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Pokoj S.,Paul Ehrlich Institute | Lauer I.,Paul Ehrlich Institute | Fotisch K.,Paul Ehrlich Institute | Himly M.,University of Salzburg | And 5 more authors.
Protein Expression and Purification | Year: 2010

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. © 2009 Elsevier Inc. All rights reserved.


Hochwallner H.,Medical University of Vienna | Schulmeister U.,Medical University of Vienna | Swoboda I.,Christian Doppler Laboratory | Focke-Tejkl M.,Christian Doppler Laboratory | And 19 more authors.
Journal of Allergy and Clinical Immunology | Year: 2010

Background: α-Lactalbumin (α-La) is a major cow's milk (CM) allergen responsible for allergic reactions in infants. Objective: We performed molecular, structural, and immunologic characterization of α-La. Methods: Recombinant α-lactalbumin (rα-La) was expressed in Escherichia coli, purified to homogeneity, and characterized by means of mass spectrometry and circular dichroism, and its allergenic activity was studied by using microarray technology, as well as in a basophil histamine release assay. IgE epitope mapping was performed with synthetic peptides. Results: According to circular dichroism analysis, rα-La represented a folded protein with a high thermal stability and refolding capacity. rα-La reacted with IgE antibodies from 57.6% of patients with CM allergy (n = 66) and induced the strongest basophil degranulation with sera from patients with CM allergy who had exhibited gastrointestinal symptoms or severe systemic reactions on CM exposure. rα-La contained sequential and conformational IgE epitopes. Superposition of IgE-reactive peptides onto the 3-dimensional structure of α-La revealed a close vicinity of the N- and C-terminal peptides within a surface-exposed patch. Conclusions: rα-La can be used for the diagnosis of patients with severe allergic reactions to CM and serves as a paradigmatic tool for the development of therapeutic strategies for CM allergy. © 2010 American Academy of Allergy, Asthma & Immunology.


Hochwallner H.,Medical University of Vienna | Schulmeister U.,Medical University of Vienna | Swoboda I.,Christian Doppler Laboratory | Balic N.,Medical University of Vienna | And 14 more authors.
Clinical and Experimental Allergy | Year: 2010

Background Cow's milk is one of the most common causes of food allergy affecting approximately 2.5% of infants in the first years of their life. However, only limited information regarding the allergenic activity of individual cow's milk allergens is available.Objective To analyse the frequency of IgE reactivity and to determine the allergenic activity of individual cow's milk allergens.Methods A nitrocellulose-based microarray, based on purified natural and recombinant cow's milk allergens was used to determine IgE reactivity profiles using sera from 78 cow's milk-sensitized individuals of varying ages. The allergenic activity of the individual allergens was tested using patients' sera for loading rat basophil leukaemia cells (RBL) expressing the α-chain of the human receptor FcεRI.Results Using the microarray and the RBL assay, cow's milk allergens were assessed for frequency of IgE recognition and allergenic activity. Moreover, the RBL assay allowed distinguishing individuals without or with mild clinical reactions from those with severe systemic or gastrointestinal symptoms as well as persons who grew out cow's milk allergy from those who did not.Conclusions Component-resolved testing using milk allergen microarrays and RBL assays seems to provide useful additional diagnostic information and may represent a basis for future forms of prophylactic and therapeutic strategies for cow's milk allergy. © 2010 Blackwell Publishing Ltd.


Schulten V.,Medical University of Vienna | Nagl B.,Medical University of Vienna | Nagl B.,Christian Doppler Laboratory | Scala E.,Center for Clinical and Experimental Allergology | And 13 more authors.
Allergy: European Journal of Allergy and Clinical Immunology | Year: 2011

Background: Nonspecific lipid transfer proteins (nsLTPs) are important food allergens. Often, patients allergic to the nsLTP in peach suffer from allergy to hazelnuts. We aimed to analyse the T-cell response to Cor a 8, the nsLTP in hazelnut and its immunological cross-reactivity with the nsLTP in peach, Pru p 3. Methods: Cor a 8-reactive T-cell lines (TCL) established from patients allergic to hazelnut and peach were stimulated with 12-mer peptides representing the complete amino acid sequence of Cor a 8 to identify its T-cell-activating regions and with Pru p 3 to investigate cellular cross-reactivity. T-cell clones specific for different major T-cell-activating regions of Pru p 3 were stimulated with Cor a 8. Both nsLTPs were subjected to endolysosomal degradation assays. Immunoglobulin E (IgE) cross-reactivity between Cor a 8 and Pru p 3 was assessed in inhibition enzyme-linked immunosorbent assay. Results: No major T-cell-activating region was found among 26 T-cell-reactive peptides identified in Cor a 8. Although generated with Cor a 8, 62% of the TCL responded more strongly to Pru p 3. This cross-reactivity was mediated by T cells specific for the immunodominant region Pru p 3 61-75. Peptide clusters encompassing this region were generated during lysosomal degradation of both nsLTPs. Cor a 8 was more rapidly degraded by lysosomal proteases than Pru p 3. Pre-incubation of sera with Pru p 3 completely abolished IgE binding to Cor a 8, which was not the case vice versa. Conclusions: T-cell reactivity to Cor a 8 is predominantly based on cross-reactivity with Pru p 3, indicating that the latter initiates sensitisation to its homolog in hazelnut. The limited allergenic potential of Cor a 8 seems to be associated with rapid lysosomal degradation during allergen processing and the lack of major T-cell-activating regions. © 2011 John Wiley & Sons A/S.


Zheng Y.-W.,Research Asia Pacific | Li J.,Guangzhou Medical College | Lai X.-X.,Research Asia Pacific | Zhao D.-Y.,Nanjing Medical University | And 7 more authors.
Chinese Medical Journal | Year: 2011

Background Allergen micro-arrays are powerful tools for screening of serum IgE-reactivity. In this study allergen micro-arrays were used to identify dominating IgE-binding allergens and cross-reactivity patterns among selected Chinese allergy patients. Methods The study was conducted using patient sera from the cities of Guangzhou, Nanjing, Chengdu and Shenyang. In total 100 sera with Dermatophagoides pteronyssinus (Der p) specific IgE-levels higher than 50 kU/L were selected for testing against 103 individual allergens. Results Among 100 selected patients, 95% showed IgE-reactivity towards house-dust mite allergens Dermatophagoides farinae (Der f) 1, Der f 2 and Der p 2 and 94% were IgE positive against Der p 1, and 60% of sera contained IgE reacting against allergen Euroglyphus maynei (Eur m) 2. IgE against cat allergen, Felisdomesticus (Fel d) 1, was seen in 20%. Only 2% showed specific IgE-reactivity to Der p 10, a panallergen belonging to the tropomyosin family. Serum IgE-reactivity towards other allergens was in general low. IgE-reactivity against pollen allergens showed geographic differences. Conclusions This study clearly confirms that group 1 and group 2 are major allergens of house dust mites. These selected house-dust mite allergy patients are close to being mono-sensitized. Der p 10 is not an important allergen for cross-reactivity. Specific IgE-sensitization towards pollen allergens is low in southern China compared to other regions. The prevalence of food and stinging insect allergens known to give rise to IgE-mediated cross-reactivity is 2% or less.


Pfiffner P.,University of Bern | Truffer R.,University of Bern | Matsson P.,Phadia AB | Rasi C.,Center for Clinical and Experimental Allergology | And 3 more authors.
Allergy: European Journal of Allergy and Clinical Immunology | Year: 2010

Background: Cross reactions are an often observed phenomenon in patients with allergy. Sensitization against some allergens may cause reactions against other seemingly unrelated allergens. Today, cross reactions are being investigated on a per-case basis, analyzing blood serum specific IgE (sIgE) levels and clinical features of patients suffering from cross reactions. In this study, we evaluated the level of sIgE compared to patients total IgE assuming epitope specificity is a consequence of sequence similarity. Methods: Our objective was to evaluate our recently published model of molecular sequence similarities underlying cross reactivity using serum-derived data from IgE determinations of standard laboratory tests. We calculated the probabilities of protein cross reactivity based on conserved sequence motifs and compared these in silico predictions to a database consisting of 5362 sera with sIgE determinations. Results: Cumulating sIgE values of a patient resulted in a median of 25-30% total IgE. Comparing motif cross reactivity predictions to sIgE levels showed that on average three times fewer motifs than extracts were recognized in a given serum (correlation coefficient: 0.967). Extracts belonging to the same motif group co-reacted in a high percentage of sera (up to 80% for some motifs). Conclusions: Cumulated sIgE levels are exaggerated because of a high level of observed cross reactions. Thus, not only bioinformatic prediction of allergenic motifs, but also serological routine testing of allergic patients implies that the immune system may recognize only a small number of allergenic structures. © 2010 John Wiley & Sons A/S.


Magni C.,University of Milan | Ballabio C.,University of Milan | Restani P.,University of Milan | Fuggetta D.,University of Milan | And 6 more authors.
Annals of Allergy, Asthma and Immunology | Year: 2010

Background Food allergy is becoming a major public health concern in recent times. Several sesame seed allergenic proteins have been identified. However, sensitization toward these proteins does not follow a common and unique pattern of clinical reactivity, as shown by the differential geographic recognition of single proteins. Objective To evaluate the sensitization profiles of 18 Italian individuals who experienced clinical symptoms after sesame seed consumption, including 4 anaphylactic reactions. Methods Using an in vitro approach, we adopted a 2-dimensional electrophoretic technique combined with immunoblotting analyses by using sera from 18 Italian sesame-allergic patients. Results We showed the prevalent and almost exclusive reactivity of the sesame 11S globulin. We shed light on the active role of the basic subunit of this globulin family. The limited accessibility of this polypeptide chain, unless the interchain disulphide bonds are cleaved, may be one of the reasons for its structural/functional stability and, thus, great potential for induction of IgE reactivity. Conclusions These results confirmed previous findings on the reactivity of the basic subunit of 11S globulin in various legume species. Moreover, this experimental approach proved to be useful for the noninvasive screening of specific reactivities in sensitized patients. © 2010 American College of Allergy, Asthma & Immunology.


Ciardiello M.A.,National Research Council Italy | Palazzo P.,Center for Clinical and Experimental Allergology | Bernardi M.L.,Center for Clinical and Experimental Allergology | Carratore V.,National Research Council Italy | And 7 more authors.
Allergy: European Journal of Allergy and Clinical Immunology | Year: 2010

Background: Mulberry (Morus spp.) is a genus comprising several species of deciduous trees whose fruits are commonly eaten in southern Europe. Subjects with severe systemic reaction have been described. The aim of this study was to isolate the allergens of this species. Methods: A nonspecific lipid transfer protein 1 (ns-LTP1) was purified from black mulberry by ion exchange and reverse phase high-performance liquid chromatography, and the primary structure was elucidated by direct protein sequencing. Its allergenic activity was evaluated in vivo by skin prick test and in vitro by Western Blot, CD203c basophil activation assay and high throughput multiplex inhibition method on immunosolid-phase allergen chip (ISAC). Results: Mulberry ns-LTP (Mor n 3) comprises 91 amino acids producing a molecular mass of 9246 Da. This protein shows high sequence identity with several allergenic ns-LTP1. Immunoblot analysis and CD203c activation assay demonstrated its allergenic activity in symptomatic subjects and in ns-LTP allergic patients who are not mulberry consumers. Immunological co-recognition was studied in vivo on a selected group of well-characterized ns-LTP allergic patients showing a high percentage of nMor n 3+ subjects (88.46%) even in patients who have never eaten mulberry before. IgE inhibition on ISAC micro-array demonstrated an almost complete cross-reactivity to nArt v 3, rCor a 8 and a very high percentage of inhibition to nPru p 3. Conclusions: Mor n 3 is the first allergen isolated in black mulberry and immunologically characterized. It displayed allergenic activity among symptomatic and nonconsumer patients and a pattern of cross-reactivity to other plant-derived LTPs. © 2009 John Wiley & Sons A/S.


Scala E.,Center for Clinical and Experimental Allergology | Alessandri C.,Center for Clinical and Experimental Allergology | Bernardi M.L.,Center for Clinical and Experimental Allergology | Ferrara R.,Center for Clinical and Experimental Allergology | And 7 more authors.
Clinical and Experimental Allergy | Year: 2010

Summary Background The availability of allergenic molecules and high-throughput microtechnologies allow the collection of a large number of IgE results at the same time in a single test. This can be carried out applying the test in the routine diagnostic work-up. Objective The aim of this study was to make a cross-sectional evaluation of the raw prevalence of IgE reactivity to allergenic molecules in serum samples from a cohort of Italian patients using an innovative tool. Methods The ISAC, a microarray system, has been used for specific IgE detection using 75 different allergenic molecules. Sera were collected from 23 077 unselected consecutive individuals complaining about any allergic disease. Results Sixteen thousand four hundred and eight of 23 077 patients had IgE to at least one of 75 allergenic molecules. The top-ranked molecules in this cohort were Cup a 1 (42.7%), Der f 2 (38.7%), and Phl p 1 (37.9%), whereas all the other allergens tested scored in a range between 36.8% and 0.04%, including the first food allergen, Pru p 3, ranked 15th (9.79%). Prevalence varied quite markedly depending on the age range considered, and showing a different behaviour in the lifetime sensitization process. Unsupervised two-way hierarchical clustering analysis generated distinctive patterns of reactivity as the result of IgE recognition of either homologous allergens belonging to different biological sources or non-homologous belonging to the same biological source. Conclusions Allergen-based microarray is a tool for the detection of IgE-related sensitization to panels of allergens and gives a more precise and comprehensive evaluation for an IgE-based epidemiology. This insight brings data for better understanding of the sensitization process. © 2010 Blackwell Publishing Ltd.

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