Allergic Reactions In Individuals Research Articles

Detection of common adulterants in olive oils by bench top 60 MHz 1H NMR with partial least squares regression

Olive oil is the oil obtained from the fruit of the olive tree, Olea europaea L., and is commonly used in cooking, cosmetics, and soaps. The high value and demand for olive oil products has led to some producers adding cheaper edible non-olive oils to olive oil to increase their profitability. Many of these adulterant oils have similar taste profiles to extra virgin olive oil; however, these can cause allergic reactions in individuals with sensitivities to the non-olive oil. Given the implications of olive oil adulteration, there is a demand for methods to identify and quantify adulterant oils in olive oil samples. This work explores the utility of low-field benchtop nuclear magnetic resonance (NMR) with a novel chemometrics workflow for the quantification of non-olive oils in authentic extra virgin olive oil samples. The measurements were obtained using a 60 MHz 1H NMR. The icoshift algorithm was utilized for alignment, and partial least squares regression models of the entire NMR spectrum were used for quantification. Here, 17 different varieties of edible non-olive oils were investigated. The models were highly linear, accurate, and precise. Most of these adulterant oils were detectable below 10% v/v in extra virgin olive oil samples.

Post hoc analysis examining symptom severity reduction and symptom absence during food challenges in individuals who underwent oral immunotherapy for peanut allergy: results from three trials

PurposePeanut allergy and its current management, involving peanut avoidance and use of rescue medication during instances of accidental exposure, are burdensome to patients and their caregivers and can be a source of stress, uncertainty, and restriction. Physicians may also be frustrated with a lack of effective and safe treatments other than avoidance in the current management of peanut allergy. Efficacy, determined using double-blind, placebo-controlled food challenges (DBPCFCs), of oral immunotherapy with peanut (Arachis hypogaea) allergen powder-dnfp (PTAH; Palforzia®) was demonstrated versus placebo in children and adolescents aged 4 to 17 years in multiple phase 3 trials; continued benefit of PTAH was shown in a follow-on trial. The DBPCFC is a reproducible, rigorous, and clinically meaningful assessment accepted by regulatory authorities to evaluate the level of tolerance as an endpoint for accidental exposures to peanut in real life. It also provides useful clinical and patient-relevant information, including the amount of peanut protein an individual with peanut allergy can consume without experiencing dose-limiting symptoms, severity of symptoms, and organs affected upon ingestion of peanut protein. We explored symptoms of peanut exposure during DBPCFCs from phase 3 and follow-on trials of PTAH to further characterize treatment efficacy from a perspective relevant to patients, caregivers, and clinicians.MethodsSymptom data recorded during screening and/or exit DBPCFCs from participants aged 4 to 17 years receiving PTAH or placebo were examined post hoc across three PTAH trials (PALISADE [ARC003], ARC004 [PALISADE follow-on], and ARTEMIS [ARC010]). The maximum peanut protein administered as a single dose during DBPCFCs was 1000 mg (PALISADE and ARTEMIS) and 2000 mg (ARC004). Symptoms were classified by system organ class (SOC) and maximum severity. Endpoints were changes in symptom severity and freedom from symptoms (ie, asymptomatic) during DBPCFC. Relative risk (RR) was calculated for symptom severity by SOC and freedom from symptoms between groups; descriptive statistics were used to summarize all other data.ResultsThe risk of any respiratory (RR 0.42 [0.30–0.60], P < 0.0001), gastrointestinal (RR 0.34 [0.26–0.44], P < 0.0001), cardiovascular/neurological (RR 0.17 [0.08–0.39], P < 0.001), or dermatological (RR 0.33 [0.22–0.50], P < 0.0001) symptoms was significantly lower in participants treated with PTAH versus placebo upon exposure to peanut at the end of the PALISADE trial (ie, exit DBPCFC). Compared with placebo-treated participants (23.4%), the majority (76.3%) of PTAH-treated participants had no symptoms at the exit DBPCFC when tested at the peanut protein dose not tolerated (ie, reactive dose) during the screening DBPCFC. Significantly higher proportions of PTAH-treated participants were asymptomatic at doses ≤ 100 mg in the exit DBPCFC compared with placebo-treated participants (PALISADE: 69.35% vs 12.10%, RR 5.73 [95% confidence interval (CI) 3.55–9.26]; P < 0.0001; ARTEMIS: 67.42% vs 13.95%, RR 4.83 [95% CI 2.28–10.25]; P < 0.0001); findings were similar at peanut protein doses ≤ 1000 mg (PALISADE: RR 15.56 [95% CI 5.05–47.94]; P < 0.0001; ARTEMIS: RR 34.74 [95% CI 2.19–551.03]; P < 0.0001). In ARC004, as the period of PTAH maintenance became longer, greater proportions of participants were asymptomatic at doses of peanut protein ≤ 1000 mg in the exit DBPCFC (from 37.63% after ~ 6 months of maintenance treatment [exit DBPCFC of PALISADE] to 45.54% after ~ 13 months and 58.06% after ~ 20 months of overall PTAH maintenance treatment).ConclusionsPTAH significantly reduced symptom severity due to exposure to peanut, which is clinically relevant. When exposed to peanut, participants with peanut allergy treated with PTAH rarely had moderate or severe respiratory or cardiovascular/neurological symptoms. Oral immunotherapy with PTAH appears to reduce frequency and severity of allergic reactions in individuals with peanut allergy after accidental exposure to peanut and may enable them and their families to have an improved quality of life.Trial registration ClinicalTrials.gov, NCT02635776, registered 17 December 2015, https://clinicaltrials.gov/ct2/show/NCT02635776?term=AR101&draw=2&rank=7; ClinicalTrials.gov, NCT02993107, registered 08 December 2016, https://clinicaltrials.gov/ct2/show/NCT02993107?term=AR101&draw=2&rank=6; ClinicalTrials.gov, NCT03201003, registered 22 June 2017, https://clinicaltrials.gov/ct2/show/NCT03201003? term = AR101&draw = 2&rank = 9

Survey for Immune Response and Allergic Reactions in Individuals with Covid Vaccination at Kannur District, Kerala, India

Vaccination is a boon for humans as it prevents the microbes to cause disease. In the similar aspect it is important to know the immunoresponse generated by the administered vaccine. Such a study may help to improve the performance of vaccines and thereby to understand its suitability for individuals of various age group and physiological conditions. A Survey of individuals for COVID-19 vaccination from the Kannur district of Kerala from India is reflecting that most of the individuals are showing immune response post vaccination by Covishield and Covaxin. Different types of immunoresponse such as fever, headache, body pain, vomiting and nausea are observed. However, few individuals are not showing any above type of immunoresponse. Also, vaccinations are given mostly to an individual without knowledge of medical history or physiology.

Lupine allergens: Clinical relevance, molecular characterization, cross-reactivity, and detection strategies.

Lupine is commonly utilized as a technological food and ingredient in a great variety of processed products (snacks, bakery, meat, and dairy products) principally owing to its nutritional value and technological properties. However, its ingestion, even at trace amounts (in the range of mg protein per kg of food), can lead to severe adverse reactions in allergic individuals. Lupine belongs to the Leguminosae family, having the conglutins (α-, β-, δ-, and γ-) as allergens, among other proteins. Cross-sensitization of lupine-sensitized individuals with other legume species, mainly peanut, can occur, but the associated clinical reactivity is still unclear. The protection of the sensitized individuals should depend on an avoidance diet, which should rely on the compliance of food labeling and, as such, on their verification by analytical methods. Food processing, such as heat treatments, has an important influence on the structural properties of lupine proteins, altering their detectability and allergenicity. In this review, different aspects related with lupine allergy are described, namely, the overall prevalence, clinical relevance, diagnosis, and treatment. The characterization of lupine allergens and their potential cross-reactivity with other legumes are critically discussed. The effects of food matrix, processing, and digestibility on lupine proteins, as well as the available analytical tools for detecting lupine at trace levels in foods, are also herein emphasized.

Phage Displayed Domain Antibodies (dAb) for Detection of Allergenic Pistachio Proteins in Foods.

Pistachio nuts (Pistacia vera) have been consumed by past and present-day civilizations because of their organoleptic characteristics and potential health benefits. However, they can also produce moderate to severe IgE-mediated reactions in allergic individuals. In this work, we report the isolation of the first recombinant antibodies against pistachio nut, produced without animal immunization, to be used in immunoassays for detection of allergenic pistachio in food products. Several phage display biopanning strategies were evaluated to screen the human-based domain antibody library (dAb) in search for pistachio-specific probes. The clone producing the PVF4 phage-dAb was finally selected, and it does not cross-react with cashew despite the phylogenetic proximity with pistachio. Western blot and matrix-assisted laser desorption/ionization tandem mass spectrometry (MALDI-TOF/TOF) analysis demonstrated that this clone recognised a unique band of ∼22 kDa related to the basic subunit of pistachio 11S globulin (allergen Pis v 2). The PVF4 phage-dAb allowed detection of pistachio in a food matrix with a limit of detection (LOD) of 3983 mg kg−1 in an indirect phage-enzyme-linked immunosorbent assay (ELISA). The ELISA method developed was used to assess applicability of the PVF4 phage-dAb for analysis of 77 commercial food products.

Phenotypic and Molecular Characterization of Risk Loci Associated With Asthma and Lung Function.

PurposeRespiratory diseases have a highly multifactorial etiology where different mechanisms contribute to the individual's susceptibility. We conducted a deep characterization of loci associated with asthma and lung function by previous genome-wide association studies (GWAS).MethodsSixteen variants were selected from previous GWAS of childhood/adult asthma and pulmonary function tests. We conducted a phenome-wide association study of these loci in 4,083 traits assessed in the UK Biobank (n = 361,194 participants). Data from the Genotype-Tissue Expression (GTEx) project were used to conduct a transcriptomic analysis with respect to tissues relevant for asthma pathogenesis. A pediatric cohort assessed with the International Study of Asthma and Allergies in Children (ISAAC) Phase II tools was used to further explore the association of these variants with 116 traits related to asthma comorbidities.ResultsOur phenome-wide association studies (PheWAS) identified 206 phenotypic associations with respect to the 16 variants identified. In addition to the replication of the phenotypes tested in the discovery GWAS, we observed novel associations related to blood levels of immune cells (eosinophils, neutrophils, monocytes, and lymphocytes) for the asthma-related variants. Conversely, the lung-function variants were associated with phenotypes related to body fat mass. In the ISAAC-assessed cohort, we observed that risk alleles associated with increased fat mass can exacerbate allergic reactions in individuals affected by allergic respiratory diseases. The GTEx-based analysis showed that the variants tested affect the transcriptomic regulation of multiple surrounding genes across several tissues.ConclusionsThis study generated novel data regarding the genetics of respiratory diseases and their comorbidities, providing a deep characterization of loci associated with asthma and lung function.

Suspected Reaction With Cephalosporin May Be a Predictive Factor for β-Lactam Allergy in Children

To determine the frequency and risk factors of confirmed allergy in patients who report β-lactam allergy and evaluate cross-reactivity between β-lactams in children with confirmed allergy.The population included 67 children evaluated at an academic allergy and immunology practice in Turkey from 2012 to 2017.All patients referred to an outpatient clinic with history of immediate reaction to β-lactam antibiotics were included. Patients completed a questionnaire and underwent workup on the basis of the European Network for Drug Allergy guidelines. Serum-specific immunoglobulin E to penicilloyl V, penicilloyl G, amoxicilloyl, and ampicilloyl were measured by UniCAP in all patients. Patients with undetectable serum results underwent skin prick tests (SPTs). Intradermal testing was performed if the SPT result was negative. A drug provocation test with suspected β-lactam was performed in patients with negative workup.β-lactam allergy was confirmed in 14.9% of patients who reported a history of allergy. All of the allergic patients had negative results on SPT and serology. Four patients with negative intradermal results had a reaction (urticaria) with the drug provocation test. Age, sex, time interval since reaction, atopy, reaction type, parental history of drug allergy, and multiple drug allergy history were not significantly different between allergic and tolerant patients. The rate of confirmed diagnosis was higher for the group that reported cephalosporin allergy compared with report of penicillin allergy (P = .03). Three patients with allergy to penicillin tolerated cefuroxime. Four patients with selective allergy to ceftriaxone tolerated cephalosporins with dissimilar side chains.Appropriate evaluation of suspected drug allergy will lead to accurate diagnoses and reduce the use of less-effective yet more-expensive alternative antibiotics.This study supports a large body of literature demonstrating that most patients who report a β-lactam allergy tolerate the drug. Neither serum immunoglobulin E testing nor SPT alone are sufficient to evaluate β-lactam allergy. Even intradermal testing may miss some truly allergic patients. The gold standard, drug provocation challenge, is required to confirm a diagnosis of allergy. The clinical history and available diagnostic results must be weighed to determine if drug provocation challenge is appropriate because reactions in allergic individuals can be potentially life-threatening.

Engineered ovalbumin-expressing regulatory T cells protect against anaphylaxis in ovalbumin-sensitized mice

Allergy is a major public health concern, the main treatment for which is symptomatic relief with anti-inflammatory drugs. A key clinical challenge is to induce specific tolerance in order to control allergen-specific memory B and T cells, and specifically block effector cell responses. Our lab recently developed antigen-specific regulatory T-cell (Treg) therapies as a treatment for adverse responses. Recently, we created a chimeric antigen receptor (CAR) approach in which we engineered a target protein antigen, ovalbumin (OVA), linked with the transmembrane and signal transduction domains, CD28-CD3ζ to directly target B cells and sensitized mast cells in an allergy model. We named this receptor “BAR” for B-cell Antibody Receptor. Murine or human Tregs, transduced with a BAR containing OVA or control Tregs expressing an unrelated antigen, were successfully expanded in vitro and tested in the murine OVA-alum allergy model with measurable titers of anti-OVA IgE. Because BAR Tregs express the target antigen and could interact with specific IgE on sensitized mast cells, we first demonstrated that intravenously injected OVA-BAR Tregs did not directly lead to a drop in temperature or release of mediators in plasma indicative of anaphylaxis. Forty-eight hours later, mice were challenged intraperitoneally with 200 μg OVA to induce an anaphylactic reaction, and temperature immediately measured for 30 min. We found that OVA-BAR Tregs protected mice from hypothermia, whereas mice given control BARs (expressing an unrelated antigen) or PBS showed substantial temperature drops indicative of anaphylaxis when systemically challenged with OVA. Importantly, this effect was also demonstrated in a passive anaphylaxis model in which mice that received anti-OVA IgE antibody were protected from hypothermia when treated with OVA-BAR Tregs prior to systemic OVA challenge. These results provide proof of principle that engineered allergen-specific T-regulatory cells can provide clinical protection against severe allergic reactions in individuals already IgE-sensitized to an allergen.

Cross-allergenicity of crustacean and the edible insect Gryllus bimaculatus in patients with shrimp allergy

Food scarcity is a serious problem for many developing as well as developed countries. Edible insects have attracted attention recently as a novel food source. Crickets are especially high in nutritional value and easy to breed and harvest. In this study, we evaluated the risk of allergic reactions associated with cricket consumption in individuals with crustacean allergy.We evaluated food allergy risk in the consumption of Gryllus bimaculatus (cricket) in patients with shrimp allergy, using enzyme-linked immunosorbent assay (ELISA) and IgE crosslinking-induced luciferase expression assay (EXiLE). Sera from individuals with shrimp allergy (positive for shrimp-specific IgE by ImmunoCAP (>0.35 UA/mL; n = 9) or without shrimp allergy (negative for shrimp-specific IgE; n = 6) were obtained.There was a strong correlation between shrimp- and Gryllus-specific IgE levels obtained by ELISA (rs = 0.99; P < 0.001). The binding of shrimp-specific IgE on shrimp allergen was dose-dependently inhibited by Gryllus allergen (0–1.0 mg/mL). There was a strong correlation between shrimp- and Gryllus-specific IgE responses, as assessed by EXiLE assays (rs = 0.89; P < 0.001). We determined that a protein of approximately 40 kDa reacted with the positive, but not negative, sera for shrimp-specific IgE by ImmunoCAP. Liquid chromatography-tandem mass spectrometry (LC–MS/MS) analysis identified the major allergen in shrimp and Gryllus to be tropomyosin.Our data suggest that the cricket allergen has the potential to induce an allergic reaction in individuals with crustacean allergy. Therefore, allergy risk and shrimp-specific IgE levels should be considered before consumption of cricket meal.

Identification, expression and characterization of the recombinant Sol g 4.1 protein from the venom of the tropical fire ant Solenopsis geminata

BackgroundFire ant venom is a complex mixture consisting of basic piperidine alkaloids, various biologically active peptides and protein components, including a variety of major allergenic proteins. Tropical fire ant Solenopsis geminata is an important stinging ant species that causes anaphylaxis and serious medical problems. Although the biological activities of allergenic venom proteins that are unique to ant venom, particularly Solenopsis 2 and 4, are still unknown, these proteins are believed to play important roles in mediating the effects of the piperidine derivatives in the venom.MethodsIn the present study, the cDNA cloning, sequencing and three-dimensional structure of Sol g 4.1 venom protein are described. The recombinant Sol g 4.1 protein (rSol g 4.1) was produced in E. coli, and its possible function as a hydrophobic binding protein was characterized by paralyzing crickets using the 50% piperidine dose (PD50). Moreover, an antiserum was produced in mice to determine the allergenic properties of Sol g 4.1, and the antiserum was capable of binding to Sol g 4.1, as determined by Western blotting.ResultsThe molecular weight of Sol g 4.1 protein is 16 kDa, as determined by SDS-PAGE. The complete cDNA is 414 bp in length and contains a leader sequence of 19 amino acids. The protein consists of six cysteines that presumably form three disulfide bonds, based on a predicted three-dimensional model, creating the interior hydrophobic pocket and stabilizing the structure. The rSol g 4.1 protein was expressed in inclusion bodies, as determined by SDS-PAGE. Dialysis techniques were used to refold the recombinant protein into the native form. Its secondary structure, which primarily consists of α-helices, was confirmed by circular dichroism analysis, and the three-dimensional model was also verified. The results of allergenic analysis performed on mice showed that the obtained protein was predicted to be allergenically active. Moreover, we report on the possible role of the Sol g 4.1 venom protein, which significantly reduced the PD50 from 0.027 to 0.013% in paralyzed crickets via synergistic effects after interactions with piperidine alkaloids.ConclusionsThe primary structure of Sol g 4.1 showed high similarity to that of venom proteins in the Solenopsis 2 and 4 family. Those proteins are life-threatening and produce IgE-mediated anaphylactic reactions in allergic individuals. The possible function of this protein is the binding of the interior hydrophobic pockets with piperidine alkaloids, as determined by the analysis of the structural model and PD50 test.

Activation of human eosinophils with palladium nanoparticles (Pd NPs): importance of the actin cytoskeleton in Pd NPs-induced cellular adhesion

Palladium (Pd) is known to be released into the environment in the fine and ultrafine (at the nanoscale) airborne particle fractions mainly from automobile catalytic converters leading to an increase human exposure to this noble metal. It was reported that Pd can induce allergic reactions in individuals exposed to it via different ways. Some studies reported an increased number of eosinophils into airways following NP exposure in vivo in rodent models of allergies and inflammation. Knowing the importance of eosinophils in allergies, asthma and other lung diseases, it is surprising to observe that the direct effect of Pd at the nanoscale in eosinophils has been poorly documented. The aim of this study was to determine how Pd NPs will affect the biology of human eosinophils. Characterization of Pd NPs by dynamic light scattering indicates the presence of some aggregates when suspended in diverse solutions used here for the different experiments. Pd NPs did not significantly induce cell necrosis and apoptosis in eosinophils (0.5–150μg/ml) as assessed by trypan blue exclusion assay, flow cytometry after staining with FITC-annexin V and propidium iodide and by morphological observations by optical microscopy. PD NPs, unlike the positive controls, did not induce reactive oxygen species (ROS) but were found to target the actin cytoskeleton, since actin was differently re-located intracellularly when compared to untreated cells as determined by fluorescence microscopy. Clearly, Pd NPs were found to increase adhesion of eosinophils onto human endothelial EA.hy926 cells. Using cytochalasin D, a cell-permeable and potent inhibitor of actin polymerization, this ability to increase adhesion was drastically reversed. Our results indicate that Pd NPs can target the cytoskeleton and increase the adhesion of human eosinophils by an actin-dependent mechanism. These findings show that human eosinophils can be activated by Pd NPs emphasizing the importance of fully investigating how these NPs could alter the biology of human cells involved in allergies, asthma and other lung diseases as well as in various other inflammatory disorders.

Cloning, structural modelling and characterization of VesT2s, a wasp venom hyaluronidase (HAase) from Vespa tropica.

BackgroundWasp venom is a complex mixture containing proteins, enzymes and small molecules, including some of the most dangerous allergens. The greater banded wasp (Vespa tropica) is well-known for its lethal venom, whose one of the major components is a hyaluronidase (HAase). It is believed that the high protein proportion and activity of this enzyme is responsible for the venom potency.MethodsIn the present study, cDNA cloning, sequencing and 3D-structure of Vespa tropica venom HAase were described. Anti-native HAase antibody was used for neutralization assay.ResultsTwo isoforms, VesT2a and VesT2b, were classified as members of the glycosidase hydrolase 56 family with high similarity (42–97 %) to the allergen venom HAase. VesT2a gene contained 1486 nucleotide residues encoding 357 amino acids whereas the VesT2b isoform consisted of 1411 residues encoding 356 amino acids. The mature VesT2a and VesT2b are similar in mass and pI after prediction. They are 39119.73 Da/pI 8.91 and 39571.5 Da/pI 9.38, respectively. Two catalytic residues in VesT2a, Asp107 and Glu109 were substituted in VesT2b by Asn, thus impeding enzymatic activity. The 3D-structure of the VesT2s isoform consisted of a central core (α/β)7 barrel and two disulfide bridges. The five putative glycosylation sites (Asn79, Asn99, Asn127, Asn187 and Asn325) of VesT2a and the three glycosylation sites (Asn1, Asn66 and Asn81) in VesT2b were predicted. An allergenic property significantly depends on the number of putative N-glycosylation sites. The anti-native HAase serum specifically recognized to venom HAase was able to neutralize toxicity of V. tropica venom. The ratio of venom antiserum was 1:12.ConclusionsThe wasp venom allergy is known to cause life-threatening and fatal IgE-mediated anaphylactic reactions in allergic individuals. Structural analysis was a helpful tool for prediction of allergenic properties including their cross reactivity among the vespid HAase.

Pathogenesis of IgE‐mediated food allergy

Food allergy is a prevalent disease for which there is no current treatment beyond careful food avoidance. Accidental exposure to foods causes reactions in allergic individuals that can range in severity from mild skin reactions to severe and life-threatening anaphylaxis, and there are no validated tools to predict severity of reactions. A greater understanding of the pathogenesis of food allergy is needed to develop prevention and treatment strategies for food allergy. In the last few years, there have been significant developments in the field of food allergy that have led to new ideas about food allergy prevention, diagnosis, and treatment. This review will discuss these recent advances in the food allergy field as well as identify gaps in our knowledge about the immune mechanisms of allergy and tolerance to foods.

Effects of venom immunotherapy on serum level of CCL5/RANTES in patients with Hymenoptera venom allergy

Hymenoptera venoms are known to cause life-threatening IgE-mediated anaphylactic reactions in allergic individuals. Venom immunotherapy is a recommended treatment of insect allergy with still the mechanism not being completely understood. We decided to assess the serum CCL5/RANTES level in patients who experienced severe anaphylactic reaction to Hymenoptera venom and to find out changes in the course of immunotherapy. Twenty patients (9 men, 11 women, mean age: 31.91 ± 7.63 years) with history of anaphylactic reaction after insect sting were included into the study. Diagnosis was made according to sIgE and skin tests. All of them were enrolled into rush venom immunotherapy with bee or wasp venom extracts (Pharmalgen, ALK-Abello, Horsholm, Denmark). Serum levels of CCL5/RANTES were measured using a commercially available ELISA kit (R&D Systems, Minneapolis, MN). CCL5/RANTES serum concentration are higher in insect venom allergic patients than in healthy controls (887.5 ± 322.77 versus 387.27 ± 85.11 pg/ml). Serum concentration of CCL5/RANTES in insect venom allergic patient was significantly reduced in the course of allergen immunotherapy already after 6 days of vaccination (887.5 ± 322.77 versus 567.32 ± 92.16 pg/ml). CCL5/RANTES serum doesn’t correlate with specific IgE. Chemokine CCL5/RANTES participates in allergic inflammation induced by Hymenoptera venom allergens. Specific immunotherapy reduces chemokine CCL5/RANTES serum level already after initial days of venom immunotherapy.

Effects of different extraction buffers on peanut protein detectability and lateral flow device (LFD) performance

The accidental uptake of peanuts can cause severe health reactions in allergic individuals. Reliable determination of traces of peanuts in food products is required to support correct labelling and therefore minimise consumers’ risk. The immunoanalytical detectability of potentially allergenic peanut proteins is dependent on previous heat treatment, the extraction capacity of the applied buffer and the specificity of the antibody. In this study a lateral flow device (LFD) for the detection of peanut protein was developed and the capacity of 30 different buffers to extract proteins from mildly and strongly roasted peanut samples as well as their influence on the test strip performance were investigated. Most of the tested buffers showed good extraction capacity for putative Ara h 1 from mildly roasted peanuts. Protein extraction from dark-roasted samples required denaturing additives, which were proven to be incompatible with LFD performance. High-pH buffers increased the protein yield but inhibited signal generation on the test strip. Overall, the best results were achieved using neutral phosphate buffers but equal detectability of differently altered proteins due to food processing cannot be assured yet for immunoanalytical methods.

Evaluation of IgE Antibodies to Recombinant Peanut Allergens in Patients with Reported Reactions to Peanut

Background: Peanut may cause severe reactions in allergic individuals. The objective was to evaluate IgE antibodies to various recombinant (r) peanut and birch pollen allergens in relation to IgE levels to whole peanut extract and severe allergic reactions to peanut. Methods: Seventy-four Swedish peanut-allergic patients (age: 14–61 years) reported previous peanut exposure and associated symptoms using a questionnaire. Their IgE reactivity to peanut, birch pollen and individual allergen components was analyzed using ImmunoCAP®. Results: Of the 48 subjects sensitized to Ara h 1, 2 or 3, 60% had peanut-specific IgE levels >15 kU_A/l, while 100% of the subjects without detectable IgE to these allergens had low peanut-specific IgE levels (<10 kU_A/l). The levels of IgE to rAra h 8, rBet v 1 and birch pollen were highly correlated (r_S = 0.94, p < 0.0001). Fifty-eight patients reported adverse reactions after accidental or deliberate peanut exposure (oral, inhalation or skin) of whom 41 had IgE to rAra h 1, 2 or 3. Symptoms of respiratory distress were associated with sensitization to Ara h 1, 2 or 3 (56 vs. 18%, p < 0.01). Two cases of anaphylaxis were reported among the individuals sensitized to Ara h 1–3. IgE to rAra h 8, rAra h 9, profilin or cross-reactive carbohydrate determinants were not associated with severe symptoms. Conclusions: The results indicate that IgE reactivity to Ara h 1, 2 and 3 is associated with severe reactions after exposure to peanut in Swedish patients.

A retrospective study to describe the incidence of moderate to severe allergic reactions to factor IX in subjects with haemophilia B

Using a patient chart review process, we conducted a retrospective study to describe the frequency of allergic reactions in individuals with haemophilia B receiving factor IX (FIX) replacement therapy. The number of allergic reactions in individuals receiving a recombinant FIX (rFIX) product (BeneFix(®)) was then compared with the number of reactions in patients receiving plasma-derived FIX (pdFIX) products. Of the 180 subjects in the study, 163 received rFIX, 88 received pdFIX; 71 received both product types. A total of seven (3.89%) subjects had a moderate or severe allergic reaction to a FIX product (95% confidence interval [CI], 1.06-6.71%). Among those receiving rFIX, four subjects (2.45%) had an allergic reaction (95% CI, 0.08-4.83%). Of individuals taking pdFIX products, three (3.41%) developed an allergic reaction (95% CI, 0-7.20%). It was noted that three (1.84%) of those taking rFIX developed an inhibitor to FIX (95% CI, 0-3.90%), while four (4.55%) of those receiving a pdFIX product developed an inhibitor (95% CI, 0.19-8.90%). Inhibitor development was frequently associated with allergic reaction. These results provide evidence that there is no difference in the frequency of allergic reactions or inhibitor development in individuals receiving rFIX compared with those receiving pdFIX concentrates. The current study and a previous study of similar design have now compared the rate of allergic reactions associated with rFIX and pdFIX concentrates has now been compared in a total of 414 subjects; this represents the largest collection of data to date on this rare complication of haemophilia B therapy.

Development of a real-time PCR method for the simultaneous detection of soya and lupin mitochondrial DNA as markers for the presence of allergens in processed food

Lupin and soya are members of the Leguminosae family which are recognised as some of the richest source of vegetable proteins. Lupin- and soya-containing products are available on the EU market and could cause severe adverse reactions in allergic individuals, even if consumed at low concentrations. In this context the development of methods for reliable detection of these allergens in food products is a useful tool for the surveillance of established legislation on food labelling within the EU. This work described the development of a duplex real-time PCR method allowing the simultaneous detection of traces of lupin and soya in processed food based on a specific TaqMan® probe designed on a mitochondrial tRNA-MET gene. A set of primers and probes was designed for the amplification of a 168 and 175bp fragment of lupin and soya mitochondrial DNA, respectively. The performance of the method was established using lupin and soya flours and cookies baked from lupin- and soya-containing dough (different concentrations and baking times). The PCR platform yielded consistent and repeatable results. The specificity of the system was tested with DNA from 28 plant species. The sensitivity of the method was suitable to detect allergenic ingredients in the low mg per kg range. Both lupin and soya at a level of 2.5mg per kg food matrix could be detected in cookies baked at 180°C for 10min. The method was successfully applied to bakery (e.g. bread) and vegetarian (e.g. non-meat sausages) food products that contain or may contain soya and/or lupin as ingredient or contaminant (according to the declaration on the product label).

Recombinant phospholipase A1 (Ves v 1) from yellow jacket venom for improved diagnosis of hymenoptera venom hypersensitivity

BackgroundHymenoptera venoms are known to cause life-threatening IgE-mediated anaphylactic reactions in allergic individuals. Proper diagnosis of hymenoptera venom allergy using venom extracts is severely affected by molecular cross-reactivities. Although non-glycosylated marker allergens would facilitate the identification of the culprit venom, the major allergen phospholipase A1 (Ves v 1) from yellow jacket venom (YJV) remained unavailable so far.MethodsExpression of Ves v 1 as wild type and enzymatically inactivated mutant and Ves v 5 in insect cells yielded soluble proteins that were purified via affinity chromatography. Functionality of the recombinant allergens was assessed by enzymatic and biophysical analyses as well as basophil activation tests. Diagnostic relevance was addressed by ELISA-based analyses of sera of YJV-sensitized patients.ResultsBoth major allergens Ves v 1 and Ves v 5 could be produced in insect cells in secreted soluble form. The recombinant proteins exhibited their particular biochemical and functional characteristics and were capable for activation of human basophils. Assessment of IgE reactivity of sera of YJV-sensitized and double-sensitized patients emphasised the relevance of Ves v 1 in hymenoptera venom allergy. In contrast to the use of singular molecules the combined use of both molecules enabled a reliable assignment of sensitisation to YJV for more than 90% of double-sensitised patients.ConclusionsThe recombinant availability of Ves v 1 from yellow jacket venom will contribute to a more detailed understanding of the molecular and allergological mechanisms of insect venoms and may provide a valuable tool for diagnostic and therapeutic approaches in hymenoptera venom allergy.

Dissecting cross-reactivity in hymenoptera venom allergy by circumvention of α-1,3-core fucosylation

Hymenoptera venom allergy is known to cause life-threatening and sometimes fatal IgE-mediated anaphylactic reactions in allergic individuals. About 30–50% of patients with insect venom allergy have IgE antibodies that react with both honeybee and yellow jacket venom. Apart from true double sensitisation, IgE against cross-reactive carbohydrate determinants (CCD) are the most frequent cause of multiple reactivities severely hampering the diagnosis and design of therapeutic strategies by clinically irrelevant test results. In this study we addressed allergenic cross-reactivity using a recombinant approach by employing cell lines with variant capacities of α-1,3-core fucosylation. The venom hyaluronidases, supposed major allergens implicated in cross-reactivity phenomena, from honeybee (Api m 2) and yellow jacket (Ves v 2a and its putative isoform Ves v 2b) as well as the human α-2HS-glycoprotein as control, were produced in different insect cell lines. In stark contrast to production in Trichoplusia ni (HighFive) cells, α-1,3-core fucosylation was absent or immunologically negligible after production in Spodoptera frugiperda (Sf9) cells. Consistently, co-expression of honeybee α-1,3-fucosyltransferase in Sf9 cells resulted in the reconstitution of CCD reactivity. Re-evaluation of differentially fucosylated hyaluronidases by screening of individual venom-sensitised sera emphasised the allergenic relevance of Api m 2 beyond its carbohydrate epitopes. In contrast, the vespid hyaluronidases, for which a predominance of Ves v 2b could be shown, exhibited pronounced and primary carbohydrate reactivity rendering their relevance in the context of allergy questionable. These findings show that the use of recombinant molecules devoid of CCDs represents a novel strategy with major implications for diagnostic and therapeutic approaches.