Search

Background: Approximately 70% of individuals allergic to birch pollen (Bet v 1.01 [Bet]) develop a secondary food allergy (e.g., hazelnut: Cor a 1.04 [Cor]), due to allergen cross‐reactivity. However, standard immunotherapy for type I allergies often does not improve the food allergy sufficiently. We analyzed the allergen‐specific and cross‐reactive suppressive capacity of primary human regulatory T cells (Treg) induced by autologous IL‐10‐modulated dendritic cells (IL‐10 DC) in vitro and in vivo. Methods: CD4+ T cells of patients with birch pollen and associated hazelnut allergies were differentiated into Bet‐specific or non‐specific induced Treg (iTreg). After Bet‐ or Cor‐specific restimulation the phenotype, proliferation, and suppressive capacity of iTreg subsets were analyzed. iTreg function was further investigated in humanized mouse models of airway and intestinal allergy, generated by engraftment of peripheral blood mononuclear cells from allergic donors into immunodeficient animals. Results: After IL‐10 DC priming and allergen‐specific restimulation (Bet or Cor), non‐specific control iTreg remained anergic, whereas Bet‐specific iTreg proliferated extensively and exhibited a regulatory phenotype (enhanced expression of CTLA‐4, PD‐1, TNFR2, IL‐10). Accordingly, activated Bet‐specific iTreg displayed a high capacity to suppress Bet‐ and Cor‐induced responder Th2 cell responses in vitro, indicating induction of both allergen‐specific (birch) and cross‐reactive tolerance (hazelnut). In vivo, the beneficial effect of Bet‐specific iTreg was verified in humanized mouse models of allergic airway and intestinal inflammation, resulting in reduced allergen‐induced clinical symptoms, and immune responses. Conclusion: Human IL‐10 DC‐induced iTreg facilitate allergen‐specific and cross‐reactive tolerance. Therefore, they are potential candidates for regulatory cell therapy in allergic and autoimmune diseases. [ABSTRACT FROM AUTHOR]

Introduction: Allergen‐specific IgE‐blocking IgG antibodies contribute to successful allergen immunotherapy (AIT), however, not much is known about their affinity. Since affinity measurements of polyclonal antibodies in serum are technically challenging we evaluated the applicability of acidic disruption of antibody‐allergen complexes by a modified ELISA protocol with monoclonal antibodies (mAbs) specific for the relevant major allergens Betv1 and Mald1. Then, AIT‐induced blocking and non‐blocking Mald1‐specific antibodies in sera from individuals with or without reduced apple allergy were compared. Methods: After testing their pH stability coated recombinant allergens were incubated with (i) mAbs diluted in PBS or human serum and (ii) sera from individuals after sublingual administration of Mald1 or Betv1 for 16 weeks. Immune complexes were exposed to buffers in the pH range of 6.4–3.4 and residual antibodies were measured. Avidity indexes (AI), defined as the pH removing 50% of antibodies, were compared to the dissociation constants (KD) of mAbs determined by surface plasmon resonance. Results: The selected pH range was applicable to disrupt allergen complexes with highly affine mAbs without compromising allergen integrity. AIs of mAbs accorded with KD values and were unaffected by epitope specificity or the presence of serum proteins. The inter‐assay variability was <4% CV. Protective Mald1‐specific IgG antibodies from individuals with reduced apple allergy showed a higher collective binding strength than that of the non‐protective antibodies of individuals without reduced apple allergy. Conclusion: Acidic disruption of allergen‐antibody complexes may be used to estimate the net‐binding force of polyclonal serum antibodies and eases the investigation of affinity‐related research questions in AIT. [ABSTRACT FROM AUTHOR]

Background: The experimental fusion protein rFlaA:Betv1 was shown to efficiently suppress allergen‐specific sensitization in mice. However, the detailed mechanism of rFlaA:Betv1‐mediated immune modulation is not fully understood. In this study, we investigated the effect of rFlaA:Betv1 on naïve murine B cells. Methods: Immune modulating capacity of rFlaA:Betv1 was screened in IL‐10 reporter mice. B cells were isolated from spleens of naïve C57Bl/6, TLR5−/−, or MyD88−/− mice, stimulated with rFlaA:Betv1 and controls, and monitored for the expression of the regulatory B cell markers CD1d, CD24, CD38, and surface IgM by flow cytometry. Secreted cytokines, antibodies, and reactivity of the induced antibodies were investigated by ELISA and intracellular flow cytometry. Suppressive capacity of rFlaA:Betv1‐stimulated B cells was tested in mDC:CD4+ T cell:B cell triple cultures. Results: Upon in vivo application of rFlaA:Betv1 into IL‐10‐GFP reporter mice, CD19+ B cells were shown to produce anti‐inflammatory IL‐10, suggesting B cells to contribute to the immune‐modulatory properties of rFlaA:Betv1. rFlaA:Betv1‐induced IL‐10 secretion was confirmed in human B cells isolated from buffy coats. In vitro stimulation of naïve murine B cells with rFlaA:Betv1 resulted in an mTOR‐ and MyD88‐dependent production of IL‐10 and rFlaA:Betv1 induced Bet v 1‐reactive IgG production, which was not observed for IgA. rFlaA:Betv1‐stimulated B cells formed a CD19+CD24+CD1d+IgM+CD38+ Breg subpopulation capable of suppressing Bet v 1‐induced TH2 cytokine secretion in vitro. Conclusion: rFlaA:Betv1 can act as a thymus‐independent B cell antigen, stimulating the mTOR‐ and MyD88‐dependent differentiation of B cells displaying a regulatory phenotype, IL‐10 secretion, antigen‐binding antibody production, and a suppressive capacity in vitro. [ABSTRACT FROM AUTHOR]

Purpose of Review: The recent introduction of edible insects in Western countries has raised concerns about their safety in terms of allergenic reactions. The characterization of insect allergens, the sensitization and cross-reactivity mechanisms, and the effects of food processing represent crucial information for risk assessment. Recent Findings: Allergic reactions to different insects and cross-reactivity with crustacean and inhalant allergens have been described, with the identification of new IgE-binding proteins besides well-known pan-allergens. Depending on the route of sensitization, different potential allergens seem to be involved. Food processing may affect the solubility and the immunoreactivity of insect allergens, with results depending on species and type of proteins. Chemical/enzymatic hydrolysis, in some cases, abolishes immunoreactivity. Summary: More studies based on subjects with a confirmed insect allergy are necessary to identify major and minor allergens and the role of the route of sensitization. The effects of processing need to be further investigated to assess the risk associated with the ingestion of insect-containing food products. [ABSTRACT FROM AUTHOR]

However, celeriac allergic patients often show sensitization to mugwort defensin Art v 1 (Table S1).6 This prompted us to investigate the potential presence and role of a defensin-related allergen in allergy to celeriac. IgE-binding capacity and allergenicity of celeriac defensin (officially named Api g 7, www.allergen.org4) was investigated by immunoblotting and ImmunoCAP testing in eight celeriac allergic patients, two celeriac-tolerant controls with mugwort pollen allergy and a non-atopic control. Celeriac allergy is often accompanied by respiratory allergy to mugwort ( I Artemisia vulgaris i ) pollen,5 which cannot be explained by IgE cross-reactivity to known homologous celeriac allergens. [Extracted from the article]

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. [ABSTRACT FROM AUTHOR]

Background: The use of hypoallergenic derivatives is considered beneficial to promote the safety and efficacy of allergen-specific immunotherapy. We aimed to assess the efficacy of reduced and alkylated (R/A) Pru p 3, a hypoallergenic folding variant of the major peach allergen, in subcutaneous immunotherapy (SCIT) using a murine model of peach allergy.Methods and Results: After sensitization with Pru p 3, BALB/c mice received SCIT with Pru p 3 or R/A Pru p 3 and were challenged with Pru p 3. SCIT with Pru p 3, but not with R/A Pru p 3, suppressed anaphylaxis upon the challenge significantly. SCIT with Pru p 3 did not suppress Pru p 3-specific IgE and IgG1 production, but enhanced IgG2a production. In contrast, SCIT with R/A Pru p 3 suppressed IgE and IgG1 production, but enhanced IgG2a production only moderately. The therapeutic efficacy of SCIT with Pru p 3 was associated with induction of IL-10 and IFN-γ.Conclusion: Hypoallergenic folding variant of Pru p 3 is not likely an efficacious therapeutic component in SCIT of peach allergy. The lower efficacy of R/A Pru p 3 might be attributed to poor antigenicity and/or weak stability due to its unfolded conformation. [ABSTRACT FROM AUTHOR]

Background: TLR ligands can promote Th1-biased immune responses, mimicking potent stimuli of viruses and bacteria.Aim: To investigate the adjuvant properties of dual TLR2/7 ligands compared to those of the mixture of both single ligands.Methods: Dual TLR2/7 ligands: CL401, CL413, and CL531, including CL264 (TLR7-ligand) and Pam2CysK4 (TLR2-ligand), were used. Immune-modulatory capacity of the dual ligands with the individual ligands alone or as a mixture in mouse BMmDCs, BMmDC:TC cocultures, or BMCMCs was compared and assessed in naïve mice and in a mouse model of OVA-induced intestinal allergy.Results: CL413 and CL531 induced BMmDC-derived IL-10 secretion, suppressed rOVA-induced IL-5 secretion from OVA-specific DO11.10 CD4+ TCs, and induced proinflammatory cytokine secretion in vivo. In contrast, CL401 induced considerably less IL-10 secretion and led to IL-17A production in BMmDC:TC cocultures, but not BMCMC IL-6 secretion, or IL-6 or TNF-α production in vivo. No immune-modulating effects were observed with single ligands. All dual TLR2/7 ligands suppressed DNP-induced IgE-and-Ag-specific mast cell degranulation. Compared to vaccination with OVA, vaccination with the mixture CL531 and OVA, significantly suppressed OVA-specific IgE production in the intestinal allergy model.Conclusions: Based on beneficial immune-modulating properties, CL413 and CL531 may have utility as potential adjuvants for allergy treatment. [ABSTRACT FROM AUTHOR]

Conjugated vaccines consisting of flagellin and antigen activate TLR5 and induce strong innate and adaptive immune responses. Objective of the present study was to gain further insight into the mechanisms by which flagellin fusion proteins mediate their immune modulating effects. In a mouse model of Ova-induced intestinal allergy a fusion protein of flagellin and Ova (rflaA:Ova) was used for intranasal and intraperitoneal vaccination. Aggregation status of flaA, Ova and flaA:Ova were compared by light scattering, uptake of fluorescence labeled proteins into mDC was analyzed, processing was investigated by microsomal digestion experiments. Mechanism of DC-activation was investigated using proteasome and inflammasome inhibitors. Immune responses of wildtype, IL-10−/−, TLR5−/− mDCs and Ova-transgenic T cells were investigated. Mucosal and i.p.-application of rflaA:Ova were able to prevent allergic sensitization, suppress disease-related symptoms, prevent body weight loss and reduction in food uptake. Intranasal vaccination resulted in strongest suppression of Ova-specific IgE production. These protective effects were associated with increased aggregation of rflaA:Ova and accompanied by tenfold higher uptake rates into mDC compared to the mixture of both proteins. Microsomal digestion showed that stimulation with rflaA:Ova resulted in faster degradation and the generation of different peptides compared to rOva. rflaA:Ova-mediated activation of mDC could be suppressed in a dose-dependent manner by the application of both inflammasome and proteasome inhibitors. Using TLR5−/− mDC the rflaA:Ova induced IL-10 secretion was shown to be TLR5 dependent. In co-cultures of IL-10−/− mDC with DO11.10 T cells the lack of rflaA:Ova-mediated IL-10 secretion resulted in enhanced levels of both TH2 (IL-4, IL-5) and TH1 (IL-2 and IFN-y) cytokines. In summary, mucosal vaccination with flaA:Ova showed strongest preventive effect. Stimulation with rflaA:Ova results in strong immune modulation mediated by enhanced uptake of the aggregated fusion protein, likely resulting in a different processing by DC as well as stronger TLR5 mediated cell activation. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Evaluates the use of optimized allergen extracts and recombinant allergens in diagnostic applications. Serological data of patients with pollen-related allergy to cherry; Association between specific immunoglobulin E responses in allergic patients and the pathomechanism of the disease condition; Characterization of various cherry allergens.

Developing new adjuvants/vaccines and better understanding their mode-of-action is an important task. To specifically improve birch pollen allergy treatment, we designed a fusion protein consisting of major birch pollen allergen Betv1 conjugated to the TLR5-ligand flagellin (rFlaA:Betv1). This study investigates the immune-modulatory effects of rFlaA:Betv1 on airway epithelial cells. LA-4 mouse lung epithelial cells were stimulated with rFlaA:Betv1 in the presence/absence of various inhibitors with cytokine- and chemokine secretion quantified by ELISA and activation of intracellular signaling cascades demonstrated by Western blot (WB). Either LA-4 cells or LA-4-derived supernatants were co-cultured with BALB/c bone marrow-derived myeloid dendritic cells (mDCs). Compared to equimolar amounts of flagellin and Betv1 provided as a mixture, rFlaA:Betv1 induced higher secretion of IL-6 and the chemokines CCL2 and CCL20 from LA-4 cells and a pronounced MAPK- and NFκB-activation. Mechanistically, rFlaA:Betv1 was taken up more strongly and the induced cytokine production was inhibited by NFκB-inhibitors, while ERK- and p38-MAPK-inhibitors only suppressed IL-6 and CCL2 secretion. In co-cultures of LA-4 cells with mDCs, rFlaA:Betv1-stimulated LA-4 cells p38-MAPK- and COX2-dependently secreted PGE2, which modulated DC responses by suppressing pro-inflammatory IL-12 and TNF-α secretion. Taken together, these results contribute to our understanding of the mechanisms underlying the strong immune-modulatory effects of flagellin-containing fusion proteins. [ABSTRACT FROM AUTHOR]

• Allergy to multi-ingredient dishes requires the identification of hidden allergens. • 2S albumin and nsLTP allergens in pizza-allergic patients present limited cross-reactivity. • Identify allergens underrepresented in extracts is essential to an accurate diagnosis. • Reducing agents can mimic the allergenic epitope changes in industrial food processing. Although pizza is one of the most popular foods in the world, allergic responses after ingesting pizza are relatively uncommon. However, precisely identifying the allergens responsible for these allergic reactions is challenging because of the high and diverse number of ingredients used in pizza preparation. In this report, we aim to identify the allergens responsible for systemic allergic reactions following ingestion of pizza in two patients. Using a skin prick by prick test (SPPT) and in vitro techniques, with natural and recombinant purified allergens from tomato and mustard seeds, we identified 2S albumin and non-specific lipid transfer proteins (nsLTP) as the proteins involved. However, IgE responses to the four nsLTPs differed before and after denaturation and reduction, thus suggesting additional complexity around nsLTP in food processing. [ABSTRACT FROM AUTHOR]

• Shrimp sensitized patients' IgE recognized several cricket proteins. • High IgE-cross-reactivity was found among shrimp and cricket tropomyosins. • Cricket tropomyosin was more stable to digestion than the shrimp homologue. • Baking enhances stability of IgE-binding proteins to gastric digestion. • Stability of cricket tropomyosin represents a risk of primary sensitization. The allergenic potency of the cricket Acheta domesticus , a promising edible insect, has never been assessed. This work aims to study the immunoreactivity of Acheta domesticus , and its cross-reactivity with the shrimp Litopenaeus vannamei , assessing the effect of cooking and gastrointestinal digestion on their allergenic properties. Different cricket proteins were detected by immunoblotting with shrimp-allergic patients' sera. Tropomyosin was identified as the most relevant IgE-binding protein, and its cross-reactivity with shrimp tropomyosin was demonstrated by ELISA. While shrimp tropomyosin showed scarce stability to gastric digestion, cricket tropomyosin withstood the whole digestion process. The sarcoplasmic calcium-binding protein, specifically detected in shrimp, showed exceptional stability to gastrointestinal digestion. IgE-binding proteins in a model of enriched baked products were partially protected from proteolysis. In conclusion, the ingestion of A. domesticus proteins poses serious concerns to the Crustacean-allergic population. The high stability of tropomyosin may represent a risk of primary sensitization and clinical cross-reactivity. [ABSTRACT FROM AUTHOR]