IgE Reactivity Research Articles

A microarray-based IgE-molecular mimicry index (IgE-MMI): A biomarker for disease severity, clinical phenotypes, and therapeutic response in atopic dermatitis?

The role of autoimmune IgE responses in atopic dermatitis (AD) is highly debated. While IgE targeting self-proteins has been extensively studied, IgE responses induced by human-homologous exogenous molecular allergens (HEMAs) remains less understood. To investigate whether IgE antibody responses to HEMAs are associated with AD, its severity, and response to dupilumab. We enrolled 3325 participants with a history of allergic diseases, including 577 (17.3%) diagnosed with AD. Serum IgE antibodies against 183 exogenous allergenic molecules were measured using the IgE microarray (Allergy Explorer-ALEX-2®, MADX, Vienna). Based on international classification criteria, participants were stratified by AD severity and clinical phenotypes. For each patient, we developed an 'IgE molecular-mimicry index' (IgE-MMI), calculated from IgE reactivity to a panel of five HEMA protein families: arginine kinase, enolase (ENO), cyclophilin (CYP), lipocalin, and MnSOD. Logistic regression was employed to assess the association between IgE to HEMAs or IgE-MMI and AD, its severity, and response to dupilumab. IgE sensitization to most HEMAs (32/48, 67%), but only to a small fraction of non-HEMAs (3/135, 2.2%), was significantly more common in patients with severe AD compared to other patient groups. The IgE-MMI was positive in 295/2748 (10.7%) of allergic patients without AD, and in 58/283 (20%), 52/134 (39%), and 86/160 (54%) of patients with remitting, moderate, or severe AD, respectively. It was strongly associated with specific phenotypes, such as flexural dermatitis (OR 8.4, 95% CI: 6.3-11.2), head and neck dermatitis (OR: 16.5, 95% CI: 7.4-37.2), and generalized eczema (OR: 8.6, 95% CI: 4.9-15.6). Poor response to dupilumab was associated with IgE antibodies to ENO (OR: 22.7, 95% CI: 1.7-302.9), but inversely associated with IgE antibodies to MnSOD (OR: 0.1, 95% CI: 0.02-0.8) and NPC-2 from dust mites (OR: 0.1, 95% CI: 0.01-0.9). IgE microarrays are useful for broadly assessing IgE to HEMAs in allergic patients. IgE reactivity to HEMAs and a positive IgE-MMI may serve as valuable biomarkers for severe AD, its clinical phenotypes, and the response to dupilumab.

Interpreting epidemiologic distribution of total and specific IgE levels for food allergy in Southern China from 2004 to 2023: understanding the mechanisms and focusing on prevention

BackgroundThe burgeoning prevalence of food allergy-related diseases is closely associated with geographical allergen distribution and societal lifestyle paradigms. This study aims to shed light on the distribution patterns of specific IgE (sIgE) and total IgE (tIgE) reactivity to common food allergens in the Southern Chinese populace.MethodsEmploying an analytical technique spanning two decades, we conducted specific IgE and total IgE on serum samples harvested from patients with food allergy-related pathologies at First Affiliated Hospital of Guangzhou Medical University from 2004 to 2023. This comprehensive examination of eight prototypical food allergens: egg white, milk, wheat, sesame, peanut, soybean, shrimp, and crab.ResultsOur analysis showed a 100% positivity rate for sIgE and an 86.54% positivity rate for tIgE. Milk had the highest positive response rate, followed by egg white and shrimp. Age-stratified data indicated that milk sensitization peaked in children aged 2 years or younger, while egg white sensitization peaked between 3 and 5 years of age. Sensitization rates for the remaining six allergens increased with age. Additionally, co-sensitization was observed between milk, egg white, crab, and shrimp with other allergens.ConclusionIn common allergens of Southern China, egg white, milk, and shrimp ascend as the dominant subjects, underlining their imperative role in food allergy pathogenesis. This landscape-wide allergenic profiling, segregated across age clusters and enhanced by co-sensitization data, augments our power for early diagnosis and strategic intervention in food allergy diseases.

Gut microbiome impact on childhood allergic rhinitis and house dust mite IgE responses.

The correlation between the gut microbiota and airway inflammation in childhood allergic rhinitis (AR), particularly concerning allergen exposure, remains insufficiently explored. This study aimed to link gut microbiota changes with house dust mite (HDM)-specific IgE responses in pediatric AR. Using metagenomic shotgun sequencing, we compared the fecal microbiota of 60 children with HDM-AR to 48 healthy controls (HC), analyzing the link to IgE reactions. We examined the effects of oral Escherichia (E.) fergusonii treatment in mice sensitized with ovalbumin and HDM on allergic symptoms, mucosal cell infiltration, Th1/Th2/Tregs balance in the spleen, serum cytokine levels, and E. fergusonii presence in feces. Children with HDM-AR have a less diverse gut microbiome and lower levels of E. fergusonii compared to controls, with a negative correlation between E. fergusonii abundance and HDM-specific IgE levels. In mice sensitized with OVA and HDM, oral administration of E. fergusonii improved allergic symptoms, reduced nasal eosinophils/mast cells infiltration and adjusted Th cell populations towards a non-allergic profile in splenic lymphocytes with exception of IFN-γ change in serum. These findings underline the potential of targeting gut microbiota, particularly E. fergusonii, in managing childhood HDM-AR, suggesting a promising approach for future interventions. The composition and distribution of gut microbiota in children with HDM-AR are significant changed. The abundance of Escherichia genus is decreased in HDM-AR children. HDM-specific IgE levels are strongly negatively associated with E. fergusonii abundance. Oral administration of E. fergusonii effectively suppresses allergic responses in murine model. These findings offer novel insights into the diagnosis and treatment of HDM-AR, which suggested that E. fergusonii holds promise as a potential therapeutic avenue for managing HDM-AR.

Identification, Purification, and Characterization of Kunitz-Type Protease Inhibitors from White-Fleshed Dragon Fruit (Selenicereus undatus) and Red-Fleshed Dragon Fruit (Selenicereus costaricensis) Seeds as Novel Food Allergens.

There have been clinical reports of allergies to dragon fruits. However, little is known about the allergens that trigger these reactions. This study aimed to investigate novel allergens in dragon fruits. The peptide mass fingerprints of two purified natural allergens were identified by LC-MS/MS with chymotrypsin proteolysis. The complete amino acid sequences of the allergens were deduced from cDNA amplicon sequences. The coding sequences of two allergens were inserted in the expression vector pColdI, and recombinant allergens were produced in Escherichia coli. Circular dichroism was performed to analyze the secondary structures of natural and recombinant allergens. Our results identified two novel allergens as Kunitz-type protease inhibitors. Recombinant allergens exhibited well-folded structures, predominantly composed of β-sheets, similar to their natural counterparts. IgE reactivity analysis with sera from ten patients primarily sensitized to dragon fruit indicated that Kunitz-type protease inhibitors are major allergens in dragon fruits. These results improve our understanding of allergen sources and provide important insights into the allergenicity of proteins from different species.

Assessment of the effect of lactic acid bacteria fermentation on IgE-/IgG-binding ability and nutritional properties of cow milk.

Cow milk (CM) is an important food source for humans, and food allergy caused by CM has attracted attention worldwide. To our knowledge, systematic studies about the effects of Lactobacillus paracasei, Lactobacillus plantarum, and Pediococcus pentosaceus on the IgE-/IgG-binding ability and nutritional properties of CM are very rare. In this study, L. paracasei, L. plantarum, and P. pentosaceus fermentation on the IgE-/IgG-binding ability was determined by Enzyme-Linked Immunosorbent Assay (ELISA), and the protein quality, amino acid profile, and color were systematically evaluated. The results showed that these LAB strains exhibited higher protein degradation ability, and the IgE reactivity reduction rate was 41.03%-60.00% and the IgG reduction rate was 29.86%-67.20%, respectively. Additionally, the nutritional value was improved obviously, and the color was altered significantly, which was conductive to develop dairy products. These findings provided a theoretical foundation for the development of hypoallergenic dairy products. PRACTICAL APPLICATION: In this study, L. paracasei, L. plantarum and P. pentosaceus could be considered as good potential candidates for solving cow milk allergy owing to their decreased IgE/IgG binding ability andimproved nutritional and sensory properties, which provide a promising strategy to develop hypoallergenic dairy products.

A Novel Quality Control Strategy for the Preparation of High-Quality Recombinant Allergens in Escherichia coli: A Case Study of Der f 2

Introduction: Recombinant allergens produced by Escherichia coli (E. coli) system play an important role in the component-resolved diagnostics of allergy and vaccine development. However, incorrect folding of recombinant allergens may affect their application. Therefore, it is very important to monitor the correct folding of recombinant allergens. Currently, there is still a lack of a quality control strategy to solve this problem. In this study, a mite allergen, Der f 2, was taken as an example to establish a novel quality control strategy, which was based on chromatography to isolate the allergen, and on enzyme-linked immunosorbent assay to verify the IgE reactivity of the isolated allergen. Methods: The nucleotide sequence encoding Der f 2 was codon-optimized and cloned into pET-28a (+) plasmid. Best conditions for the expression of Der f 2 in E. coli were sought. The inclusion body of Der f 2 was denatured and purified by nickel affinity chromatography. Refolding processes were compared using glutathione redox system. The fully and partially folded proteins were separated by anion exchange chromatography, and the IgE reactivity of the isolated proteins was verified by indirect enzyme-linked immunosorbent assay. Results: An optimized 387 bp segment of the Der f 2 coding gene was successfully expressed in E. coli. Best induction conditions included preinduction bacterial density with absorbance value at 600 nm was 0.6, 1 mM isopropyl beta-d-thiogalactopyranoside at 28°C for 4 h. The Der f 2 protein after refolding was separated by chromatography and two fractions were obtained. The first fraction was identified as monomer protein and the second as aggregate by size-exclusion chromatography. Indirect enzyme-linked immunosorbent assay also confirmed that the first fraction showed higher IgE reactivity. Conclusion: In this study, a novel quality control strategy based on chromatographic separation and IgE reactivity monitoring was established in the case of mite Der f 2, which systematically evaluated the effectiveness of multiple preparation methods for the first time. It is faster and more convenient when compared with the existing methods such as size-exclusion chromatography. This strategy laid a foundation for the stable application of recombinant allergens produced by E. coli in component-resolved diagnostics and the development of molecular vaccines in the future.

Allergenic potency of various foods of mammalian origin in patients with α-Gal syndrome.

The α-Gal syndrome (AGS) is an emerging allergy to mammalian food caused by IgE-mediated reactions to the carbohydrate galactose-α-1,3-galactose (α-Gal). Mammalian food sources contain α-Gal, but the amount differs. The objective of this study was to investigate the allergenic potency of various foods of mammalian origin among AGS patients. Twenty-six AGS patients were included. Food extracts from innards, lean meats, processed meat products, milk, and whey were analyzed. Immunoblot, ELISA, immunofluorescence, and basophil activation test were used to determine the α-Gal content, characterize IgE binding, and assess foods' allergenicity. The determined amount of α-Gal, IgE reactivity to food extracts, and food extract potencies to activate patients' basophils correlated well with each other. Pork and beef kidney showed the highest allergenicity. Beef liver and bacon showed allergenicity comparable to that of lean meats. Game meat seemed to have a higher allergenic potency than meats from farm-raised animals. The processed meat products liver pâté and black pudding, despite lower α-Gal content, demonstrated moderate allergenicity. Milk showed the lowest allergenicity. IgE reactivity to food extracts was highly similar for all patients and strongly dominated by the α-Gal epitope. The allergenic potency of mammalian meat depends on the origin of the meat, the different cuts, and type of processing, with innards posing the greatest risk to AGS patients. Even processed mammalian meat constitutes a risk. Dairy products show the lowest risk. This study highlights the importance of analyzing even more foods to improve the management of AGS.

Tolerance of peanuts and tree nuts in Spanish children with exclusive sensitization to lipid transfer proteins.

Allergy to peanuts and tree nuts is a common cause of food allergy in Spain, with lipid transfer proteins (LTP) being the most frequently recognized panallergen. LTP sensitization often leads to multiple food group sensitivities, resulting in overly restrictive diets that hinder patient's quality of life. This study aimed to assess the tolerance of peanuts and tree nuts (hazelnuts and walnuts) in children sensitized to LTP, potentially mitigating the need for such diets. This prospective study enrolled individuals diagnosed with allergy to peanuts, hazelnuts, or walnuts. Data were collected from medical records, including demographics and clinical history. Allergological assessment comprised skin prick tests using commercial extracts and the nuts in question, alongside measurements of total and specific IgE to nuts and their primary molecular components. Participants showing positive LTP sensitization without sensitization to seed storage proteins underwent open oral nut challenges. A total of 75 individuals labeled as allergic to peanuts, 44 to hazelnuts, and 51 to walnuts were included. All of them underwent an open oral provocation test with the incriminated nut, showing a high tolerance rate. Peanut was tolerated by 98.6% of patients, 97.72% tolerated hazelnut, and 84.3% tolerated walnut. The findings suggest that the majority of patients allergic to peanuts, hazelnuts, or walnuts, due to LTP sensitization and lacking IgE reactivity to seed storage proteins, can tolerate these nuts. This supports the need for personalized nut tolerance assessments to avoid unnecessary dietary restrictions.

Non-Specific Lipid Transfer Protein Amb a 6 Is a Source-Specific Important Allergenic Molecule in Ragweed Pollen.

Pollen from common ragweed is an important allergen source worldwide and especially in western and southern Romania. More than 100 million patients suffer from symptoms of respiratory allergy (e.g., rhinitis, asthma) to ragweed pollen. Among the eleven characterized allergens, Amb a 6 is a non-specific lipid transfer protein (nsLTP). nsLTPs are structurally stable proteins in pollen and food from different unrelated plants capable of inducing severe reactions. The goal of this study was to produce Amb a 6 as a recombinant and structurally folded protein (rAmb a 6) and to characterize its physicochemical and immunological features. rAmb a 6 was expressed in Spodoptera frugiperda Sf9 cells as a secreted protein and characterized by mass spectrometry and circular dichroism (CD) spectroscopy regarding molecular mass and fold, respectively. The IgE-binding frequency towards the purified protein was evaluated using sera from 150 clinically well-characterized ragweed-allergic patients. The allergenic activities of rAmb a 6 and the nsLTP from the weed Parietaria judaica (Par j 2) were evaluated in basophil activation assays. rAmb a 6-specific IgE reactivity was associated with clinical features. Pure rAmb a 6 was obtained by insect cell expression. Its deduced molecular weight corresponded to that determined by mass spectrometry (i.e., 10,963 Da). rAmb a 6 formed oligomers as determined by SDS-PAGE under non-reducing conditions. According to multiple sequence comparisons, Amb a 6 was a distinct nsLTP with less than 40% sequence identity to currently known plant nsLTP allergens, except for nsLTP from Helianthus (i.e., 52%). rAmb a 6 is an important ragweed allergen recognized by 30% of ragweed pollen allergic patients. For certain patients, rAmb a 6-specific IgE levels were higher than those specific for the major ragweed allergen Amb a 1 and analysis also showed a higher allergenic activity in the basophil activation test. rAmb a 6-positive patients suffered mainly from respiratory symptoms. The assumption that Amb a 6 is a source-specific ragweed allergen is supported by the finding that none of the patients showing rAmb a 6-induced basophil activation reacted with Par j 2 and only one rAmb a 6-sensitized patient had a history of plant food allergy. Immunization of rabbits with rAmb a 6 induced IgG antibodies which strongly inhibited IgE binding to rAmb a 6. Our results demonstrate that Amb a 6 is an important source-specific ragweed pollen allergen that should be considered for diagnosis and allergen-specific immunotherapy of ragweed pollen allergy.

Preoperative survey to evaluate the patients' allergy list and its relevance to perioperative care.

Perioperative hypersensitivity and allergic reactions can result in significant morbidity and mortality. For routine anesthetic care, allergies are determined from a review of the electronic medical record supplemented by a detailed patient history. Although the electronic medical record is generally assumed to be accurate, it may be that allergies are erroneously listed or not based on sound medical practice. The purpose of the current study is to evaluate allergies listed in the electronic medical record of children presenting for surgery and determine their origin, authenticity, and impact on perioperative care. Eligible patients included those presenting for a surgical procedure in the main operating room, who were ≤ 21 years of age, with a drug allergy listed on the EMR. Prior to intraoperative care, an electronic survey questionnaire containing questions related to medication allergies was provided to a guardian or parent. Two anesthesiology physicians reviewed the survey responses to determine the validity of any reported allergies. A second electronic survey was given postoperatively to the attending anesthesiologist to determine whether the documented allergy impacted anesthetic care. The study cohort included 250 patients, ranging in age from 5 to 14 years (median age 9 years). All of the patients had at least one allergy listed on the electronic medical record. Seventy of the 250 patients (28%) had more than one drug allergy listed for a total of 351 medication allergies. The majority of the listed allergies were related to antibiotics including 155 (44%) from the penicillin family, 26 (7%) cephalosporins, 16 (5%) sulfonamides, and 36 (10%) other antimicrobial agents. Other commonly listed allergies were 27 (8%) nonsteroidal anti-inflammatory agents and 15 (4%) opioids. The remaining 76 (22%) included a miscellaneous list of other medications. On further review of the allergies, the survey was completed for 301 medications. After physician review, 135 of 301 (45%) responses were considered consistent with IgE reactions "true allergy," 73 (24%) were deemed less relevant to IgE reactions "unlikely true allergy," and 93 (31%) were not related to IgE reactions "not an allergy." Care alterations during surgery were uncommon regardless of whether the issue was assessed as a true allergy (11%), unlikely to be a true allergy (3%), or not a true allergy (13%). A significant portion of the documented allergies in children are not true allergies, but rather recognized adverse effects (apnea from an opioid, renal failure from an NSAIDs) or other nonallergic concerns (gastrointestinal upset such as nausea). Erroneously listed allergies may lead to unnecessary alterations in patient care during perioperative care. A careful analysis of the allergy list on the EMR should be supplemented by a thorough patient history with specific questions related to the drug allergy. Once this is accomplished, the allergy listed should be updated to avoid its erroneous impact on perioperative care.

The cytoskeletal protein profilin is an important allergen in saltwort (Salsola kali).

Pollen from Salsola kali, i.e., saltwort, Russian thistle, is a major allergen source in the coastal regions of southern Europe, in Turkey, Central Asia, and Iran. S. kali-allergic patients mainly suffer from hay-fever (i.e., rhinitis and conjunctivitis), asthma, and allergic skin symptoms. The aim of this study was to investigate the importance of individual S. kali allergen molecules. Sal k 1, Sal k 2, Sal k 3, Sal k 4, Sal k 5, and Sal k 6 were expressed in Escherichia coli as recombinant proteins containing a C-terminal hexahistidine tag and purified by nickel affinity chromatography. The purity of the recombinant allergens was analyzed by SDS-PAGE. Their molecular weight was determined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, and their fold and secondary structure were studied by circular dichroism (CD) spectroscopy. Sera from clinically well-characterized S. kali-allergic patients were used for IgE reactivity and basophil activation experiments. S. kali allergen-specific IgE levels and IgE levels specific for the highly IgE cross-reactive profilin and the calcium-binding allergen from timothy grass pollen, Phl p 12 and Phl p 7, respectively, were measured by ImmunoCAP. The allergenic activity of natural S. kali pollen allergens was studied in basophil activation experiments. Recombinant S. kali allergens were folded when studied by CD analysis. The sum of recombinant allergen-specific IgE levels and allergen-extract-specific IgE levels was highly correlated. Sal k 1 and profilin, reactive with IgE from 64% and 49% of patients, respectively, were the most important allergens, whereas the other S. kali allergens were less frequently recognized. Specific IgE levels were highest for profilin. Of note, 37% of patients who were negative for Sal k 1 showed IgE reactivity to Phl p 12, emphasizing the importance of the ubiquitous cytoskeletal actin-binding protein, profilin, for the diagnosis of IgE sensitization in S. kali-allergic patients. rPhl p 12 and rSal k 4 showed equivalent IgE reactivity, and the clinical importance of profilin was underlined by the fact that profilin-monosensitized patients suffered from symptoms of respiratory allergy to saltwort. Accordingly, profilin should be included in the panel of allergen molecules for diagnosis and in molecular allergy vaccines for the treatment and prevention of S. kali allergy.

Development of a low allergenic product for patients with milk allergy and assessment of its specific IgE reactivity.

Milk oral immunotherapy is the riskiest and most unpredictable form of oral immunotherapy. We aimed to produce a low allergenic product than conventional once baked-cake/muffin, to develop indirect in-house ELISA to check the tolerance status with milk products and evaluate IgE reactivity of patients' sera via western blotting (WB) and indirect in-house ELISA. A low allergenic product named biscotti-twice baked-cake was developed, and the total protein concentration was determined. The protein content was studied by SDS-PAGE and proteomics. Milk-specific IgE (sIgE) binding assays were performed by WB and indirect in-house ELISA by using patients' sera. Casein band intensity was observed to be lower in the biscotti-twice baked-cake than in the once baked-cake (p = .014). Proteomics analysis and αS1-casein measurement showed that the lowest intensity of casein was found in biscotti. The low binding capacity of milk sIgE to biscotti compared with once baked-cake was shown by WB (p = .0012) and by indirect in-house ELISA (p = .0001). In the ROC analysis, the area under the curve (AUC) of the in-house ELISA IgE was comparable with Uni-CAP milk and casein sIgE. The AUC of the in-house ELISA IgE for cake (0.96) and biscotti (1) was slightly better than Uni-CAP milk sIgE (0.94; 0.97) and casein sIgE (0.96; 0.97), respectively. The low allergenicity of the newly developed low allergenic product "biscotti-twice baked-cake" has been demonstrated by invitro experiments. Biscotti could be a safe treatment option than once baked-cake/muffin in patients who are reactive to once baked-milk products.

Cross-Reactivity of Ragweed Pollen Calcium-Binding Proteins and IgE Sensitization in a Ragweed-Allergic Population from Western Romania.

Ragweed pollen allergy is the most common seasonal allergy in western Romania. Prolonged exposure to ragweed pollen may induce sensitization to pan-allergens such as calcium-binding proteins (polcalcins) and progression to more severe symptoms. We aimed to detect IgE sensitization to recombinant Amb a 9 and Amb a 10 in a Romanian population, to assess their potential clinical relevance and cross-reactivity, as well as to investigate the relation with clinical symptoms. rAmb a 9 and rAmb a 10 produced in Escherichia coli were used to detect specific IgE in sera from 87 clinically characterized ragweed-allergic patients in ELISA, for basophil activation experiments and rabbit immunization. Rabbit rAmb a 9- and rAmb a 10-specific sera were used to detect possible cross-reactivity with rArt v 5 and reactivity towards ragweed and mugwort pollen extracts. The results showed an IgE reactivity of 25% to rAmb a 9 and 35% to rAmb a 10. rAmb a 10 induced basophil degranulation in three out of four patients tested. Moreover, polcalcin-negative patients reported significantly more skin symptoms, whereas polcalcin-positive patients tended to report more respiratory symptoms. Furthermore, both rabbit antisera showed low reactivity towards extracts and showed high reactivity to rArt v 5, suggesting strong cross-reactivity. Our study indicated that recombinant ragweed polcalcins might be considered for molecular diagnosis.

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.

Effects of enzymatic hydrolysis combined with pressured heating on tree nut allergenicity

Hazelnut, pistachio and cashew are tree nuts with health benefits but also with allergenic properties being prevalent food allergens in Europe. The allergic characteristics of these tree nuts after processing combining heat, pressure and enzymatic digestion were analyzed through in vitro (Western blot and ELISA) and in vivo test (Prick-Prick). In the analyzed population, the patients sensitized to Cor a 8 (nsLTP) were predominant over those sensitized against hazelnut seed storage proteins (Sprot, Cor a 9 and 14), which displayed higher IgE reactivity. The protease E5 effectively hydrolyzed proteins from hazelnut and pistachio, while E7 was efficient for cashew protein hydrolysis. When combined with pressured heating (autoclave and Controlled Instantaneous Depressurization (DIC)), these proteases notably reduced the allergenic reactivity. The combination of DIC treatment before enzymatic digestion resulted in the most effective methodology to drastically reduce or indeed eliminate the allergenic capacity of tree nuts.

Identification, Characterization, Cloning, and Cross-Reactivity of Zan b 2, a Novel Pepper Allergen of 11S Legumin.

Protein from Sichuan peppers can elicit mild to severe allergic reactions. However, little is known about their allergenic proteins. We aimed to isolate, identify, clone, and characterize Sichuan pepper allergens and to determine its allergenicity and cross-reactivities. Sichuan pepper seed proteins were extracted and then analyzed by SDS-PAGE. Western blotting was performed with sera from Sichuan pepper-allergic individuals. Proteins of interest were purified using hydrophobic interaction chromatography and gel filtration and further analyzed by analytical ultracentrifugation, circular dichroism spectroscopy, and mass spectrometry (MS). Their coding region was amplified in the genome. IgE reactivity and cross-reactivity of allergens were evaluated by dot blot, enzyme-linked immunosorbent assay (ELISA), and competitive ELISA. Western blot showed IgE binding to a 55 kDa protein. This protein was homologous to the citrus proteins and has high stability and a sheet structure. Four DNA sequences were cloned. Six patients' sera (60%) showed specific IgE reactivity to this purified 11S protein, which was proved to have cross-reactivation with extracts of cashew nuts, pistachios, and citrus seeds. A novel allergen in Sichuan pepper seeds, Zan b 2, which belongs to the 11S globulin family, was isolated and identified. Its cross-reactivity with cashew nuts, pistachios, and citrus seeds was demonstrated.

The primary ciliary dyskinesia-related genetic risk score is associated with susceptibility to adult-onset asthma.

Disturbance of mucociliary clearance is an important factor in the pathogenesis of asthma. We hypothesized that common variants in genes responsible for ciliary function may contribute to the development of asthma with certain phenotypes. Three independent adult Japanese populations (including a total of 1,158 patients with asthma and 2,203 non-asthmatic healthy participants) were studied. First, based on the ClinVar database (https://www.ncbi.nlm.nih.gov/clinvar/), we selected 12 common single-nucleotide polymorphisms (SNPs) with molecular consequences (missense, nonsense, and 3'-untranslated region mutation) in 5 primary ciliary dyskinesia (PCD)-related genes and calculated a PCD-genetic risk score (GRS) as a cumulative effect of these PCD-related genes. Second, we performed a two-step cluster analysis using 3 variables, including PCD-GRS, forced expiratory volume in 1 second (%predicted FEV1), and age of asthma onset. Compared to adult asthma clusters with an average PCD-GRS, clusters with high and low PCD-GRS had similar overall characteristics: adult-onset, female predominance, preserved lung function, and fewer features of type 2 immunity as determined by IgE reactivity and blood eosinophil counts. The allele frequency of rs1530496, a SNP representing an expression quantitative trait locus (eQTL) of DNAH5 in the lung, showed the largest statistically significant difference between the PCD-GRS-High and PCD-GRS-Low asthma clusters (p = 1.4 x 10-15). Genes associated with PCD, particularly the common SNPs associated with abnormal expression of DNAH5, may have a certain influence on the development of adult-onset asthma, perhaps through impaired mucociliary clearance.

Food-dependent exercise-induced allergic reactions in Lipid Transfer Protein (LTP) hypersensitive subjects: new data and a critical reappraisal.

Background. Lipid transfer protein is the main cause of both primary food allergy and food-dependent exercise-induced allergic reactions (FDEIAR) in Italy. What characterizes LTP-hypersensitive patients with FDEIAR is still unclear. We investigated the key characteristics of LTP-hypersensitive patients with or without FDEIAR in a large cohort of individuals sensitized to this allergen. Methods. 1,203 food-allergic patients, diagnosed on the basis of unequivocal clinical history and presence of circulating food allergen-specific IgE were studied. Serum IgE reactivity was assessed using the Allergen ExplorerALEX® system (Macroarray Diagnostics, Vienna, Austria). Association of specific IgE reactivities with FDEIAR was investigated, and patients with and without FDEIAR sensitized to LTP were compared. Results. 116 subjects (9.6%) had FDEIAR. Among these, 77 (66.3%) were LTP-reactors and 16 (13.8%) were sensitized to Tri a 19 (omega-5-gliadin). Different LTPs and omega-5-gliadin emerged as the sole allergens clearly associated with FDEIAR. Severity of allergic reactions was paralleled the level of specific IgE to LTPs. Patients with FDEIAR showed significantly lower IgE levels than their counterparts with food allergy at rest, and displayed nearly identical IgE levels regardless of the severity of allergic reactions induced by exercise. Conclusions. FDEIAR are associated with specific allergens. Specific IgE levels in LTP-hypersensitive patients with FDEIAR show an intermediate titer between those simply sensitized and those showing classic food allergy.

Identification of allergens from Aspergillus fumigatus-Potential association with lung damage in asthma.

Component-resolved diagnosis allows detection of IgE sensitization having the advantage of reproducibility and standardization compared to crude extracts. The main disadvantage of the traditional allergen identification methods, 1- or 2-dimensional western blotting and screening of expression cDNA libraries with patients' IgEs, is that the native structure of the protein is not necessarily maintained. We used a novel immunoprecipitation technique in combination with mass spectrometry to identify new allergens of Aspergillus fumigatus. Magnetic Dynabeads coupled with anti-human IgE antibodies were used to purify human serum IgE and subsequently allergens from A. fumigatus protein extract. Of the 184 proteins detected by subsequent mass peptide fingerprinting, a subset of 13 were recombinantly expressed and purified. In a panel of 52 A. fumigatus-sensitized people with asthma, 23 non-fungal-sensitized asthmatics and 18 healthy individuals, only the former showed an IgE reaction by immunoblotting and/or ELISA. We discovered 11 proteins not yet described as A. fumigatus allergens, with fructose-bisphosphate aldolase class II (FBA2) (33%), NAD-dependent malate dehydrogenase (31%) and Cu/Zn superoxide dismutase (27%) being the most prevalent. With respect to these three allergens, native versus denatured protein assays indicated a better recognition of the native proteins. Seven of 11 allergens fulfilled the WHO/IUIS criteria and were accepted as new A. fumigatus allergens. In conclusion, we introduce a straightforward method of allergen identification from complex allergenic sources such as A. fumigatus by immunoprecipitation combined with mass spectrometry, which has the advantage over traditional methods of identifying allergens by maintaining the structure of the proteins.

Tick bite-induced alpha-gal syndrome and immunologic responses in an alpha-gal deficient murine model.

Alpha-Gal Syndrome (AGS) is a delayed allergic reaction due to specific IgE antibodies targeting galactose-α-1,3-galactose (α-gal), a carbohydrate found in red meat. This condition has gained significant attention globally due to its increasing prevalence, with more than 450,000 cases estimated just in the United States alone. Previous research has established a connection between AGS and tick bites, which sensitize individuals to α-gal antigens and elevate the levels of specific IgE. However, the precise mechanism by which tick bites influence the host's immune system and contribute to the development of AGS remains poorly understood. This study investigates various factors related to ticks and the host associated with the development of AGS following a tick bite, using mice with a targeted disruption of alpha-1,3-galactosyltransferase (AGKO) as a model organism. Lone-star tick (Amblyomma americanum) and gulf-coast tick (Amblyomma maculatum) nymphs were used to sensitize AGKO mice, followed by pork meat challenge. Tick bite site biopsies from sensitized and non-sensitized mice were subjected to mRNA gene expression analysis to assess the host immune response. Antibody responses in sensitized mice were also determined. Our results showed a significant increase in the total IgE, IgG1, and α-gal IgG1 antibodies titers in the lone-star tick-sensitized AGKO mice compared to the gulf-coast tick-sensitized mice. Pork challenge in Am. americanum -sensitized mice led to a decline in body temperature after the meat challenge. Gene expression analysis revealed that Am. americanum bites direct mouse immunity toward Th2 and facilitate host sensitization to the α-gal antigen. This study supports the hypothesis that specific tick species may increase the risk of developing α-gal-specific IgE and hypersensitivity reactions or AGS, thereby providing opportunities for future research on the mechanistic role of tick and host-related factors in AGS development.