Mediator Release Assay Research Articles

Rapid induction of allergen-blocking IgG in dogs vaccinated with plant-based, Der f 2-expressing bioparticles.

Allergen-carrying virus-like particles are effective and safe means of allergen immunotherapy (AIT) in rodent models. To study the development of allergen-blocking immunoglobulin (Ig)G in dogs injected with Der f 2-carrying enveloped plant-based bioparticles (eBPs). Laboratory beagle dogs were injected intradermally (ID) or subcutaneously (SC) with Der f 2-eBP three times at 2-week intervals. A basophil mediator release assay was used to compare the reactivity of Der f 2-eBPs to that of recombinant Der f 2. Allergen-specific IgG serum levels were determined by immunoblotting and ELISA. The allergen-blocking potential of postvaccination IgG was assessed by Pet Allergy Xplorer (PAX) macroarray and basophil mediator release inhibition assays. The amount of Der f 2 eBPs needed to induce basophil activation was 1000-fold higher than that of the soluble natural allergen. In both immunisation groups, eBP injections caused no adverse events and induced Der f 2-specific IgG, first detected on Day (D)14 and peaking on D41. The co-incubation of sera with a Der f 2-IgE-rich canine serum pool resulted in a mean PAX inhibition of 70% (ID) to 80% (SC) on D41. For both groups, the inhibition of basophil mediator release reached 75% on D28 and D41. The percentage inhibition of PAX and mediator release correlated significantly with Der f 2 IgG levels. Intradermal and subcutaneous injections of Der f 2-eBPs were safe and increased Der f 2-specific IgG. The clinical benefit of immunotherapy will be evaluated in future trials enrolling atopic dogs allergic to house dust mites.

Heterogenous Induction of Blocking Antibodies against Ragweed Allergen Molecules by Allergen Extract-Based Immunotherapy Vaccines.

Currently, allergen-specific immunotherapy (AIT) for ragweed allergy is still based on natural allergen extracts. This study aimed to analyse the ability of four commercially available AIT vaccines (CLUSTOID, TYRO-SIT, POLLINEX Quattro Plus and Diater Depot) regarding their ability to induce IgG antibodies against ragweed pollen allergens in rabbits. Accordingly, the IgG reactivity of AIT-induced rabbit sera was tested for ten different ragweed pollen allergens (Amb a 1, 3, 4, 5, 6, 8, 9, 10, 11 and 12) by an ELISA. Furthermore, the ability of rabbit AIT-specific sera to block allergic patients' IgE binding to relevant ragweed allergens (Amb a 1, 4, 6, 8 and 11) and to inhibit allergen-induced basophil activation was evaluated by an IgE inhibition ELISA and a mediator release assay. Only two AIT vaccines (Diater Depot > CLUSTOID) induced relevant IgG antibody levels to the major ragweed allergen Amb a 1. The IgG responses induced by the AIT vaccines against the other ragweed allergens were low and highly heterogeneous. Interestingly, the kinetics of IgG responses were different among the AIT vaccines and even within one AIT vaccine (Diater Depot) for Amb a 1 (long-lasting) versus Amb a 8 and Amb a 11 (short-lived). This could be due to variations in allergen contents, the immunogenicity of the allergens, and different immunization protocols. The IgE inhibition experiments showed that rabbit AIT-specific sera containing high allergen-specific IgG levels were able to inhibit patients' IgE binding and prevent the mediator release with Diater Depot. The high levels of allergen-specific IgG levels were associated with their ability to prevent the recognition of allergens by patients' IgE and allergen-induced basophil activation, indicating that the measurement of allergen-induced IgG could be a useful surrogate marker for the immunological efficacy of vaccines. Accordingly, the results of our study may be helpful for the selection of personalized AIT vaccination strategies for ragweed-allergic patients.

Insect Cell-Expressed Major Ragweed Allergen Amb a 1.01 Exhibits Similar Allergenic Properties to Its Natural Counterpart from Common Ragweed Pollen.

Common ragweed pollen allergy has become a health burden worldwide. One of the major allergens in ragweed allergy is Amb a 1, which is responsible for over 90% of the IgE response in ragweed-allergic patients. The major allergen isoform Amb a 1.01 is the most allergenic isoform in ragweed pollen. So far, no recombinant Amb a 1.01 with similar allergenic properties to its natural counterpart (nAmb a 1.01) has been produced. Hence, this study aimed to produce a recombinant Amb a 1.01 with similar properties to the natural isoform for improved ragweed allergy management. Amb a 1.01 was expressed in insect cells using a codon-optimized DNA construct with a removable N-terminal His-Tag (rAmb a 1.01). The recombinant protein was purified by affinity chromatography and physicochemically characterized. The rAmb a 1.01 was compared to nAmb a 1.01 in terms of the IgE binding (enzyme-linked immunosorbent assay (ELISA), immunoblot) and allergenic activity (mediator release assay) in well-characterized ragweed-allergic patients. The rAmb a 1.01 exhibited similar IgE reactivity to nAmb a 1.01 in different IgE-binding assays (i.e., IgE immunoblot, ELISA, quantitative ImmunoCAP inhibition measurements). Furthermore, the rAmb a 1.01 showed comparable dose-dependent allergenic activity to nAmb a 1.01 regarding basophil activation. Overall, the results showed the successful expression of an rAmb a 1.01 with comparable characteristics to the corresponding natural isoform. Our findings provide the basis for an improvement in ragweed allergy research, diagnosis, and immunotherapy.

Prospective assessment of pre-existing and de novo anti-HLA IgE in kidney, liver, lung and heart transplantation.

Antibody mediated rejection (ABMR) is a major factor limiting outcome after organ transplantation. Anti-HLA donor-specific antibodies (DSA) of the IgG isotype are mainly responsible for ABMR. Recently DSA of the IgE isotype were demonstrated in murine models as well as in a small cohort of sensitized transplant recipients. In the present study, we aimed to determine the frequency of pre-existing and de novo anti-HLA IgE antibodies in a cohort of 105 solid organ transplant recipients. We prospectively measured anti-HLA IgE antibodies in a cohort of kidney (n=60), liver, heart and lung (n=15 each) transplant recipients before and within one-year after transplantation, employing a single-antigen bead assay for HLA class I and class II antigens. Functional activity of anti-HLA IgE antibodies was assessed by an in vitro mediator release assay. Antibodies of the IgG1-4 subclasses and Th1 and Th2 cytokines were measured in anti-HLA IgE positive patients. Pre-existing anti-HLA IgE antibodies were detected in 10% of renal recipients (including 3.3% IgE-DSA) and in 4.4% of non-renal solid organ transplant recipients (heart, liver and lung cohort). Anti-HLA IgE occurred only in patients that were positive for anti-HLA IgG, and most IgE positive patients had had a previous transplant. Only a small fraction of patients developed de novo anti-HLA IgE antibodies (1.7% of kidney recipients and 4.4% of non-renal recipients), whereas no de novo IgE-DSA was detected. IgG subclass antibodies showed a distinct pattern in patients who were positive for anti-HLA IgE. Moreover, patients with anti-HLA IgE showed elevated Th2 and also Th1 cytokine levels. Serum from IgE positive recipients led to degranulation of basophils in vitro, demonstrating functionality of anti-HLA IgE. These data demonstrate that anti-HLA IgE antibodies occur at low frequency in kidney, liver, heart and lung transplant recipients. Anti-HLA IgE development is associated with sensitization at the IgG level, in particular through previous transplants and distinct IgG subclasses. Taken together, HLA specific IgE sensitization is a new phenomenon in solid organ transplant recipients whose potential relevance for allograft injury requires further investigation.

Biological activity of human IgE monoclonal antibodies targeting Der p 2, Fel d 1, Ara h 2 in basophil mediator release assays.

Human Immunoglobulin E monoclonal antibodies (hIgE mAb) are unique tools for investigating IgE responses. Here, the biological activity of hIgE mAb, derived from immortalized B cells harvested from the blood of allergic donors, targeting three allergens (Der p 2, Fel d 1 and Ara h 2) was investigated. Three Der p 2-, three Fel d 1- and five Ara h 2-specific hIgE mAb produced by human B cell hybridomas, were combined in pairs and used to passively sensitize humanized rat basophilic leukemia cells and compared with sensitization using serum pools. Sensitized cells were stimulated with corresponding allergens (recombinant or purified), allergen extracts or structural homologs, having 40-88% sequence similarity, and compared for mediator (β-hexosaminidase) release. One, two and eight pairs of Der p 2-, Fel d 1- and Ara h 2-specific hIgE mAb, respectively, produced significant mediator release (>50%). A minimum hIgE mAb concentration of 15-30 kU/L and a minimum antigen concentration between 0.01-0.1 µg/mL were sufficient to induce a pronounced mediator release. Individual sensitization with one Ara h 2-specific hIgE mAb was able to induce crosslinking independently of a second specific hIgE mAb. Der p 2- and Ara h 2-specific mAb showed a high allergen specificity when compared to homologs. Mediator release from cells sensitized with hIgE mAb was comparable to serum sensitization. The biological activity of hIgE mAb reported here provides the foundation for novel methods of standardization and quality control of allergen products and for mechanistic studies of IgE-mediated allergic diseases, using hIgE mAb.

Physicochemical and immunological characterization of Amb a 12, a novel ragweed (Ambrosia artemisiifolia) pollen allergen

BackgroundRagweed is an invasive plant in Europe, causing hay fever and asthma in allergic patients. Climate change is predicted to increase expansion and allergenicity. Elevated NO2 induced upregulation of a new allergen in ragweed pollen, an enolase, Amb a 12. Objectiveof this study was producing ragweed enolase as a recombinant protein and characterizing its physicochemical and immunological features. MethodsAmb a 12 was designed for E. coli and insect cell expression. Physicochemical features were determined by mass spectrometry, circular dichroism measurements and enzymatic activity assay. Immunological characteristics were determined in ELISA, in a mediator release assay and by investigation of association with clinical symptoms. Common allergen sources were screened for similar proteins. ResultsRagweed enolase was produced as a 48 kDa protein forming oligomers in both expression systems, showing differences in secondary structure content and enzymatic activity depending on expression system. IgE frequency and allergenicity were low regardless of expression system. Enolase-specific serum bound to similar sized molecules in mugwort, timothy grass and birch pollen, as well as food allergen sources, while highest IgE inhibition was achieved with peach pulp extract. ConclusionsAmb a 12 had high sequence similarity and comparable IgE frequency to enolase allergens from different sources. 50 kDa proteins were found in other pollen and food allergen sources, suggesting that enolases might be pan-allergens in pollen and plant foods.

Ragweed Major Allergen Amb a 11 Recombinant Production and Clinical Implications

Ragweed pollen is highly allergenic and elicits type I hypersensitivity reactions in the exposed populations. Amb a 11 is a recently discovered component of this pollen, and its biological role in allergy is still being researched. In our study, ragweed allergy patients were recruited prospectively over a three-year period; a comprehensive questionnaire was administered, and sera were collected and stored. The production of recombinant Amb a 11 was achieved in parallel with patients' recruitment. The gene coding for mature protein was inserted in E. coli and in Sf9 Spodoptera frugiperda cells. The recombinant allergens (designated eAmb a 11 and iAmb a 11) were tested for His-tag presence in Western blot. IgE reactivity was evaluated in 150 patients' sera for both recombinant allergen forms in ELISA, with 5 positive sera being tested further by hRBL (humanized rat basophilic leukemia) hexosaminidase release assay. Both allergen forms were proven to be IgE-reactive His-tagged proteins, with an extensive overlap of positive sera (92 toward the former recombinant allergen, 100 toward the latter) and an overall Amb a 11 sensitization prevalence estimated at 68.67%. The hRBL mediator release assay revealed a significant, slightly weaker effect of recombinant allergens when compared with nAmb a 1. Sensitization to this major allergen appears to be associated with more severe asthma symptoms (OR = 4.71, 95% CI = 1.81-12.21). In conclusion, recombinant Amb a 11 is a bona fide allergen, which is IgE-reactive and an inducer of hRBL degranulation. It is an important IgE-reactive component from ragweed pollen, with high IgE sensitization prevalence in the sample population and allergenicity of the recombinant allergen comparable to Amb a 1.

Structural and functional analyses of antibodies specific for modified core N-glycans suggest a role in TH 2 responses.

Immune responses to N-glycan structures from allergens and parasites are often associated with pronounced, high affinity IgE reactivities. Cross-reactive carbohydrate determinants (CCDs) are constituted by modified N-glycan core structures and represent the most frequently recognized epitopes in allergic immune responses. Although recently accepted as potentially allergenic epitopes, the biological and clinical relevance as well as structural and functional characteristics of CCD-specific antibodies remain elusive. In order to gain structural insights into the recognition of CCDs, two specific antibody fragments were isolated from a leporid immune repertoire library and converted into human/leporid IgE and IgG formats. The antibody formats were assessed by ELISA and surface plasmon resonance, structural and functional analyses were performed by X-ray crystallography, mediator release, and ELIFAB assays. The recombinant IgE exhibited highly specific interactions with different types of CCDs on numerous CCD-carrying glycoproteins. Crystal structures of two CCD-specific antibodies, one of which in complex with a CCD-derived disaccharide emphasize that mechanisms of core glycan epitope recognition are as specific as those governing protein epitope recognition. The rIgE triggered immediate cellular responses via FcεRI cross-linking and mediated facilitated antigen presentation by binding of IgE/antigen complexes to CD23, a process that also could be blocked by IgG of allergic patients. Our study provides evidence for the relevance of N-glycan recognition in TH 2 responses and corroborates that IgE and IgG antibodies to ubiquitous carbohydrate epitopes can be equivalent to those directed against proteinaceous epitopes with implications for diagnostic and immunotherapeutic concepts.

Humanized Mediator Release Assay as a Read-Out for Allergen Potency.

Mediator release assays analyze in vitro immunoglobulin E (IgE)-mediated degranulation and secretion of mediators by effector cells, such as mast cells and basophils, upon stimulation with serial dilutions of putative allergens. Therefore, these assays represent an essential tool that mimics the in vivo degranulation process, which occurs upon allergen exposure in sensitized patients or in skin prick tests. Additionally, these assays are usually employed to investigate the allergenic potential of proteins and the reactivity of patients' sera's reactivity. Herein, we describe a simple 2-day protocol using an immortalized rat basophil leukemia cell line transfected and humanized with the human high-affinity IgE plasma-membrane receptor (FcεRI). This variant of the mediator release assay is a robust, sensitive, and reproducible in vitro cell-based system without the need to immobilize the antigen to solid matrices. The protocol consists of the following steps: (1) complement inactivation of human sera, (2) harvesting, seeding, and passive sensitization of the cells, (3) stimulation with antigen to cause mediator release, and (4) measuring of β-hexosaminidase activity as a surrogate for the released inflammatory mediators, such as histamine. The assay represents a useful tool to assess the capacity of the allergen-IgE cross-linking to trigger cell degranulation and can be implemented to standardize allergen extracts, to compare patients' reactivity to minor or major allergens and to allergenic extracts (pollen, cat dander, etc.), to investigate the potency of allergen homologs, isoforms, and fold-variants (e.g., hypoallergenicity), as well as the effects of ligands on the allergenic activity. A more recent application includes the use of the assay to monitor the treatment efficacy in the course of allergen immunotherapy.

Humanized Mediator Release Assay as a Read-Out for Allergen Potency

Humanized Mediator Release Assay as a Read-Out for Allergen Potency

A Novel Isoallergen Dau c 1.0401 in Carrot: Stability, Allergenicity, and Comparison with Other Isoallergens

Around 25% of food allergic persons in Central Europe suffer from carrot allergy caused by the major carrot allergen Dau c 1. Three different isoallergens, Dau c 1.01, Dau c 1.02 and Dau c 1.03 are identified. However, information about the qualitative and quantitative composition of natural (n)Dau c 1 is scarce. The new carrot allergen Dau c 1.0401 is identified on the mRNA and protein level by RT-PCR and mass spectrometry. It displays only around 60% sequence identity to the other known Dau c 1 isoallergens. NMR and CD-spectra are typical for a well-folded protein containing both α-helices and β-strands. It showed a poor refolding capacity after incubation at 95 °C. IgE-binding is impaired in immunoblots, whereas in inhibition assays IgE binding to soluble Dau c 1.0401 is detected and it clearly provoked a response in mediator release assays. Dau c 1.0401 is a new isoallergen which contributes to the allergenicity of carrots. The absence of immunoreactivity in immobilized assays indicates that IgE binding is impaired when the protein is blotted on a solid phase. Altogether, the results point out that its allergenicity can be reduced upon carrot processing.

Analysis of the allergenicity and B cell epitopes in tropomyosin of shrimp (Litopenaeus vannamei) and correlation to cross-reactivity based on epitopes with fish (Larimichthys crocea) and clam (Ruditapes philippinarum)

Tropomyosin (TM) is a highly conserved protein that considered as the major allergen of crustacean and mollusk species, while, fish-TM also shares high homology with low allergenicity. In this study, the amino acid sequence, B cell epitopes and allergenicity of shrimp (Litopenaeus vannamei), which is widely consumed, were evaluated by using immunoinformatic tools, dot-blot, enzyme-linked immunosorbent assay (ELISA) and mediator release assay. Meanwhile, cross-reactivity of allergic epitopes of fish-TM, shrimp-TM and clam-TM were assessed. Results showed that three IgE-binding epitopes (X1: 47–61, QKRMQQLENDLDQVQ; X2: 97–108, EDLERSEERLNT and X3: 244–257, RSVQKLQKEVDRLE) of shrimp-TM also exhibited degranulation ability. In comparison with epitopes from shrimp-TM, those from clam-TM showed high cross-reactivity (>80%) and degranulation ability, while those from fish-TM showed low cross-reactivity (<20%). These findings would apply a new understanding of the cross-reactivity of TM from fish, shrimp and clam in terms of allergenic epitopes.

Similar Allergenicity to Different Artemisia Species Is a Consequence of Highly Cross-Reactive Art v 1-Like Molecules.

Background and objectives: Pollens of weeds are relevant elicitors of type I allergies. While many Artemisia species occur worldwide, allergy research so far has only focused on Artemisia vulgaris. We aimed to characterize other prevalent Artemisia species regarding their allergen profiles. Materials and Methods: Aqueous extracts of pollen from seven Artemisia species were characterized by gel electrophoresis and ELISA using sera from mugwort pollen-allergic patients (n = 11). The cDNA sequences of defensin–proline-linked proteins (DPLPs) were obtained, and purified proteins were tested in a competition ELISA, in rat basophil mediator release assays, and for activation of Jurkat T cells transduced with an Art v 1-specific TCR. IgE cross-reactivity to other allergens was evaluated using ImmunoCAP and ISAC. Results: The protein patterns of Artemisia spp. pollen extracts were similar in gel electrophoresis, with a major band at 24 kDa corresponding to DPLPs, like the previously identified Art v 1. Natural Art v 1 potently inhibited IgE binding to immobilized pollen extracts. Six novel Art v 1 homologs with high sequence identity and equivalent IgE reactivity were identified and termed Art ab 1, Art an 1, Art c 1, Art f 1, Art l 1, and Art t 1. All proteins triggered mediator release and cross-reacted at the T cell level. The Artemisia extracts contained additional IgE cross-reactive molecules from the nonspecific lipid transfer protein, pectate lyase, profilin, and polcalcin family. Conclusions: Our findings demonstrate that DPLPs in various Artemisia species have high allergenic potential. Therefore, related Artemisia species need to be considered to be allergen elicitors, especially due to the consideration of potential geographic expansion due to climatic changes.

Development of a progesterone-specific IgE assay for diagnosing patients with suspected progestogen hypersensitivity

BackgroundProgesterone hypersensitivity (PH) manifests as a spectrum of allergic symptoms during the luteal phase of the menstrual cycle. Confirming progesterone-specific immunoglobulin E (IgE; sIgE) by skin testing is unreliable because of irritant responses. ObjectiveTo develop a progesterone sIgE assay to assist in diagnosing PH. MethodsA progesterone–bovine serum albumin (BSA) conjugate was characterized and used to analyze sera collected from women in our center with suspected PH in a 1-batch enzyme-linked immunosorbent assay (ELISA) to establish high, low and negative cut-points. Sera collected from healthy nonatopic female subjects and from women with classical PH symptoms were included as negative and positive controls, respectively. Values exceeding the average negative control (OD + 3× the standard deviation) were considered positive. These cut-points were subsequently used to establish positive and negative results for serum from women with suspected PH received from other centers. A subset of high positive sera was used for ELISA-inhibition and in a beta-hexosaminidase mediator release assay to evaluate the specificity and functional relevance of progesterone-specific serum IgE, respectively. The numbers of true negative, false negative, true positive, and false positive samples were determined. ResultsThe direct progesterone sIgE ELISA results ranged from high positive to low positive and negative compared with healthy nonatopic control sera. Enzyme-linked immunosorbent assay inhibition and beta-hexosaminidase mediator release confirmed specificity and functional relevance of progesterone-sIgE, respectively. The sensitivity, specificity positive predictive values and negative predictive values were found to be 82%, 100%, 86%, and 100%, respectively, using the mediator release assay results as the gold standard. ConclusionThis assay has a good specificity and positive predictive value for screening women with suspected PH for progesterone sIgE.

Chemically modified peanut extract shows increased safety while maintaining immunogenicity.

Peanuts are most responsible for food-induced anaphylaxis in adults in developed countries. An effective and safe immunotherapy is urgently needed. The aim of this study was to investigate the immunogenicity, allergenicity, and immunotherapeutic efficacy of a well-characterized chemically modified peanut extract (MPE) adsorbed to Al(OH)3 . Peanut extract (PE) was modified by reduction and alkylation. Using sera of peanut-allergic patients, competitive IgE-binding assays and mediator release assays were performed. The immunogenicity of MPE was evaluated by measuring activation of human PE-specific T-cell lines and the induction of PE-specific IgG in mice. The safety and efficacy of MPE adsorbed to Al(OH)3 was tested in two mouse models by measuring allergic manifestations upon peanut challenge in peanut-allergic mice. Compared to PE, the IgE-binding and capacity to induce allergic symptoms of MPE were lower in all patients. PE and MPE displayed similar immunogenicity invivo and invitro. In mice sensitized to PE, the threshold for anaphylaxis (drop in BT) upon subcutaneous challenge with PE was 0.01mg, while at 0.3mg MPE no allergic reaction occurred. Anaphylaxis was not observed when PE and MPE were fully adsorbed to Al(OH)3 . Both PE and MPE+Al(OH)3 showed to be efficacious in a model for immunotherapy. In our studies, an Al(OH)3 adsorbed MPE showed reduced allergenicity compared to unmodified PE, while the efficacy of immunotherapy is maintained. The preclinical data presented in this study supports further development of modified peanut allergens for IT.

Germination results in reduced allergenicity of peanut by degradation of allergens and resveratrol enrichment

Abstract The germination process may produce bioactive compounds that could attenuate the antigenicity of peanut. However, it is poorly understood whether germinated peanut causes less severe immunogenic responses and if resveratrol is beneficial for the prevention of peanut allergy. In this study, allergen content and resveratrol production were measured during the germination process. Immunoreactivity was evaluated by immunoblotting and ELISA assay. The allergic activities of germinated peanut were determined by the mediator-release assay using RBL-2H3 cells. The results showed that the total protein content and the most allergenic proteins, Ara h 1, 3, and 2/6, were significantly degraded after germination. The IgE reactivity of five day-germinated peanut was significantly decreased as determined by immunoassay and mediator release assay. A lower concentration (1 nmol/L) resveratrol had an inhibitory effect on β‑hexosaminidase release. The degradation of allergens and resveratrol enrichment may be responsible for the lower allergenicity of germinated peanut.

Does clinical outcome of birch pollen immunotherapy relate to induction of blocking antibodies preventing IgE from allergen binding? A pilot study monitoring responses during first year of AIT

BackgroundThe clinical benefit of allergen-specific immunotherapy (AIT) involves induction of blocking antibodies. It is not clear if these antibodies function via steric hindrance alone or a combination of levels, avidities, and epitope specificities, and clinical outcome cannot be predicted. We aim to in-depth characterize serum antibody profiles during birch pollen AIT, investigate therapy-induced antibodies for their capacity to block IgE binding to Bet v 1 and correlate data with clinical outcomes.MethodsImmune responses of five birch pollen allergic patients were monitored during the first year of AIT by nasal provocation tests (NPTs), ImmunoCAP, immunoblots, direct and avidity enzyme-linked immunosorbent assays, mediator release assays, facilitated antigen binding (FAB) assays, and inhibition mediator release assays.ResultsThere was no correlation between NPT results and therapy-induced changes in levels (IgE, IgG, IgA, IgM), avidities, or mediator release potency of Bet v 1-specific antibodies. In FAB assays, blocking antibodies initiated upon AIT were shown to prevent formation of Bet v 1-IgE complexes of an indicator serum pool and significantly correlated with clinical readout. Inhibition mediator release assays using patient-specific IgE for passive sensitization revealed therapy-induced blocking capacities with very good correlation to NPT results. Notably, this assay was the only one to detect a non-responder during treatment in this pilot study.ConclusionsClinical outcome of AIT depends on induction of blocking antibodies able to prevent the patient’s own IgE from allergen binding. Monitoring of clinical efficacy seems to be best achieved using the inhibition mediator release assay, as development of relevant blocking antibodies can be verified in a patient-tailored manner.

Endolysosomal protease susceptibility of Amb a 1 as a determinant of allergenicity

summary In the absence of extrinsic adjuvants, purified Amb a 1 induced a strong Th2-polarized immune response in mice. This high allergenic activity seems to be linked to the allergen’s susceptibility to endolysosomal proteolysis.

Thermal processing effects on the IgE-reactivity of cashew and pistachio

Thermal processing can modify the structure and function of food proteins and may alter their allergenicity. This work aimed to elucidate the influence of moist thermal treatments on the IgE-reactivity of cashew and pistachio. IgE-western blot and IgE-ELISA were complemented by Skin Prick Testing (SPT) and mediator release assay to determine the IgE cross-linking capability of treated and untreated samples. Moist thermal processing diminished the IgE-binding properties of both nuts, especially after heat/pressure treatment. The wheal size in SPT was importantly reduced after application of thermally-treated samples. For cashew, heat/pressure treated-samples still retain some capacity to cross-link IgE and degranulate basophils, however, this capacity was diminished when compared with untreated cashew. For pistachio, the degranulation of basophils after challenge with the harshest heat/pressure treatment was highly decreased. Boiling produced more variable results, however this treatment applied to both nuts for 60 min, led to an important decrease of basophil degranulation.

Orai and TRPC channel characterization in FcεRI-mediated calcium signaling and mediator secretion in human mast cells.

Inappropriate activation of mast cells via the Fcε RI receptor leads to the release of inflammatory mediators and symptoms of allergic disease. Calcium influx is a critical regulator of mast cell signaling and is required for exocytosis of preformed mediators and for synthesis of eicosanoids, cytokines and chemokines. Studies in rodent and human mast cells have identified Orai calcium channels as key contributors to Fcε RI‐initiated mediator release. However, until now the role of TRPC calcium channels in Fcε RI‐mediated human mast cell signaling has not been published. Here, we show evidence for the expression of Orai 1,2, and 3 and TRPC1 and 6 in primary human lung mast cells and the LAD2 human mast cell line but, we only find evidence of functional contribution of Orai and not TRPC channels to Fcε RI‐mediated calcium entry. Calcium imaging experiments, utilizing an Orai selective antagonist (Synta66) showed the contribution of Orai to Fcε RI‐mediated signaling in human mast cells. Although, the use of a TRPC3/6 selective antagonist and agonist (GSK‐3503A and GSK‐2934A, respectively) did not reveal evidence for TRPC6 contribution to Fcε RI‐mediated calcium signaling in human mast cells. Similarly, inactivation of STIM1‐regulated TRPC1 in human mast cells (as tested by transfecting cells with STIM1‐KK 684‐685 EE ‐ TRPC1 gating mutant) failed to alter Fcε RI‐mediated calcium signaling in LAD2 human mast cells. Mediator release assays confirm that Fcε RI‐mediated calcium influx through Orai is necessary for histamine and TNF α release but is differentially involved in the generation of cytokines and eicosanoids.