37 research outputs found

    Identification and molecular characterization of allergenic nsLTP from durum wheat (Triticum turgidum)

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    Background: Common wheat (Triticum aestivum) and durum wheat (T. turgidum) are both involved in Baker's asthma (BA) and food allergy (FA) including wheat-dependent exercise-induced asthma (WDEIA). However, allergens in durum wheat have not been described, and the over-expression of T. turgidum non-specific lipid-transfer protein (nsLTPs) is considered to increase resistance to phytopathogens. Objective: To identify and assess the allergenicity of nsLTP from T. turgidum. Methods: Recombinant T. turgidum nsLTP Tri tu 14 was generated and tested for structural integrity (circular dichroism-spectroscopy) and purity (SDS-PAGE). Thirty-two wheat allergic patients were enrolled: 20 Spanish patients (BA) with positive bronchial challenge to wheat flour, and 12 Italian patients (wheat FA/WDEIA) with positive double-blind placebo-controlled food challenge/open food challenge (OFC) to pasta. IgE values to wheat, Tri tu 14, Tri a 14 (T. aestivum) and Pru p 3 (P. persica) were determined by ImmunoCAP testing. Allergenic potency (in vitro mediator release) and IgE cross-reactivity were investigated. Results: Tri tu 14 was found to share 49% and 52% amino acid identity with Tri a 14 and Pru p 3, respectively. Among 25 Tri a 14 CAP positive sera, 23 (92%) were reactive to wheat extract, 22 (88%) to Tri tu 14 and 20 (80%) to Pru p 3. The correlation between Tri a 14 and Tri tu 14 specific IgE levels was r = 0.97 (BA) and r = 0.93 (FA/WDEIA), respectively. FA/WDEIA patients showed higher specific IgE values to Tri tu 14 and Pru p 3 than BA patients. Tri tu 14 displayed allergenic activity by mediator release from effector cells and IgE cross-reactivity with Pru p 3. The degree of IgE cross-reactivity between the two wheat nsLTPs varied between individual patients. Conclusions and Clinical Relevance: Sensitization to Tri tu 14 likely appears to be more important in wheat FA/WDEIA than in BA. Over-expression of Tri tu 14 in wheat would represent a risk for patients with nsLTP-mediated FA

    Does the food ingredient pectin provide a risk for patients allergic to non-specific lipid-transfer proteins?

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    Pectin, a dietary fiber, is a polysaccharide that is widely used in food industry as a gelling agent. In addition, prebiotic and beneficial immunomodulatory effects of pectin have been demonstrated, leading to increased importance as food supplement. However, as cases of anaphylactic reactions after consumption of pectin-supplemented foods have been reported, the present study aims to evaluate the allergy risk of pectin. This is of particular importance since most of the pectin used in the food industry is extracted from citrus or apple pomace. Both contain several allergens such as non-specific lipid transfer proteins (nsLTPs), known to induce severe allergic reactions, which could impair the use of pectins in nsLTP allergic patients. Therefore, the present study for the first time was performed to analyze residual nsLTP content in two commercial pectins using different detection methods. Results showed the analytical sensitivity was diminished by the pectin structure. Finally, spiking of pectin with allergenic peach nsLTP Pru p 3 led to the conclusion that the potential residual allergen content in both pectins is below the threshold to induce anaphylactic reactions in nsLTP-allergic patients. This data suggests that consumption of the investigated commercial pectin products provides no risk for inducing severe reactions in nsLTP-allergic patients

    Scientific and regulatory evaluation of mechanistic in silico drug and disease models in drug development: building model credibility

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    The value of in silico methods in drug development and evaluation has been demonstrated repeatedly and convincingly. While their benefits are now unanimously recognized, international standards for their evaluation, accepted by all stakeholders involved, are still to be established. In this white paper, we propose a risk-informed evaluation framework for mechanistic model credibility evaluation. To properly frame the proposed verification and validation activities, concepts such as context of use, regulatory impact and risk-based analysis are discussed. To ensure common understanding between all stakeholders, an overview is provided of relevant in silico terminology used throughout this paper. To illustrate the feasibility of the proposed approach, we have applied it to three real case examples in the context of drug development, using a credibility matrix currently being tested as a quick-start tool by regulators. Altogether, this white paper provides a practical approach to model evaluation, applicable in both scientific and regulatory evaluation contexts

    Evaluation of the Allergenicity Potential of TcPR-10 Protein from Theobroma cacao

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    Background: The pathogenesis related protein PR10 (TcPR-10), obtained from the Theobroma cacao-Moniliophthora perniciosa interaction library, presents antifungal activity against M. perniciosa and acts in vitro as a ribonuclease. However, despite its biotechnological potential, the TcPR-10 has the P-loop motif similar to those of some allergenic proteins such as Bet v 1 (Betula verrucosa) and Pru av 1 (Prunus avium). The insertion of mutations in this motif can produce proteins with reduced allergenic power. The objective of the present work was to evaluate the allergenic potential of the wild type and mutant recombinant TcPR-10 using bioinformatics tools and immunological assays. Methodology/Principal Findings: Mutant substitutions (T10P, I30V, H45S) were inserted in the TcPR-10 gene by sitedirected mutagenesis, cloned into pET28a and expressed in Escherichia coli BL21(DE3) cells. Changes in molecular surface caused by the mutant substitutions was evaluated by comparative protein modeling using the three-dimensional structure of the major cherry allergen, Pru av 1 as a template. The immunological assays were carried out in 8-12 week old female BALB/c mice. The mice were sensitized with the proteins (wild type and mutants) via subcutaneous and challenged intranasal for induction of allergic airway inflammation. Conclusions/Significance: We showed that the wild TcPR-10 protein has allergenic potential, whereas the insertion of mutations produced proteins with reduced capacity of IgE production and cellular infiltration in the lungs. On the other hand, in vitro assays show that the TcPR-10 mutants still present antifungal and ribonuclease activity against M. perniciosa RNA. In conclusion, the mutant proteins present less allergenic potential than the wild TcPR-10, without the loss of interesting biotechnological properties. (Résumé d'auteur

    Abstracts from the Food Allergy and Anaphylaxis Meeting 2016

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    Immunoglobulin E reactivity and allergenic potency of Morus papyrifera (paper mulberry) pollen

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    Contains fulltext : 144000.pdf (publisher's version ) (Open Access)BACKGROUND: Paper mulberry (Morus papyrifera) pollen is considered to be one of the most clinically relevant aeroallergens in Pakistan. To date, the allergenicity of the pollen has not been investigated. OBJECTIVE: To characterize the sensitization profile of mulberry-allergic patients and the proteins of paper mulberry pollen contributing to pollinosis in the Pakistani population. METHODS: Proteins were extracted from mulberry pollen using different protocols. Immunoglobulin (Ig) E binding proteins to mulberry pollen was determined by ImmunoCAP testing and immunoblotting using sera from 29 mulberry pollen-allergic patients with positive skin prick test results to mulberry pollen antigens. The histamine release assay was performed in vitro to determine the allergenic potency of pollen extracts and a partially purified mulberry pollen allergen. The protein was identified using N-terminal sequencing and matrix-assisted laser desorption/ionization-time of flight spectrometry (MALDI-TOF/TOF). RESULTS: IgE sensitization to mulberry pollen was confirmed by positive ImmunoCAP results to pollen from Morus alba (white mulberry) in 23 out of 29 mulberry pollen-allergic patients. A 10-kDa protein from the paper mulberry pollen extract was considered a major allergen, along with additional IgE-reactive proteins. Sera from 79% of the patients reacted to the 10-kDa allergen, which showed substantial capacity to trigger histamine release in 3 out of 4 patients. N-terminal sequencing and MALDI-TOF/TOF yielded an amino acid sequence with no homology to known proteins. CONCLUSIONS: Mulberry-allergic patients are sensitized to multiple mulberry pollen allergens. We identified a novel 10-kDa protein that was a major allergen and should be further investigated for diagnostic and therapeutic purposes

    Recombinant allergens Pru av 1 and Pru av 4 and a newly identified lipid transfer protein in the vitro diagnosis of cherry allergy

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    Background: In central and northern Europe food allergy to fruits of the Rosaceae family is strongly associated with birch pollinosis because of the existence of IgE cross-reactive homologous allergens in birch pollen and food. By contrast, in the Mediterranean population allergic reactions to these fruits frequently are not related to birch pollen allergy and are predominantly elicited by lipid transfer proteins (LTPs), Objective: We sought to determine the prevalence of IgE sensitization to the recombinant cherry allergens Pru av 1 and Pru av 4 in comparison with cherry extract within a representative group of patients who were allergic to cherries recruited in Germany and to compare the relevance of IgE to cherry LTPs in Italian patients. Methods: Recombinant Pru av 1 and rPru av 4 were available from earlier studies. The cDNA of the cherry LTPs was obtained by using a PCR-cloning strategy. The protein was expressed in Escherichia coli and purified by means of metal chelate affinity chromatography. Sera from 101 German patients with birch pollinosis and oral allergy syndrome to cherry and sera from 7 Italian patients with cherry allergy were investigated by using enzyme allergosorbent tests for IgE reactivity with cherry extract, rPru av 1, rPru av 4, and the recombinant cherry LTP. Inhibition experiments were performed to compare the IgE reactivity of natural and recombinant cherry LTPs and to investigate potential cross-reactivity with birch pollen allergens, Results: The LTP from cherry comprises 91 amino acids and a 26 amino acid signal peptide, The mature cherry LTP shows high amino acid sequence identity with allergenic LTPs from peach (Pru p 3, 88%), apricot (Pru ar 3, 86%), and maize (Zea m 14, 59%) and displays no IgE cross-reactivity with birch pollen. The IgE prevalences in the German patients were as follows: LTP, 3 of 101 (3%); rPru av 1, 97 of 101 (96.0%); rPru av 4, 16 of 101 (16.2%); and cherry extract, 98 of 101 (97%), All 7 Italian patients had IgE against the cherry LTP. Conclusions: Recombinant allergens are useful tools for a more accurate in vitro IgE-based diagnosis of cherry allergy, Taken together, they mimic the allergenic activity of cherry extract, having slightly higher biologic activity. Sensitization to the cherry LTP is relevant for a minority of patients recruited in Germany, but our data indicate that it may be a major allergen in Italy
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