RefLab ApS

Copenhagen, Denmark

RefLab ApS

Copenhagen, Denmark

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Blanc F.,French National Institute for Agricultural Research | Vissers Y.M.,Wageningen University | Adel-Patient K.,French National Institute for Agricultural Research | Rigby N.M.,UK Institute of Food Research | And 15 more authors.
Molecular Nutrition and Food Research | Year: 2011

Scope: Roasting rather than boiling and Maillard modifications may modulate peanut allergenicity. We investigated how these factors affect the allergenic properties of a major peanut allergen, Ara h 1. Methods and results: Ara h 1 was purified from either raw (N-Ara h 1) or roasted (R-Ara h 1) peanuts. Boiling (100°C 15min; H-Ara h 1) resulted in a partial loss of Ara h 1 secondary structure and formation of rod-like branched aggregates with reduced IgE-binding capacity and impaired ability to induce mediator release. Glycated Ara h 1 (G-Ara h 1) formed by boiling in the presence of glucose behaved similarly. However, H- and G-Ara h1 retained the T-cell reactivity of N-Ara h 1. R-Ara h 1 was denatured, comprised compact, globular aggregates, and showed no evidence of glycation but retained the IgE-binding capacity of the native protein. Conclusion: Ara h 1 aggregates formed by boiling were morphologically distinct from those formed by roasting and had lower allergenic activity. Glycation had no additional effect on Ara h 1 allergenicity compared with heating alone. Taken together with published data on the loss of Ara h 2/6 from boiled peanuts, this supports the hypothesis that boiling reduces the allergenicity of peanuts. © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.


Vissers Y.M.,Wageningen University | Iwan M.,University of Warmia and Mazury | Adel-Patient K.,French National Institute for Agricultural Research | Stahl Skov P.,RefLab ApS | And 11 more authors.
Clinical and Experimental Allergy | Year: 2011

Background Peanuts are often consumed after roasting, a process that alters the three-dimensional structure of allergens and leads to Maillard modification. Such changes are likely to affect their allergenicity. Objective We aimed to establish the effect of thermal treatment mimicking the roasting process on the allergenicity of Ara h 1 and a mix of 2S albumins from peanut (Ara h 2/6). Methods Ara h 1 and Ara h 2/6 were purified from raw peanuts and heated in a dry form for 20min at 145°C in the presence (R+g) or absence (R-g) of glucose, and soluble proteins were then extracted. Sera obtained from 12 well-characterized peanut-allergic patients were used to assess the IgE binding and degranulation capacities of the allergens. Results Extensive heating at low moisture resulted in the hydrolysis of both Ara h 1 and Ara h 2/6. However, in contrast to Ara h 2/6, soluble R+g Ara h 1 formed large aggregates. Although the IgE-binding capacity of R+g and R-g Ara h 1 was decreased 9000- and 3.6-fold, respectively, compared with native Ara h 1, their capacity to elicit mediator release was increased. Conversely, both the IgE-binding capacity and the degranulation capacity of R-g Ara h 2/6 were 600-700-fold lower compared with the native form, although the presence of glucose during heating significantly moderated these losses. Conclusions and Clinical Relevance Extensive heating reduced the degranulation capacity of Ara h 2/6 but significantly increased the degranulation capacity of Ara h 1. This observation can have important ramifications for component-resolved approaches for diagnosis and demonstrates the importance of investigating the degranulation capacity in addition to IgE reactivity when assessing the effects of food processing on the allergenicity of proteins. © 2011 Blackwell Publishing Ltd.


Huber H.,Biomay AG | Swoboda I.,Medical University of Vienna | Rigby N.,UK Institute of Food Research | Jensen B.M.,Copenhagen University | And 38 more authors.
International Archives of Allergy and Immunology | Year: 2015

Background: The FAST (food allergy-specific immunotherapy) project aims at developing safe and effective subcutaneous immunotherapy for fish allergy, using recombinant hypoallergenic carp parvalbumin, Cyp c 1. Objectives: Preclinical characterization and good manufacturing practice (GMP) production of mutant Cyp (mCyp) c 1. Methods:Escherichia coli-produced mCyp c 1 was purified using standard chromatographic techniques. Physicochemical properties were investigated by gel electrophoresis, size exclusion chromatography, circular dichroism spectroscopy, reverse-phase high-performance liquid chromatography and mass spectrometry. Allergenicity was assessed by ImmunoCAP inhibition and basophil histamine release assay, immunogenicity by immunization of laboratory animals and stimulation of patients' peripheral blood mononuclear cells (PBMCs). Reference molecules were purified wild-type Cyp c 1 (natural and/or recombinant). GMP-compliant alum-adsorbed mCyp c 1 was tested for acute toxicity in mice and rabbits and for repeated-dose toxicity in mice. Accelerated and real-time protocols were used to evaluate stability of mCyp c 1 as drug substance and drug product. Results: Purified mCyp c 1 behaves as a folded and stable molecule. Using sera of 26 double-blind placebo-controlled food-challenge-proven fish-allergic patients, reduction in allergenic activity ranged from 10-to 5,000-fold (1,000-fold on average), but with retained immunogenicity (immunization in mice/rabbits) and potency to stimulate human PBMCs. Toxicity studies revealed no toxic effects and real-time stability studies on the Al(OH)3-adsorbed drug product demonstrated at least 20 months of stability. Conclusion: The GMP drug product developed for treatment of fish allergy has the characteristics targeted for in FAST: i.e. hypoallergenicity with retained immunogenicity. These results have warranted first-in-man immunotherapy studies to evaluate the safety of this innovative vaccine. © 2015 S. Karger AG, Basel.


Vissers Y.M.,Wageningen University | Blanc F.,French National Institute for Agricultural Research | Skov P.S.,RefLab ApS | Johnson P.E.,UK Institute of Food Research | And 14 more authors.
PLoS ONE | Year: 2011

Background: Peanut allergy is one of the most common and severe food allergies, and processing is known to influence the allergenicity of peanut proteins. We aimed to establish the effect of heating and glycation on the IgE-binding properties and biological activity of 2S albumins (Ara h 2/6) from peanut. Methodology/Principal Findings: Native Ara h 2/6 was purified from raw peanuts and heated in solution (15 min, 110°C) in the presence or absence of glucose. Ara h 2 and 6 were also purified from roasted peanut. Using PBMC and sera from peanut-allergic patients, the cellular proliferative potency and IgE reactivity (reverse EAST inhibition) and functionality (basophil degranulation capacity) of allergens were assessed. Heating Ara h 2/6 at 110°C resulted in extensive denaturation, hydrolysis and aggregation of the protein, whilst Ara h 2 and 6 isolated from roasted peanut retained its native conformation. Allergen stimulation of PBMC induced proliferation and Th2 cytokine secretion which was unaffected by thermal processing. Conversely, IgE reactivity and functionality of Ara h 2/6 was decreased by heating. Whilst heating-glycation further reduced the IgE binding capacity of the proteins, it moderated their loss of histamine releasing capacity. Ara h 2 and 6 purified from roasted peanut demonstrated the same IgE reactivity as unheated, native Ara h 2/6. Conclusions/Significance: Although no effect of processing on T-cell reactivity was observed, heat induced denaturation reduced the IgE reactivity and subsequent functionality of Ara h 2/6. Conversely, Ara h 2 and 6 purified from roasted peanut retained the structure and IgE reactivity/functionality of the native protein which may explain the allergenic potency of this protein. Through detailed molecular study and allergenicity assessment approaches, this work then gives new insights into the effect of thermal processing on structure/allergenicity of peanut proteins. © 2011 Vissers et al.


De Moira A.P.,University of Cambridge | Fitzsimmons C.M.,University of Cambridge | Jones F.M.,University of Cambridge | Wilson S.,University of Cambridge | And 8 more authors.
Journal of Infectious Diseases | Year: 2014

Background: The poor correlation between allergen-specific immunoglobulin E (asIgE) and clinical signs of allergy in helminth infected populations suggests that helminth infections could protect against allergy by uncoupling asIgE from its effector mechanisms. We investigated this hypothesis in Ugandan schoolchildren coinfected with Schistosoma mansoni and hookworm. Methods: Skin prick test (SPT) sensitivity to house dust mite allergen (HDM) and current wheeze were assessed pre-anthelmintic treatment. Nonspecific (anti-IgE), helminth-specific, and HDM-allergen-specific basophil histamine release (HR), plus helminth- and HDM-specific IgE and IgG4 responses were measured pre- and post-treatment. Results: Nonspecific- and helminth-specific-HR, and associations between helminth-specific IgE and helminthspecific HR increased post-treatment. Hookworm infection appeared to modify the relationship between circulating levels of HDM-IgE and HR: a significant positive association was observed among children without detectable hookworm infection, but no association was observed among infected children. In addition, hookworm infection was associated with a significantly reduced risk of wheeze, and IgG4 to somatic adult hookworm antigen with a reduced risk of HDM-SPT sensitivity. There was no evidence for S. mansoni infection having a similar suppressive effect on HDM-HR or symptoms of allergy. Conclusions: Basophil responsiveness appears suppressed during chronic helminth infection; at least in hookworm infection, this suppression may protect against allergy. © The Author 2014.


Grant
Agency: European Commission | Branch: FP7 | Program: CP-FP | Phase: HEALTH-2007-1.4-3 | Award Amount: 4.16M | Year: 2008

The FAST project aims at the development of safe and effective treatment of food allergies. It targets persistent and severe allergy to fish and fruit. Besides persistence and severity, this choice is based on prevalence and the importance of these foods for a healthy diet. Classical allergen-specific immunotherapy (SIT) for treatment of food allergy using subcutaneous injections with food extracts has proven to be effective but too dangerous due to anaphylactic side-effects. FAST will therefore develop a safe alternative by replacing food extracts with hypo-allergenic recombinant major allergens, the active ingredients of SIT. Both severe fish and fruit allergy are caused by a single major allergen, parvalbumin for fish and lipid transfer protein for fruit. This makes development of a novel biotechnological product feasible. Two approaches will be evaluated for achieving hypoallergenicity, i.e. site-directed mutagenesis and chemical modification. The most promising hypo-allergenic versions of parvalbumin and lipid transfer protein will be produced under GMP conditions. After pre-clinical testing (toxicology testing and efficacy in mouse models), Phase I and II randomized double-blind placebo-controlled multi-center clinical trials will be performed. Two routes of administration will be evaluated, subcutaneous in case of fish and sublingual in case of fruit. The primary read-out will be the double-blind placebo-controlled food challenge. To understand the underlying immune mechanisms of subcutaneous and sublingual immunotherapy, these trials will be accompanied by in depth serological and cellular immune analyses, allowing identification of novel biomarkers for monitoring treatment efficacy. FAST will improve the quality of life of food allergic patients by providing a safe and effective curative treatment that will end their dependence on avoidance and rescue medication.


Falkencrone S.,University of Southern Denmark | Poulsen L.K.,Copenhagen University | Bindslev-Jensen C.,University of Southern Denmark | Woetmann A.,Copenhagen University | And 9 more authors.
Allergy: European Journal of Allergy and Clinical Immunology | Year: 2013

Background IgE-mediated activation of mast cells has been reported to induce the release of tumour necrosis alpha (TNF-α), which may display autocrine effects on these cells by inducing the generation of the tissue remodelling protease matrix metalloproteinase-9 (MMP-9). While mast cells and basophils have been shown to express complementary and partially overlapping roles, it is not clear whether a similar IgE/TNF-α/MMP-9 axis exists in the human basophil. The purpose of this study was thus to investigate whether IgE-mediated activation of human basophils induces TNF-α and MMP-9 release. Methods Human peripheral blood mononuclear cells (PBMC), isolated basophils and monocytes were stimulated up to 21 h with anti-IgE. Mediator releases were assessed by ELISA, and surface expressions of mediators were detected by flow cytometry. Upregulation of cytokine production was detected by Western blot and polymerase chain reaction (PCR). Results IgE-mediated activation of basophils induced the synthesis and release of both TNF-α and MMP-9 from PBMC. In contrast, IgE-mediated activation of purified basophils induced the release and cellular expression of TNF-α but not MMP-9. Isolated monocytes did not release MMP-9 upon anti-IgE stimulation, but MMP-9 release was induced by stimulating monocytes with supernatants from activated basophils, and this release was inhibited by anti-TNF-α neutralizing antibodies. Conclusion Our results strongly indicate that human basophils release TNF-α following IgE-dependent activation and that this cytokine subsequently stimulates MMP-9 release from monocytes. These findings support a direct involvement of basophils in inflammation as well as suggesting a role for the basophil in tissue remodelling. © 2013 John Wiley & Sons A/S. Published by Blackwell Publishing Ltd.

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