminal sequencing and characterized by IgE binding and IgE inhibition assays using additional sera from 8 subjects with sensitization to rice and/or a history of hypersensitivity symptoms after rice ingestion.
Prick-to-prick tests were positive to raw and cooked rice (basmati rice and long-grain rice) and preparations of different rice extracts. Specific IgE against rice (f9) was 1.87 kU(A)/l. The BAT showed specific IgE-mediated activation of basophils after stimulation with rice extracts. Four IgE-reactive rice proteins with an apparent molecular weight of 49, 52, 56 and 98 kDa were identified. Interestingly, only binding to the 56-kDa glycoprotein was at least partially independent from cross-reactive carbohydrate determinants (CCD), whereas IgE binding to the other rice proteins was completely inhibited by pre-incubation with the CCD MUXF derived from bromelain.
Yet unidentified high-molecular-weight allergens from rice seeds, predominantly a 56-kDa glycoprotein, seem to be responsible for anaphylaxis after consumption of rice in a German patient.
International Archives of Allergy and Immunology 12/2011; 158(1):9-17. · 2.40 Impact Factor
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ABSTRACT: Food allergy is caused by primary (class 1) food allergens, e.g. Bos d 5 (cow's milk) and Cor a 8 (hazelnut) or secondary (class 2) food allergens, e.g. Mal d 1 (apple). The latter cannot sensitize susceptible individuals but can cause allergy due to immunological cross-reactivity with homologous respiratory allergens. Here, we studied the effects of food matrix on gastrointestinal proteolysis, epithelial transport and in vivo absorption of class 1 and class 2 food allergens.
Mal d 1 lost its IgE-reactivity immediately after simulated gastric digestion whereas Bos d 5 and Cor a 8 did not. Only Cor a 8 maintained IgE-binding capacity after simulated intestinal proteolysis. The presence of hazelnut and peanut extracts, which served as protein-rich model food matrices, delayed gastrointestinal degradation and reduced epithelial transport rates of all allergens through CaCo-2 monolayers. Finally, IgE-reactive allergens were assessed at different time points in sera from rats fed with all three allergens with or without hazelnut extract. The levels of all allergens peaked 2 h after animals were fed without matrix and increased over 8 h after feeding.
A protein-rich food matrix delays gastrointestinal digestion and epithelial transport of food allergens and thereby may affect their sensitizing capacity and clinical symptoms.
Molecular Nutrition & Food Research 10/2011; 55(10):1484-91. · 4.30 Impact Factor
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ABSTRACT: The Toll-like receptor (TLR) 5 agonist flagellin is associated with immunomodulatory functions.
We sought to investigate whether Listeria monocytogenes-derived flagellin A (flaA) can modulate ovalbumin (OVA)-specific T-cell responses and prevent OVA-induced intestinal allergy.
Bone marrow-derived myeloid dendritic cells from BALB/c, C57BL/6, or TLR signaling-deficient (MyD88(-/-)) mice were stimulated with rOVA, rflaA, rflaA plus rOVA, or a recombinant fusion protein consisting of rflaA and rOVA (rflaA:OVA). The immunomodulating properties of rflaA plus rOVA and rflaA:OVA were investigated by means of DC-T-cell coculture with CD4(+) T cells from OVA-T-cell receptor transgenic or OVA/alum-immunized mice. rflaA:OVA was applied as a prophylactic and therapeutic vaccine in a murine model of intestinal allergy.
rflaA:OVA induced upregulation of TLR5 and dose-dependent IL-6 and IL-10 secretion by myeloid dendritic cells. IL-10 contributed to repressing IL-4 and IFN-γ secretion by OVA-T-cell receptor transgenic CD4(+) T cells. Moreover, rflaA:OVA suppressed CD4(+) T cells derived from T(H)2-biased mice on OVA/alum immunization. In a murine model of intestinal allergy, prophylactic vaccination with rflaA:OVA reduced T-cell activation. Protection from intestinal allergy included suppression of OVA-specific IgE while inducing OVA-specific IgG(2a). Equimolar amounts of rflaA or rOVA provided alone or as a mixture did not have comparable effects. Moreover, therapeutic vaccination was shown to reduce allergic symptoms and T-cell activation in the spleen.
The rflaA:OVA fusion protein showed strong TLR-mediated immunomodulating capacities probably attributed by the proximity of adjuvant and allergen, leading to the prevention of intestinal allergy in a murine disease model. Therefore recombinant flaA:allergen fusion proteins are promising vaccine candidates for intervention in patients with IgE-mediated allergy.
The Journal of allergy and clinical immunology 08/2011; 128(6):1340-1348.e12. · 9.17 Impact Factor
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ABSTRACT: Pathogenesis-related protein-10 (PR10) is a ubiquitous small plant protein induced by microbial pathogens and abiotic stress that adversely contributes to the allergenic potency of many fruits and vegetables, including carrot. In this plant, two highly similar genes encoding PR10 isoforms have been isolated and designated as allergen Dau c 1.01 and Dau c 1.02. The aim of the study was to generate PR10-reduced hypoallergenic carrots by silencing either one of these genes in transgenic carrots by means of RNA interference (RNAi). The efficiency of gene silencing by stably expressed hairpin RNA (hnRNA) was documented by means of quantitative RT-PCR (qPCR) and immunoblotting. Quantification of the residual protein revealed that PR10 accumulation was strongly decreased compared with untransformed controls. Treatment of carrot plants with the PR protein-inducing chemical salicylic acid resulted in an increase of PR10 isoforms only in wild-type but not in Dau c 1-silenced mutants. The decrease of the allergenic potential in Dau c 1-silenced plants was sufficient to cause a reduced allergenic reactivity in patients with carrot allergy, as determined with skin prick tests (SPT). However, simultaneous silencing of multiple allergens will be required to design hypoallergenic carrots for the market. Our findings demonstrate the feasibility of creating low-allergenic food by using RNAi. This constitutes a reasonable approach to allergen avoidance.
Transgenic Research 06/2011; 20(3):547-56. · 2.75 Impact Factor
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Masako Toda,
Gerald Reese,
Gabriele Gadermaier,
Veronique Schulten,
Iris Lauer,
Matthias Egger,
Peter Briza,
Stefanie Randow,
Sonja Wolfheimer,
Valencia Kigongo,
Maria Del Mar San Miguel Moncin,
Kay Fötisch,
Barbara Bohle,
Stefan Vieths, Stephan Scheurer
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ABSTRACT: Allergen-specific immunotherapy for food allergies, including peach allergy, has not been established. Use of allergens with reduced allergenic potential and preserved immunogenicity could improve the safety and efficacy of allergen-specific immunotherapy.
We sought to create a hypoallergenic derivative of the major peach allergen Pru p 3 and to characterize its biochemical and immunologic properties.
A Pru p 3 folding variant generated by means of reduction and alkylation was investigated for structural integrity and stability to gastrointestinal enzymes. IgE reactivity and allergenic potency were determined by means of immunoblotting, ELISA, and in vitro mediator release assay with sera from patients with peach allergy. T-cell immunogenicity was investigated by using human allergen-specific T cells and CBA/J mice immunized with either native Pru p 3 (nPru p 3) or reduced and alkylated (R/A) Pru p 3. Pru p 3 processing by endolysosomal fractions of dendritic cells and antigenicity was examined in mice.
Unfolding of Pru p 3 reduced its high resistance to gastrointestinal proteolysis and almost completely abrogated its IgE reactivity and allergenic potency. However, R/A Pru p 3 was capable of stimulating human and murine T cells. Endolysosomal degradation of R/A Pru p 3 was accelerated in comparison with nPru p 3, but similar peptides were generated. IgG and IgE antibodies raised against nPru p 3 showed almost no cross-reactivity with R/A Pru p 3. Moreover, the antigenicity of R/A Pru p 3 was strongly reduced.
Unfolded Pru p 3 showed reduced allergenicity and antigenicity and preserved T-cell immunogenicity. The hypoallergenic variant of Pru p 3 could be a promising vaccine candidate for specific immunotherapy of peach allergy.
The Journal of allergy and clinical immunology 05/2011; 128(5):1022-30.e1-7. · 9.17 Impact Factor
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Lien Q Le,
Vera Mahler, Stephan Scheurer,
Kay Foetisch,
Yvonne Braun,
Daniela Weigand,
Ernesto Enrique,
Jonas Lidholm,
Kathrin E Paulus,
Sophia Sonnewald,
Stefan Vieths,
Uwe Sonnewald
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ABSTRACT: Gene silencing of Lyc e 1 leads to reduced allergenicity of tomato fruits but impaired growth of transgenic tomato plants. The aim of the study was to restore growth of Lyc e 1-deficient tomato plants while retaining reduced allergenicity by simultaneous complementation of profilin deficiency by expression of nonallergenic yeast profilin. Transgenic plants were generated and tested by RT-PCR and immunoblotting; allergenicity of yeast profilin and transgenic fruits was investigated by IgE binding, basophil activation, and skin-prick tests. Lyc e 1 content of transgenic tomato fruits was <5% of that of wild-type plants, causing significantly reduced IgE antibody binding. Simultaneous coexpression of yeast profilin restored growth and biomass production almost to wild-type levels. Yeast profilin, sharing 32.6% amino acid sequence identity with Lyc e 1, displayed low IgE-binding capacity and allergenic potency. Among 16 tomato-allergic patients preselected for sensitization to Lyc e 1, none showed significant reactivity to yeast profilin. Yeast profilin did not induce mediator release, and coexpression of yeast profilin did not enhance the allergenicity of Lyc e 1-reduced fruits. Simultanous coexpression of yeast profilin allows silencing of tomato profilin and generation of viable plants with Lyc e 1-deficient tomato fruits. Therefore, a novel approach to allergen avoidance, genetically modified foods with reduced allergen accumulation, can be generated even if the allergen fulfills an essential cellular function in the plant. In summary, our findings of efficiently complementing profilin-deficient tomato plants by coexpression of low allergenic yeast profilin demonstrate the feasibility of creating low-allergenic food even if the allergen fulfills essential cellular functions.
The FASEB Journal 12/2010; 24(12):4939-47. · 5.71 Impact Factor
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ABSTRACT: Toll-like receptor ligands are immune-modulatory components linking innate and adaptive immune responses and are considered to be promising vaccine components. Objective of this study was to investigate the adjuvant activity of Listeria monocytogenesis-derived TLR5-ligand flagellin A (flaA) genetically fused to ovalbumin (Ova, major chicken white egg allergen) in a murine in vitro system. Recombinant flaA, rOva, and a fusion protein of rflaA and rOva (rflaA:Ova) were over-expressed in Escherchia coli and purified by FPLC. LPS depletion was confirmed by LAL test. TLR5-binding was evaluated by human and murine TLR5-transgenic HEK 293 cells. The immune-modulatory effect of rflaA:Ova and rflaA:Ova modified by reduction and alkylation on purified BALB/c bone marrow-derived myeloid (mDC) and plasmacytoid dendritic cells (pDC) was investigated by flow cytometry and intracellular cytokine staining (ICS). Dose-dependent IL-8 secretion from transgenic HEK 293 cells confirmed binding of rflaA and rflaA:Ova molecules to human and murine TLR5. Recombinant flaA showed similar biological reactivity to TLR5-ligand fliC derived from Salmonella typhimurium applied as positive control. Compared to rflaA, both rflaA:Ova preparations induced higher expression of maturation markers (CD40, CD69, CD80, and CD86) on mDC, whereas only CD69 and CD40 were upregulated on pDC. Moreover, IL-6 and IL-10 production by mDC was enhanced upon stimulation with rflaA:Ova constructs in comparison to an equimolar mixture of both proteins whereas pDC did not show secretion of the investigated cytokines. Any immunological effects of LPS can be excluded by depletion of endotoxins and the lack of IL-10 production upon proteinase K digestion of rflaA:Ova. In summary, the rflaA:Ova fusion proteins showed an enhanced immune modulating capacity in comparison to rflaA or the mixture of rflaA and antigen. Since the rflaA:Ova fusion proteins induce strong IL-10 induction they are considered as potential vaccine candidates to improve allergen-specific immunotherapy.
Molecular Immunology 10/2010; 48(1-3):341-50. · 2.90 Impact Factor
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ABSTRACT: Green bean (GB) has been reported to cause allergic reactions after ingestion, contact or inhalation of particles deriving from processing or cooking. Up-to-date no food allergens have been fully characterized in GB.
To characterize the GB major allergen(s) on a molecular level and to verify the involvement of non-specific lipid transfer proteins (nsLTPs) in GB allergy.
We recruited 10 Spanish patients reporting adverse reactions to GB. Skin prick tests, specific IgE detection and oral provocation were performed. Two nsLTP cDNAs were cloned from GB and over-expressed in Pichia pastoris. The recombinant LTPs (rLTPs) were characterized by circular dichroism spectroscopy and IgE-binding assays (immunoblotting and ELISA) with the patients' sera. Three natural LTPs (nLTPs) were further purified from GB fruit by chromatography. In vitro histamine release test was applied to compare the allergenic potency of rLTPs and nLTPs.
Oral provocation test confirmed GB allergy. A 10kDa protein in GB extract was recognized by 80% of the sera and identified as nsLTP. The two rLTPs (named LTP1a and LTP1b), share 61.3% aa identity and present the typical nsLTP-like secondary structure. The IgE-binding and histamine release assays provided evidence that rLTPs and nLTPs possess different allergenic potency.
nsLTP (Pha v 3) is the major allergen in GB and constitute a potential risk for patients affected by LTP-syndrome. GB encodes for several LTPs with different immune reactivity.
Molecular Immunology 02/2010; 47(7-8):1561-8. · 2.90 Impact Factor
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ABSTRACT: Whether the observed clinical pattern of non-specific lipid transfer protein (nsLTP)-mediated food allergies is attributable to a primary sensitization by Pru p 3 from peach and subsequent cross-reactivity with Rosaceae- and non-Rosaceae-derived foods expressing homologous allergens is still unclear.
To investigate the allergenic properties of nsLTPs from Rosaceae and non-Rosaceae foods.
In peach-, cherry- or hazelnut-allergic patients, prevalence of sensitization, IgE-binding capacity, cross-reactivity and allergenic potency of Pru p 3 was compared with Pru av 3 (cherry) and Cor a 8 (hazelnut).
Frequency of sensitization to corresponding nsLTPs was 88, 85, and 77% in peach-, hazelnut- and cherry-allergic patients, respectively. Concomitant allergic reactions to cherry and hazelnut were reported in 51 and 44% of peach-allergic patients, respectively. In contrast to cherry allergy, hazelnut allergy was not strictly associated to peach allergy. Sensitization to Cor a 8 or Pru av 3 was strongly correlated with IgE reactivity to Pru p 3, even when subjects tolerated peach. Specific IgE was highest for Rosaceae LTPs, and cross-inhibition experiments confirmed a stronger IgE-binding capacity of Pru p 3 than Cor a 8. The biological potency of Pru p 3 and Pru av 3 was similar but stronger for both nsLTPs than that of Cor a 8.
Clinical cross-reactivity of food-allergic patients in the Mediterranean area is likely attributed to a primary sensitization to Pru p 3 and serological cross-reactivity with homologous food nsLTPs. In comparison to Cor a 8, Rosaceae nsLTPs showed a stronger IgE-binding capacity and allergenic potency indicating a different epitope pattern.
International Archives of Allergy and Immunology 01/2010; 153(4):335-46. · 2.40 Impact Factor
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ABSTRACT: : An IgE-mediated allergy against a lipid-transfer protein of grapes was the cause of repeated severe anaphylaxis in a patient after consumption of grapes, wine, and raisins.
: Although the patient was aware of her grape allergy, avoidance proved difficult and accidental anaphylaxis occurred. Furthermore, wine allergy in a wine-growing district means a non-negligible restriction of quality of life.
: Although there is little data on specific oral tolerance induction (SOTI) in lipid-transfer protein (LTP) allergy, SOTI with increasing doses starting from approximately 20 mg of grapes was done. For follow-up, skin tests, grape-specific IgE and IgG4, basophil activation tests, and immunoblotting were performed.
: Within 3 days the patient reached tolerance to the daily maintenance dose of 20 g of grapes (about 3 grape pieces) without anaphylaxis symptoms. Two months later, a controlled challenge with a total of 66.5 mL of white wine was tolerated. Grape-specific IgE stayed stable at 2.37 kU/L (class 2) and grape-specific IgG4 was first detectable 21 months after SOTI. Prick-to-prick skin tests continued to be positive to grapes, to raisins, and to white and red wine. The basophil activation test still showed strong IgE-mediated activation of basophils after stimulation with grape extract. Immunoblotting still detected IgE binding to a 8-kDa protein.
: We performed SOTI in a patient with severe IgE-mediated allergy against the LTP Vit v 1 of grapes and reduced the risk of anaphylaxis because of accidental intake of any kind of grapes. However, underlying mechanisms of SOTI and maintenance of the established tolerance are still not known.
World Allergy Organization Journal 01/2010; 3(1):1-5.
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Anne Ilchmann,
Sven Burgdorf, Stephan Scheurer,
Zoe Waibler,
Ryoji Nagai,
Anne Wellner,
Yasuhiko Yamamoto,
Hiroshi Yamamoto,
Thomas Henle,
Christian Kurts,
Ulrich Kalinke,
Stefan Vieths,
Masako Toda
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ABSTRACT: The Maillard reaction occurs between reducing sugars and proteins during thermal processing of foods. It produces chemically glycated proteins termed advanced glycation end products (AGEs). The glycation structures of AGEs are suggested to function as pathogenesis-related immune epitopes in food allergy.
This study aimed at defining the T-cell immunogenicity of food AGEs by using ovalbumin (OVA) as a model allergen.
AGE-OVA was prepared by means of thermal processing of OVA in the presence of glucose. Activation of OVA-specific CD4(+) T cells by AGE-OVA was evaluated in cocultures with bone marrow-derived murine myeloid dendritic cells (mDCs) as antigen-presenting cells. The uptake mechanisms of mDCs for AGE-OVA were investigated by using inhibitors of putative cell-surface receptors for AGEs, as well as mDCs deficient for these receptors.
Compared with the controls (native OVA and OVA thermally processed without glucose), AGE-OVA enhanced the activation of OVA-specific CD4(+) T cells on coculture with mDCs, indicating that the glycation of OVA enhanced the T-cell immunogenicity of the allergen. The mDC uptake of AGE-OVA was significantly higher than that of the controls. We identified scavenger receptor class A type I and II (SR-AI/II) as a mediator of the AGE-OVA uptake, whereas the receptor for AGEs and galectin-3 were not responsible. Importantly, the activation of OVA-specific CD4(+) T cells by AGE-OVA was attenuated on coculture with SR-AI/II-deficient mDCs.
SR-AI/II targets AGE-OVA to the MHC class II loading pathway in mDCs, leading to an enhanced CD4(+) T-cell activation. The Maillard reaction might thus play an important role in the T-cell immunogenicity of food allergens.
The Journal of allergy and clinical immunology 10/2009; 125(1):175-83.e1-11. · 9.17 Impact Factor
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ABSTRACT: Non-specific lipid-transfer proteins (nsLTP) from food and pollen are clinically important allergens, especially in patients recruited from the Mediterranean area. For the use of recombinant nsLTPs in allergy diagnosis and preclinical allergy studies the preparation of nsLTPs in a properly folded and biologically active form is required. Using hazelnut nsLTP (Cor a 8) as a model allergen, heterologous over-expression in Escherichia coli and Pichia pastoris was compared. Recombinant Cor a 8 derived from E. coli and P. pastoris was purified by IMAC and SEC or ammonium sulphate precipitation followed by IEC and SEC, respectively. The recombinant proteins were characterized with regard to IgE-binding by immunoblotting and ELISA, structure by N-terminal sequencing, CD-spectroscopy and LS and to their biological activity using an in vitro basophil histamine release assay. Purification of hazelnut nsLTP from bacterial lysate under native conditions resulted in a low yield of Cor a 8. In addition, the preparation contained non-IgE-reactive aggregations besides the IgE-reactive monomer. In contrast, the yield of rCor a 8 produced in P. pastoris was approximately 270-fold higher and impurities with oligomers have not been detected. Purified monomeric Cor a 8 from bacteria and yeast showed similar IgE-antibody reactivity and secondary structures, and both were capable of inducing histamine release from basophils. In summary, P. pastoris is superior to E. coli as expression system for the production of large quantities of soluble, properly folded, and biologically active rCor a 8.
Protein Expression and Purification 10/2009; 69(1):68-75. · 1.59 Impact Factor
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Véronique Schulten,
Astrid Radakovics,
Christina Hartz,
Adriano Mari,
Sonia Vazquez-Cortes,
Montserrat Fernandez-Rivas,
Iris Lauer,
Beatrice Jahn-Schmid,
Thomas Eiwegger, Stephan Scheurer,
Barbara Bohle
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ABSTRACT: Pru p 3, the nonspecific lipid transfer protein from peach, is an important plant food allergen that frequently induces systemic reactions.
We sought to analyze the allergic T-cell response to Pru p 3.
PBMCs from Italian and Spanish patients with peach allergy were stimulated with purified natural Pru p 3. Allergen-specific T-cell lines were used to identify T-cell epitopes of Pru p 3. Pru p 3-specific T-cell clones (TCCs) were analyzed for allergen-induced secretion of IL-4, IFN-gamma, and IL-10 and expression of the integrin beta7, a receptor critical for gut homing.
No difference in T-cell responses of Italian and Spanish patients was found. Among several T cell-activating regions, Pru p 3(13-27), Pru p 3(34-48), Pru p 3(43-57), and Pru p 3(61-75) were most frequently recognized in 18 Pru p 3-specific T-cell lines. The majority of 32 Pru p 3-specific TCCs belonged to the T(H)2 subset. In contrast to TCCs specific for other plant food and pollen allergens, only a limited number of Pru p 3-specific TCCs produced significant amounts of IL-10. The expression of integrin beta7 on Pru p 3-specific TCCs was comparable with that observed on peanut-specific TCCs and higher compared with that seen in different pollen-specific TCCs.
The T-cell response to Pru p 3 is dominated by T(H)2 cells presumably primed in the gut. The identification of relevant T cell-activating regions provides a basis for engineering hypoallergenic variants of Pru p 3 with less IgE binding and retained T-cell stimulatory capacity for safe immunotherapy of peach allergy.
The Journal of allergy and clinical immunology 05/2009; 124(1):100-7. · 9.17 Impact Factor
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ABSTRACT: Allergen microarrays are under development for a component-resolved diagnosis of Type I (IgE-mediated) allergies. Here we report an improved microarray coupled to microfluidics for the detection of allergen specific immunoglobulin E (IgE). The signal intensity for IgE detection in serum has been improved by using glass slides coated with a novel poly[DMA-co-NAS] brush copolymer which is able to immobilize allergens in their native conformation and by carrying out the incubation step in dynamic conditions. The assay, fully automated, was performed in a microcell, using a software-controlled fluidic processor, to bring assay reagents on the surface of the array. Microfluidics turns the binding between serum immunoglobulins and immobilized allergens from a diffusion-limited to a kinetic-limited process by ensuring an efficient mixing of serum samples on the surface of the microarray. As a result of this, the binding of high affinity IgE antibodies is enhanced whereas that of low affinity IgG antibodies, which are present at higher concentration, is impaired paving the way to more accurate and sensitive results.
Proteomics 04/2009; 9(8):2098-107. · 4.43 Impact Factor
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ABSTRACT: Food allergies are a major health concern in industrialized countries. Since a specific immunotherapy for food allergies is not available in clinical routine praxis till now, reduction of allergens in foods, either by food processing or genetic engineering are strategies to minimize the risk of adverse reactions for food allergic patients. This review summarizes biotechnological approaches, especially the RNA interference (RNAi) technology, for the reduction of selected allergens in plant foods. So far, only a limited number of reports showing proof-of-concept of this methodology are available. Using RNAi an impressive reduction of allergen accumulation was obtained which was stable in the next generations of plants. Since threshold doses for most food allergens are not known, the beneficial effect has to be evaluated by oral challenge tests in the future. The article critically addresses the potential and limitations of genetic engineering, as well as of alternative strategies to generate "low allergic" foods.
Frontiers in Bioscience 02/2009; 14:59-71. · 3.52 Impact Factor
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The Journal of allergy and clinical immunology 10/2008; 122(6):1229-31. · 9.17 Impact Factor
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ABSTRACT: Several hazelnut allergens with different clinical relevance and crossreactive properties have been identified and characterized so far. The aim of this study was to develop protocols for producing relatively large amounts of three recombinant hazelnut allergens Cor a 1.04, Cor a 2, and Cor a 8 in a folded and immunologically active form. The availability of well-characterized, pure recombinant allergens will improve diagnostic in vitro tests for food allergy, by allowing a highly sensitive component resolved diagnosis. Depending on the individual hazelnut allergen, protocols for heterologous production - either as fusion or nonfusion protein - were developed to obtain homogenous protein batches. The resulting proteins were purified by a two-step FPLC method and their IgE antibody reactivity was verified. Identity was verified by N-terminal sequencing and MALDI-TOF-MS analysis. Their secondary and tertiary structure was controlled by circular dichroism (CD)-spectroscopy and NMR analysis. Decisions on the strategies for expression and purification of allergens on a large scale were made on a case by case basis: Preparation of rCor a 1.04 and rCor a 2 as fusion proteins in E. coli from inclusion bodies resulted in approximately 10 mg pure protein per liter whereas rCor a 8 expression in yeast as nonfusion protein yielded 30 mg/L.
Molecular Nutrition & Food Research 09/2008; 52 Suppl 2:S262-71. · 4.30 Impact Factor
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ABSTRACT: IgE sensitisation to non-specific lipid transfer proteins (nsLTP), e.g., Pru p 3 the major allergen from peach and most important allergenic LTP, is strongly associated with severe symptoms in food allergic patients. Lac s 1, a member of the nsLTP protein family, was recently identified as major allergen in lettuce (Lactuca sativa), but has not yet been investigated on the molecular basis.
Molecular characterisation and immunological comparison of Lac s 1 to peach allergen Pru p 3.
Lac s 1 cDNA was cloned by RT-PCR and natural (n) Lac s 1 was purified by a two-step chromatography. Protein structure was verified by N-terminal sequencing, mass spectrometry, and circular dichroism spectroscopy. Immunoblotting, ImmunoCAP, and competitive IgE binding experiments were performed to study the IgE sensitisation pattern and cross-reactivity with Pru p 3. Allergenic potency was analysed by histamine release assay.
Twenty-nine lettuce allergic patients, with or without concomitant peach allergy, and 19 peach allergic patients without lettuce allergy were included in this study. IgE reactivity to lettuce was due to mono-sensitisation to Lac s 1 or cross-reactive glycan structures. Two Lac s 1 isoforms were identified which showed amino acid identity (aa-id) of 62% to each other, up to 66% to Pru p 3, and 72% to the N-terminal peptide of plane pollen LTP Pla a 3. The prevalence of IgE binding to nLac s 1 was 90% using lettuce extract in immunoblotting experiments. Enhanced sensitivity was observed in ImmunoCAP using purified nLac s 1 in comparison to extracts (93% versus 76%). Although IgE sensitisation to Lac s 1 and Pru p 3 was strongly associated, the two LTPs showed different IgE binding properties. Sensitisation to LTPs does not necessarily reflect the clinical disease, but Lac s 1 was capable of triggering histamine release as shown by positive skin test results in Lac s 1 mono-sensitised patients and by in vitro mediator release assays.
Purified nLac s 1 will enhance the sensitivity in component resolved diagnosis of lettuce allergy. Similar to other cross-reactive food allergies, exclusive testing of IgE reactivities to LTP cannot be used as biomarker for clinical relevance. Our data provide indirect evidence that Pru p 3 might act as the primary sensitising agent in patients allergic to both lettuce and peach.
Molecular Immunology 05/2007; 44(11):2820-30. · 2.90 Impact Factor
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ABSTRACT: Lipid transfer proteins (LTPs) are relevant allergens in certain plants. The role of the LTP of Hevea brasiliensis in the latex-fruit syndrome is widely unknown.
To study IgE reactivity with recombinant Hevea LTP in sera of fruit-allergic adults with and without natural rubber latex (NRL) allergy.
An LTP-specific complementary DNA of H brasiliensis leaves was amplified, subcloned into the pMAL expression system, and analyzed. The recombinant protein was coupled to ImmunoCAP, and the IgE-binding properties were studied in sera of 10 NRL-allergic patients without symptoms to fruit and 48 atopic patients with fruit allergy. Eleven of these 48 patients were also allergic to NRL, 14 displayed sensitization to NRL without symptoms on NRL exposure so far, and 23 had neither symptoms nor IgE antibodies to NRL.
After expression in Escherichia coli, a soluble maltose-binding protein-rHev b 12 fusion protein was isolated and coupled to ImmunoCAP to determine rHev b 12 specific IgE reactivity. rHev b 12 specific IgE binding was found in 3 fruit-allergic patients with NRL sensitization (0.68, 0.88, and 0.96 kU/L) and in 3 fruit-allergic patients without NRL sensitization (1.58, 2.25, and 2.27 kU/L). The remaining 52 serum samples and all maltose-binding protein control test results were negative (< 0.35 kU/L).
In these patients, rHev b 12 specific IgE reactivity seems to result from common cross-reactive epitopes with some of the fruit LTPs tested and underscores only an involvement in co-recognition. No clinical relevance of IgE binding to the LTP of H brasiliensis in association with NRL allergy was detected.
Annals of allergy, asthma & immunology: official publication of the American College of Allergy, Asthma, & Immunology 12/2006; 97(5):643-9. · 2.83 Impact Factor
