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Allergen detection methods support food labeling and quality assessment at the allergen component level of allergen preparations used for allergy diagnosis and immunotherapy (AIT). Commonly applied enzyme-linked immunosorbent assay (ELISA) requires animal antibodies but potentially shows batch variations. We developed synthetic aptamers as alternative binders in allergen detection to meet the replacement, reduction, and refinement (3R) principle on animal protection in science. ssDNA aptamers were specifically selected against the major peanut allergen Ara h 1 and identified by next-generation sequencing. Application in various detection systems (ELISA-like assays, western blot, and surface plasmon resonance) was demonstrated. The ELISA-like assay comprised a sensitivity of 10 ng/mL Ara h 1, comparable to published antibody-based ELISA, and allowed Ara h 1 detection in various peanut flours, similar to those used in peanut AIT as well as in processed food. This ELISA-like aptamer-based assay proofs antibody-free allergen detection for food labeling or quality assessment of diagnostic and therapeutic allergen products.

Scope: Considering the increasing numbers of patients suffering from food allergy (FA) as well as the great variety of novel foods and food compositions, an unmet need exists for the development of preclinical approaches to characterize the allergenic potential of proteins. The aim of our study was to evaluate the allergenicity of different food allergens in a rat model., Methods: Brown Norway rats were sensitized to protein extracts (RuBisCO, apple, soy, peanut, garden pea) or ovalbumin (OVA) combined with Bordetella pertussis and aluminium hydroxide, followed by oral allergen challenges., Results: Allergen-specific serum immunoglobulin production and the proliferation of mononuclear cells from spleen confirmed sensitization. To assess functional alterations in the gut, intestinal permeability was measured, which increased in sensitized and challenged animals compared to non-sensitized controls. Allergens with high allergenic potential (peanut, OVA, soy) caused a stronger immunological response than allergens with low allergenic potential, such as RuBisCO and apple. Moreover, the immunological responses were reduced when using boiled instead of raw soy and pea proteins., Conclusion: This model mimics key features of FA and facilitates investigating the allergenicity of allergens in novel food or food compositions in vivo., (© 2014 S. Karger AG, Basel.)

It has been hypothesized that high environmental exposure to peanut allergens may be a potent risk factor for cutaneous sensitization. Therefore, we wanted to investigate whether peanut proteins are detectable in house dust of different household areas. Peanut levels of dust samples were measured with ELISA. Overall, peanut was detectable in 19 of 21 households in the eating area and/or in bed. The frequency of peanut consumption correlated with peanut levels. Forty-eight hours after intentional peanut consumption, peanut levels were highly increased. Nevertheless, further research is required to prove whether peanut allergen in house dust can cause sensitization via skin., (© 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Background: Cow's milk allergy (CMA) is one of the most common causes of food allergy in the first years of life. Fortunately, the majority of children with CMA develop clinical tolerance with time. However, no good individual markers exist to predict whether this will occur. Therefore, a prognosis to identify children with persistent CMA at diagnosis would be helpful., Objective: In this study, we sought to assess whether measurement of IgE to individual allergens of cow's milk (CM) would separate patients with persistent CMA from those who became clinically tolerant to CM over time., Methods: A total of 52 patients ranging from 3 months to 114 months of age with proven CMA by DBPCFC were followed over time. From these 52 patients, 32 (61.5%) patients became tolerant in the analysed time period. All patients were rechallenged at least once, some were rechallenged two or three times. Serum was analysed prior to each challenge for specific IgE, IgG and IgG4 binding to crude CM protein as well as to individual allergens of CM., Results: The individual likelihood of outgrowing CMA significantly correlates with a low level of CM-specific IgE as well as a low level of specific IgE to α-lactalbumin, β-lactoglobulin (Bos d5.0102), κ-casein and α(s1) -casein. No significant correlation was found for IgE levels to total casein, lactoferrin, β-casein and β-lactoglobulin (Bos d5.0101) as well as IgG and IgG4 levels to α-lactalbumin, β-lactoglobulin and total casein., Conclusions: CM-specific IgE is a good prognostic marker for persistence of CMA. In addition, component-resolved diagnostic showed similar results. However, in our view, the rising laboratory costs do not justify a measurement on a daily basis. Additional determination of specific IgG or IgG4 levels was not useful in predicting tolerance development in our study population., (© 2012 Blackwell Publishing Ltd.)

Dendritic cells (DCs) play an important role in directing naive T cells towards a Th1/Th2 or regulatory T cells (Treg) cell phenotype. In this context, interleukin (IL)-10 has been shown to exhibit immune regulatory capacities. The aim of this study was to delineate the influence of high-IL-10-producing DCs on DC-T-cell interactions in inhibiting allergen-induced airway inflammation and hyperreactivity in a murine model of allergic airway disease. Bone marrow-derived dendritic cells (BMDCs) were generated from hemopoietic progenitors by culture with granulocyte-macrophage colony-stimulating factor (GM-CSF), and stimulated with ovalbumin (OVA) +/- lipopolysaccharide (LPS). The effects of ovalbumin-pulsed BMDCs on cytokine production by allergen-specific naive T cells were studied in vitro. The development of airway inflammation in Balb/c mice was determined after intranasal administration of BMDCs in vivo. LPS stimulation of BMDCs strongly enhanced IL-10 production. Coculture of LPS-modulated DCs exhibiting increased IL-10 production with allergen-specific naive T cells reduced the production of interferon (IFN)-gamma and IL-5, but enhanced the production of IL-10. After blockade with anti-IL-10 plus anti-IL-10-receptor antibodies, the level of IFN-gamma and IL-5 production by cocultured T cells was restored, underlining the regulatory function of IL-10. Intranasal administration of high-IL-10-producing LPS-stimulated, OVA-primed BMDCs prior to repetitive airway allergen challenges resulted in an even enhanced airway inflammation. These data demonstrate that increased IL-10 production by DCs may be a critical element for T-cell activation and differentiation in the context of allergen-induced immune responses in vitro. However, this DC modulation did not translate into suppression of allergic airway disease in vivo.

Background: New preventive strategies against the development of allergic diseases focus on potentially immunomodulatory components, such as bacterial LPSs. Optimal time frames for initiating immunomodulation to receive a sufficient effect against allergen sensitization are still unclear., Objective: Using a mouse model, we investigated the influence of prenatal LPS exposure on later allergen-mediated sensitization and airway inflammation in the offspring., Methods: Pregnant BALB/c mice were repeatedly exposed to aerosolized LPS (LPS Escherichia coli; 3x per week, day 7 of gestation time up to delivery). Some of the offspring were further exposed to aerosolized LPS before allergen sensitization with ovalbumin (OVA; administered intraperitoneally day 28 up to day 42) and OVA airway challenges (days 56-58). Positive control animals were placebo exposed to PBS instead of LPS, and negative control animals were first placebo exposed and later placebo sensitized with PBS instead of OVA., Results: Compared with positive control animals, prenatal LPS exposure suppressed (1) allergen-specific sensitization (IgE production), (2) eosinophilic airway inflammation (reduced numbers of eosinophils in bronchoalveolar lavage fluids), and (3) in vivo airway reactivity in response to methacholine. These effects occurred only when prenatal was combined with further postnatal LPS exposure. Suppression of allergen-mediated inflammatory responses was associated with increased Toll-like receptor and T-bet expression by lung tissues and a shift toward predominantly T(H)1 immune responses in spleen cells cultured with OVA in vitro., Conclusion: Prenatal initiated and postnatal sustained LPS exposure increased endotoxin susceptibility and prevented later allergen sensitization in offspring through inhibition of T(H)2 immune responses., Clinical Implications: Immunomodulation with bacterial compounds during gestation time might be an effective mode for first-step primary prevention against allergic diseases.

Background: Transcriptional factors of the signal transducer and activator of transcription (STAT) family play an important role in orchestrating immune reactions., Objective: The aim of the current study was to investigate the role of STAT-1 in murine allergen-induced sensitization and development of airway inflammation (AI) and airway hyperreactivity (AHR), cardinal features of bronchial asthma., Methods: BALB/c mice were systemically sensitized to ovalbumin and challenged with ovalbumin through the airways. Decoy oligonucleotide (ODN) specific for STAT-1 was applied once locally to the airways of sensitized animals before allergen airway challenges., Results: Single application of decoy ODN markedly and significantly reduced numbers of eosinophils and lymphocytes in bronchoalveolar lavage fluids compared with those seen in sensitized and challenged animals receiving mutant control ODN. Associated with this decrease in eosinophilic AI were significantly reduced levels of IL-5 in BAL fluid, of CD40 expression in peribronchial infiltrates, and of vascular cell adhesion molecule 1 expression in vascular endothelial cells, respectively. In addition, development of AHR after allergen sensitization and airway challenges was effectively abolished after local STAT-1 decoy ODN treatment., Conclusion: The data indicate that a decoy ODN neutralizing STAT-1 effectively inhibits allergen-induced AI and AHR, probably by attenuating upregulation of costimulatory and adhesion molecules, and suggest a significant role of STAT-1 in asthma pathology.

Background Microarray-based component-resolved diagnostics (CRD) has become an accepted tool to detect allergen-specific IgE sensitization towards hundreds of allergens in parallel from one drop of serum. Nevertheless, specificity and sensitivity as well as a simultaneous detection of allergen-specific IgG4, as a potential parameter for tolerance development, remain to be optimized. Objective We applied the recently introduced silicon chip coated with a functional polymer named copoly(DMA-NAS-MAPS) to the simultaneous detection of food allergen-specific IgE and IgG4, and compared it with ImmunoCAP and ImmunoCAP ISAC. Inter- and intraslide variation, linearity of signal and working range, sensitivity and application of internal calibrations for IgE and IgG4 were assessed. Methods Native and recombinant allergenic proteins from hen's egg and cow's milk were spotted on silicon chips coated with copoly(DMA-NAS-MAPS) along with known concentrations for human IgE and IgG4. A serum pool and 105 patient samples were assessed quantitatively and semi-quantitatively with the ImmunoCAP and ImmunoCAP ISAC and correlated with IgE- and IgG4-specific fluorescence on silicon microarrays. Results Allergen-specific IgE and IgG4 were detected in parallel using two fluorescent dyes with no crosstalk. Results from the ImmunoCAP correlated better with microarray fluorescence than with ImmunoCAP ISAC except for the allergen ovomucoid. The working range of the silicon microarray for total hen's egg-specific IgE was comparable to the range of 0.1 to >100 kUA/L of the ImmunoCAP system, whereas for total cow's milk, the silicon microarray was less sensitive. Detectable allergen-specific IgG4 could be determined only for low concentrations, but still correlated positively with ImmunoCAP results. Conclusions We confirmed the ability of the polymer coated silicon microarray to be comparably sensitive to the ImmunoCAP ISAC for various food allergens. This suggests that the copoly(DMA-NAS-MAPS) microarray is a low-cost, self-producible alternative to the commercial ImmunoCAP ISAC in allergy research. [ABSTRACT FROM AUTHOR]

Background: Hen's egg (HE) allergy is a common disease in childhood. HE-specific serum IgE has been correlated with the outcome of oral food challenge tests, and diagnostic decision points have been described as helpful but still not sufficient to reduce the requirement for oral food challenges. The aim of the study was to correlate HE-specific IgE, IgG and IgG4 levels with the outcome of double-blind, placebo-controlled food challenges (DBPCFC) in patients with suspected HE allergy to improve diagnostic procedures. Methods: HE-specific IgE, IgG, and IgG4 levels were compared between 150 children with suspected HE allergy based on sensitization and/or patient's history who underwent DBPCFC. Sixty-six patients were HE-allergic (HE-sensitized with a positive DBPCFC), 48 HE-sensitized but tolerant (negative DBPCFC), and 36 patients were nonsensitized and tolerant (negative DBPCFC). Prior to food challenge HE-specific serum IgE, IgG, and IgG4 were measured with the Phadia CAP-system. Results: HE-specific IgE was significantly higher in HE-allergic patients than in clinically tolerant ones. However, there was no difference in HE-specific IgG and IgG4 concentrations between the patient groups. Conclusion: A proposed cut-off level of 12 kU/l IgE would identify children above this level correctly as HE-allergic. The level of HE-specific IgG or IgG4 in serum of children with suspected HE allergy does not add any additional information in the diagnostic procedure of HE allergy. For diagnostic purposes, specific IgG or IgG4 should not routinely be tested. [ABSTRACT FROM AUTHOR]

Background Allergen-induced bronchial asthma is a chronic airway disease that involves the interplay of various genes with environmental factors triggering different inflammatory pathways. Objective The aim of this study was to identify possible mediators of airway inflammation (AI) in a model of allergic AI via microarray comparisons and to analyse one of these mediators, Lipocalin2 (Lcn2), for its role in a murine model of allergic airway disease. Methods Gene microarrays were used to identify genes with at least a twofold increase in gene expression in the lungs of two separate mouse strains with high and low allergic susceptibility, respectively. Validation of mRNA data was obtained by Western blotting, followed by functional analysis of one of the identified genes, Lcn2, in mice with targeted disruption of specific gene expression. Epithelial cell cultures were undertaken to define induction requirements and possible mechanistic basis of the results observed in the Lcn2 knock-out mice. Results Lcn2 was up-regulated upon allergen sensitization and airway challenges in lung tissues of both mouse strains and retraced on the protein level in bronchoalveolar lavage fluids. Functional relevance was assessed in mice genetically deficient for Lcn2, which showed enhanced airway resistance and increased AI associated with decreased apoptosis of lung inflammatory cells, compared with wild-type controls. Similarly, application of Lcn2-blocking antibodies before airway challenges resulted in increased inflammation and reduced apoptosis. Conclusion These data indicate a protective role for Lcn2 in allergic airway disease, suggesting a pro-apoptotic effect as the underlying mechanism. Cite this as: A. M. Dittrich, M. Krokowski, H.-A. Meyer, D. Quarcoo, A. Avagyan, B. Ahrens, S. M. Kube, M. Witzenrath, C. Loddenkemper, J. B. Cowland and E. Hamelmann, Clinical & Experimental Allergy, 2010 (40) 1689-1700. [ABSTRACT FROM AUTHOR]

Background Infection with influenza virus has been associated with seemingly opposing effects on the development of asthma. However, there are no data about the effects of mucosal vaccination with inactivated influenza on the inception of allergic asthma. Objective To assess the immunological effects of inhaled inactivated influenza vaccine, using two different types of flu vaccines, on the inception of allergic sensitization and allergen-mediated airway disease in a mouse model. Methods BALB/c mice were intranasally or intratracheally vaccinated with whole or split influenza virus vaccine (days −1 or −1, 27) before systemic sensitization with ovalbumin (OVA) (days 1, 14) and repeated airway allergen challenges (days 28–30). Allergen sensitization (IgE serum levels), airway inflammation (differential cells in bronchoalveolar lavage fluid) and airway hyper-reactivity (AHR) ( in vivo lung function) were analysed. Results The intranasal instillation of whole influenza vaccine before allergen sensitization significantly reduced the serum levels of total and OVA-specific IgE as well as allergen-induced AHR. Prevention was due to an allergen-specific shift from a predominant T helper (Th)2- towards a Th1-immune response. Application of split influenza vaccine did not show the same preventive effect. Conclusion Intranasal administration of inactivated whole influenza vaccine reduced subsequent allergen sensitization and prevented allergen-induced AHR. Our results show that the composition of the influenza vaccine has a major influence on subsequent development of allergen-induced sensitization and AHR, and suggest that mucosal inactivated whole influenza vaccination may represent a step towards the development of a preventive strategy for atopic asthma. [ABSTRACT FROM AUTHOR]

Background Microarray-based component-resolved diagnostics (CRD) has become an accepted tool to detect allergen-specific IgE sensitization towards hundreds of allergens in parallel from one drop of serum. Nevertheless, specificity and sensitivity as well as a simultaneous detection of allergen-specific IgG4, as a potential parameter for tolerance development, remain to be optimized. Objective We applied the recently introduced silicon chip coated with a functional polymer named copoly(DMA-NAS-MAPS) to the simultaneous detection of food allergen-specific IgE and IgG4, and compared it with ImmunoCAP and ImmunoCAP ISAC. Inter- and intraslide variation, linearity of signal and working range, sensitivity and application of internal calibrations for IgE and IgG4 were assessed. Methods Native and recombinant allergenic proteins from hen's egg and cow's milk were spotted on silicon chips coated with copoly(DMA-NAS-MAPS) along with known concentrations for human IgE and IgG4. A serum pool and 105 patient samples were assessed quantitatively and semi-quantitatively with the ImmunoCAP and ImmunoCAP ISAC and correlated with IgE- and IgG4-specific fluorescence on silicon microarrays. Results Allergen-specific IgE and IgG4 were detected in parallel using two fluorescent dyes with no crosstalk. Results from the ImmunoCAP correlated better with microarray fluorescence than with ImmunoCAP ISAC except for the allergen ovomucoid. The working range of the silicon microarray for total hen's egg-specific IgE was comparable to the range of 0.1 to >100 kUA/L of the ImmunoCAP system, whereas for total cow's milk, the silicon microarray was less sensitive. Detectable allergen-specific IgG4 could be determined only for low concentrations, but still correlated positively with ImmunoCAP results. Conclusions We confirmed the ability of the polymer coated silicon microarray to be comparably sensitive to the ImmunoCAP ISAC for various food allergens. This suggests that the copoly(DMA-NAS-MAPS) microarray is a low-cost, self-producible alternative to the commercial ImmunoCAP ISAC in allergy research. [ABSTRACT FROM AUTHOR]