Identification of a vicilin-like major allergen from Prosopis juliflora exhibiting cross- reactivity with legume food allergens.

BACKGROUND: Prosopis juliflora is a clinically relevant allergic sensitizer worldwide and shares cross-reactivity with allergens from several tree pollen and food. The present study aims to purify and immunobiochemically characterize a major allergen from Prosopis pollen. The allergen was further investigated for its cross-reactivity with legume allergens. METHODS: Prosopis extract was fractionated by Q Sepharose and Superdex 75 gel filtration column to purify the allergen. Specific IgE against purified protein was estimated via ELISA and immunoblot. The protein was subjected to mass spectrometric analysis. Glycan characterization was performed by Schiff staining and lectin binding assay followed by deglycosylation studies. The functional activity of the purified protein was evaluated by the basophil activation test. Cross-reactivity was assessed by inhibition studies with legume extracts. RESULTS: A 35 kDa protein was purified and showed 75% IgE reactivity with the patients' sera by ELISA and immunoblot. Glycan characterization of protein demonstrated the presence of terminal glucose and mannose residues. A reduction of 40% and 27% in IgE binding was observed upon chemical and enzymatic deglycosylation of the protein, respectively. The glycoprotein allergen upregulates the expression of CD203c on basophils which was significantly reduced upon deglycosylation, signifying its biological ability to activate the effector cells. The identified protein shared significant homology with Lup an 1 from the lupine bean. Immunoblot inhibition studies of the purified allergen with legume extracts underlined high cross-reactive potential. Complete inhibition was observed with peanut and common bean, while up to 70% inhibition was demonstrated with soy, black gram, chickpea, and lima bean. CONCLUSION: A 35 kDa vicilin-like major allergen was isolated from P. juliflora. The protein possesses glycan moieties crucial for IgE binding and basophil activation. Furthermore, the purified protein shows homology with Lup an 1 and exhibits cross-reactivity with common edible legume proteins.

Preclinical Efficacy and Anti-Inflammatory Mechanisms of Action of the Bruton Tyrosine Kinase Inhibitor Rilzabrutinib for Immune-Mediated Disease.

Bruton tyrosine kinase (BTK) is expressed in B cells and innate immune cells, acting as an essential signaling element in multiple immune cell pathways. Selective BTK inhibition has the potential to target multiple immune-mediated disease pathways. Rilzabrutinib is an oral, reversible, covalent BTK inhibitor designed for immune-mediated diseases. We examined the pharmacodynamic profile of rilzabrutinib and its preclinical mechanisms of action. In addition to potent and selective BTK enzyme and cellular activity, rilzabrutinib inhibited activation and inflammatory activities of B cells and innate cells such as macrophages, basophils, mast cells, and neutrophils, without cell death (in human and rodent assay systems). Rilzabrutinib demonstrated dose-dependent improvement of clinical scores and joint pathology in a rat model of collagen-induced arthritis and demonstrated reductions in autoantibody-mediated FcγR signaling in vitro and in vivo, with blockade of rat Arthus reaction, kidney protection in mouse Ab-induced nephritis, and reduction in platelet loss in mouse immune thrombocytopenia. Additionally, rilzabrutinib inhibited IgE-mediated, FcεR-dependent immune mechanisms in human basophils and mast cell-dependent mouse models. In canines with naturally occurring pemphigus, rilzabrutinib treatment resulted in rapid clinical improvement demonstrated by anti-inflammatory effects visible within 2 wk and all animals proceeding to complete or substantial disease control. Rilzabrutinib is characterized by reversible covalent BTK binding, long BTK residence time with low systemic exposure, and multiple mechanistic and biological effects on immune cells. Rilzabrutinib's unique characteristics and promising efficacy and safety profile support clinical development of rilzabrutinib for a broad array of immune-mediated diseases.

Identification of peroxidase-1 and beta-glucosidase as cross-reactive wheat allergens in grass pollen-related wheat allergy.

BACKGROUND: Some patients with wheat-dependent exercise-induced anaphylaxis (WDEIA) or wheat allergy showed negative ω-5 gliadin-specific IgE test and high level of grass pollen-specific IgE. It was presumed that these patients developed allergic reaction upon cross-reaction of their IgE antibodies raised against grass pollen allergens to wheat allergens. This study aimed to clarify clinical characteristics and wheat allergens of this phenotype of WDEIA/wheat allergy, which were tentatively diagnosed as grass pollen-related wheat allergy (GPWA). METHODS: A total of six patients with GPWA were enrolled, and controls were 17 patients with grass pollen allergy but no episode of wheat allergy, and 29 patients with other wheat allergies: 18 with conventional WDEIA and 11 with hydrolyzed wheat protein allergy. Sensitization to wheat proteins was determined by basophil activation test (BAT). IgE-binding proteins in wheat flour were identified by immunoblotting followed by mass spectrometry. Wheat allergen-specific IgE tests were established by CAP-FEIA system. RESULTS: All the six patients with GPWA were sensitized to water-soluble wheat proteins in BAT and IgE-immunoblotting, and peroxidase-1 (35 kDa) and beta-glucosidase (60 kDa) were identified as specific IgE-binding wheat proteins. The binding of patient IgE to these proteins was inhibited by pre-incubation of patient sera with grass pollen. The peroxidase-1- and beta-glucosidase-specific IgE tests identified three and four of six patients with GPWA, respectively, but only two of 29 controls, indicating high specificity of these tests. CONCLUSIONS: Peroxidase-1 and beta-glucosidase are specific wheat allergens for GPWA among grass pollen allergy and other types of wheat-induced food allergies.

Baicalin induces Mrgprb2-dependent pseudo-allergy in mice.

Baicalin, a component of traditional Chinese medicine, is one of the main compounds present in Scutellaria baicalensis Georgi. Pseudo-allergy induced by the injection of these medicines is a frequent adverse drug reaction. Therefore, elucidation of the anaphylactoid reaction of baicalin and its underlying mechanisms are important. Mast cells are primary effectors of allergic reactions, including pseudo-allergy. Studies have shown that Mrgprx2 in human mast cells is a specific receptor that is crucial for pseudo-allergic drug reactions, Mrgprb3 is the rat ortholog of human Mrgprx2, which in mice is designated as Mrgprb2. Here, we aimed to investigate baicalin-induced pseudo-allergy and the association of Mrgprb3 and Mrgprb2 with this effect. We examined the allergenic effect of baicalin on RBL-2H3 cells and Mrgprb3-knockdown RBL-2H3 cells. Mrgprb2-expressing HEK293 cells and Mrgprb2-knockout mice were used to evaluate the role of Mrgprb2 in baicalin-induced allergy. Baicalin was found to dose-dependently induce pseudo-allergy both in vitro and in vivo. RBL-2H3 cells were activated by baicalin, whereas in Mrgprb3-knockout RBL-2H3 cells, baicalin showed a negligible effect on cell activation. Furthermore, baicalin activated the Mrgprb2-expressing HEK293 cells. Our data showed that baicalin did not induce allergy in Mpgprb2-knockout mice. We conclude that baicalin induces pseudo-allergy via Mrgprb2 in mice.

Streptococcal H2O2 inhibits IgE-triggered degranulation of RBL-2H3 mast cell/basophil cell line by inducing cell death.

Mast cells and basophils are central players in allergic reactions triggered by immunoglobulin E (IgE). They have intracellular granules containing allergic mediators (e.g., histamine, serotonin, inflammatory cytokines, proteases and ß-hexosaminidase), and stimulation by IgE-allergen complex leads to the release of such allergic mediators from the granules, that is, degranulation. Mast cells are residents of mucosal surfaces, including those of nasal and oral cavities, and play an important role in the innate defense system. Members of the mitis group streptococci such as Streptococcus oralis, are primary colonizers of the human oral cavity. They produce hydrogen peroxide (H2O2) as a by-product of sugar metabolism. In this study, we investigated the effects of streptococcal infection on RBL-2H3 mast cell/basophil cell line. Infection by oral streptococci did not induce degranulation of the cells. Stimulation of the RBL-2H3 cells with anti-dinitrophenol (DNP) IgE and DNP-conjugated human serum albumin triggers degranulation with the release of ß-hexosaminidase. We found that S. oralis and other mitis group streptococci inhibited the IgE-triggered degranulation of RBL-2H3 cells. Since mitis group streptococci produce H2O2, we examined the effect of S. oralis mutant strain deficient in producing H2O2, and found that they lost the ability to suppress the degranulation. Moreover, H2O2 alone inhibited the IgE-induced degranulation. Subsequent analysis suggested that the inhibition of degranulation was related to the cytotoxicity of streptococcal H2O2. Activated RBL-2H3 cells produce interleukin-4 (IL-4); however, IL-4 production was not induced by streptococcal H2O2. Furthermore, an in vivo study using the murine pollen-induced allergic rhinitis model suggested that the streptococcal H2O2 reduces nasal allergic reaction. These findings reveal that H2O2 produced by oral mitis group streptococci inhibits IgE-stimulated degranulation by inducing cell death. Consequently, streptococcal H2O2 can be considered to modulate the allergic reaction in mucosal surfaces.

Cinnamon extract inhibits allergen-specific immune responses in human and murine allergy models.

BACKGROUND: Ceylon cinnamon has been shown to possess anti-inflammatory properties in many diseases including allergic inflammation. OBJECTIVE: The aim of this study was to analyse in more detail the effects of cinnamon extract (CE) and its major compounds p-cymene and trans-cinnamaldehyde (CA) on allergen-specific immune responses in vitro and in vivo. METHODS: Therefore, monocyte-derived mature dendritic cells (DC) from grass or birch pollen allergic donors were pulsed with the respective allergen in the presence or absence of CE, p-cymene, CA or the solvent ethanol and co-cultured with autologous CD4+ T cells. Furthermore, basophil activation test was performed with or without CE or ethanol treatment. For the in vivo experiments, BALB/c mice were immunized with ovalbumin (OVA) and orally treated with CE or ethanol. RESULTS: Addition of CE, p-cymene or CA, but not ethanol significantly inhibited DC maturation and subsequent allergen-specific T cell proliferation as well as Th1 and Th2 cytokine production. Sulphidoleukotriene release and CD63 expression by basophils were also significantly diminished after addition of CE. In vivo, treatment of OVA-sensitized mice with CE led to a significant shift from OVA-specific IgE towards IgG2a production and to a strong inhibition of OVA-specific proliferation. Moreover, airway inflammation as well as anaphylaxis after intranasal or systemic allergen challenge was significantly reduced in CE-treated mice. Furthermore, topical application of CE prevented calcipotriol-induced atopic dermatitis-like inflammation in these mice. CONCLUSIONS AND CLINICAL RELEVANCE: Taken together, our data indicate that the anti-inflammatory effect of cinnamon might be exploited for treatment of allergic inflammation, which needs to be further investigated.

Agaricus blazei-Based Mushroom Extract Supplementation to Birch Allergic Blood Donors: A Randomized Clinical Trial.

Since Agaricus blazei Murill (AbM) extract reduced specific IgE and ameliorated a skewed Th1/Th2 balance in a mouse allergy model, it was tested in blood donors with self-reported, IgE-positive, birch pollen allergy and/or asthma. Sixty recruited donors were randomized in a placebo-controlled, double-blinded study with pre-seasonal, 7-week, oral supplementation with the AbM-based extract AndosanTM. Before and after the pollen season, questionnaires were answered for allergic rhino-conjunctivitis, asthma, and medication; serum IgE was measured, and Bet v 1-induced basophil activation was determined by CD63 expression. The reported general allergy and asthma symptoms and medication were significantly reduced in the AbM compared to the placebo group during pollen season. During the season, there was significant reduction in specific IgE anti-Bet v 1 and anti-t3 (birch pollen extract) levels in the AbM compared with the placebo group. While the maximal allergen concentrations needed for eliciting basophil activation before the season, changed significantly in the placebo group to lower concentrations (i.e., enhanced sensitization) after the season, these concentrations remained similar in the AndosanTM AbM extract group. Hence, the prophylactic effect of oral supplementation before the season with the AbM-based AndosanTM extract on aeroallergen-induced allergy was associated with reduced specific IgE levels during the season and basophils becoming less sensitive to allergen activation.

Multiple roles of Bet v 1 ligands in allergen stabilization and modulation of endosomal protease activity.

BACKGROUND: Over 100 million people worldwide suffer from birch pollen allergy. Bet v 1 has been identified as the major birch pollen allergen. However, the molecular mechanisms of birch allergic sensitization, including the roles of Bet v 1 and other components of the birch pollen extract, remain incompletely understood. Here, we examined how known birch pollen-derived molecules influence the endolysosomal processing of Bet v 1, thereby shaping its allergenicity. METHODS: We analyzed the biochemical and immunological interaction of ligands with Bet v 1. We then investigated the proteolytic processing of Bet v 1 by endosomal extracts in the presence and absence of ligands, followed by a detailed kinetic analysis of Bet v 1 processing by individual endolysosomal proteases as well as the T-cell epitope presentation in BMDCs. RESULTS: We identified E1 phytoprostanes as novel Bet v 1 ligands. Pollen-derived ligands enhanced the proteolytic resistance of Bet v 1, affecting degradation kinetics and preferential cleavage sites of the endolysosomal proteases cathepsin S and legumain. E1 phytoprostanes exhibited a dual role by stabilizing Bet v 1 and inhibiting cathepsin protease activity. CONCLUSION: Bet v 1 can serve as a transporter of pollen-derived, bioactive compounds. When carried to the endolysosome, such compounds can modulate the proteolytic activity, including its processing by cysteine cathepsins. We unveil a paradigm shift from an allergen-centered view to a more systemic view that includes the host endolysosomal enzymes.

Pru p 7 sensitization is a predominant cause of severe, cypress pollen-associated peach allergy.

BACKGROUND: Peach is a common elicitor of food allergic reactions. Peach-induced immediate reactions may occur as benign pollen-food syndromes, usually due to birch pollen-related PR-10 cross-reactivity in temperate climates, and as potentially severe primary food allergies, predominantly related to nsLTP Pru p 3 in Mediterranean regions. The newly described peach allergen Pru p 7 has gained recent attention as a potential peach allergy severity marker. Sensitization to Pru p 7 and its allergenic homologues of the gibberellin-regulated protein family occurs in areas with high Cupressaceae tree pollen exposure. OBJECTIVE: We sought to investigate the distribution, clinical characteristics and molecular associations of Pru p 7 sensitization among subjects with suspected peach allergy in different regions of France. METHODS: Subjects with suspected peach allergy (n = 316) were included. Diagnostic work-up was performed according to current guidelines, including open food challenge when required. IgE antibody measurements and competition experiments were performed using the ImmunoCAP assay platform. RESULTS: Sensitization to Pru p 7 was present in 171 (54%) of all subjects in the study and in 123 of 198 (62%) diagnosed as peach allergic, more than half of whom were sensitized to no other peach allergen. Frequency and magnitude of Pru p 7 sensitization were associated with the presence of peach allergy, the clinical severity of peach-induced allergic reactions and the level of cypress pollen exposure. Cypress pollen extract completely outcompeted IgE binding to Pru p 7. Pru p 7 was extremely potent in basophil activation tests. CONCLUSION AND CLINICAL RELEVANCE: A subtype of Cupressaceae pollinosis, characterized by Pru p 7 sensitization, can be an underlying cause of severe peach allergy.

Profilin-mediated food-induced allergic reactions are associated with oral epithelial remodeling.

BACKGROUND: In areas of high exposure to grass pollen, allergic patients are frequently sensitized to profilin, and some experience severe profilin-mediated food-induced reactions. This specific population of patients is ideal to study the relationship between respiratory and food allergies. OBJECTIVE: We sought to determine the role of oral mucosal epithelial barrier integrity in profilin-mediated allergic reactions. METHODS: Thirty-eight patients with profilin allergy stratified into mild or severe according to their clinical history and response to a profilin challenge test and 6 nonallergic subjects were recruited. Oral mucosal biopsies were used for measurement of CD11c, CD3, CD4, tryptase, claudin-1, occludin, E-cadherin, and vascular endothelial growth factor A levels; Masson trichrome staining; and POSTN, IL33, TPSAB, TPSB, and CMA gene expression analysis by using quantitative RT-PCR. Blood samples were used for basophil activation tests. RESULTS: Distinct features of the group with severe allergy included the following: (1) impaired epithelial integrity with reduced expression of claudin-1, occludin, and E-cadherin and decreased numbers of epithelial cells, which is indicative of acanthosis, higher collagen deposition, and angiogenesis; (2) inflammatory immune response in the mucosa, with an increased number of CD11c+ and CD4+ infiltrates and increased expression of the cytokine genes POSTN and IL33; and (3) a 10-fold increased sensitivity of basophils to profilin. CONCLUSIONS: Patients with profilin allergy present with significant damage to the oral mucosal epithelial barrier, which might allow profilin penetration into the oral mucosa and induction of local inflammation. Additionally, severely allergic patients presented with increased sensitivity of effector cells.

Periostin deletion suppresses late-phase response in mouse experimental allergic conjunctivitis.

BACKGROUND: To investigate the potential roles of periostin (POSTN), an extracellular matrix preferentially expressed in Th2-skewed conditions in the pathophysiology of allergic conjunctivitis. METHODS: The roles of POSTN in ragweed-induced experimental allergic conjunctivitis (RW-EAC) were evaluated using both POSTN-knockout (KO) and congenic BALB/c wild-type mice. Histological analysis was carried out to enumerate eosinophils/basophils in the conjunctival tissue. Th2 cytokine expression was evaluated by quantitative polymerase chain reaction (Q-PCR), and microarray analysis was performed to elucidate genes differentially expressed in POSTN-KO and wild-type mice in the RW-EAC model. RESULTS: Upregulation of POSTN expression and eosinophil infiltration was observed in subconjunctival tissue of RW-EAC in the wild-type mice. The number of infiltrating eosinophils in the conjunctivae of RW-EAC was diminished in POSTN-KO mice compared to wild-type mice. Q-PCR analysis of conjunctival tissue showed induction of Th2 cytokine (Ccl5, Il4, Il5, Il13) expression in the RW-EAC and attenuated Ccl5, Il4, Il13 mRNA expression in the conjunctivae of the RW-EAC using POSTN-KO mice. Microarray analysis and immunohistochemical analysis showed diminished basophil marker (Mcpt8) expression and reduced numbers of infiltrating basophils in the conjunctivae of RW-EAC in POSTN-KO mice. CONCLUSIONS: POSTN expression in conjunctival tissue plays an indispensable role in the late-phase reaction of the RW-EAC model by facilitating eosinophil/basophil infiltration and augmenting Th2 cytokine expression.

Chemically modified peanut extract shows increased safety while maintaining immunogenicity.

BACKGROUND: 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 . METHODS: 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. RESULTS: 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 in vivo and in vitro. In mice sensitized to PE, the threshold for anaphylaxis (drop in BT) upon subcutaneous challenge with PE was 0.01 mg, while at 0.3 mg 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. CONCLUSION: 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.

Non-Digestible Oligosaccharides Can Suppress Basophil Degranulation in Whole Blood of Peanut-Allergic Patients.

Background: Dietary non-digestible oligosaccharides (NDOs) have a protective effect against allergic manifestations in children at risk. Dietary intervention with NDOs promotes the colonization of beneficial bacteria in the gut and enhances serum galectin-9 levels in mice and atopic children. Next to this, NDOs also directly affect immune cells and low amounts may reach the blood. We investigated whether pre-incubation of whole blood from peanut-allergic patients with NDOs or galectin-9 can affect basophil degranulation. Methods: Heparinized blood samples from 15 peanut-allergic adult patients were pre-incubated with a mixture of short-chain galacto-oligosaccharides and long-chain fructo-oligosaccharides (scGOS/lcFOS), scFOS/lcFOS, or galectin-9 (1 or 5 µg/mL) at 37°C in the presence of IL-3 (0.75 ng/mL). After 2, 6, or 24 h, a basophil activation test was performed. Expression of FcεRI on basophils, plasma cytokine, and chemokine concentrations before degranulation were determined after 24 h. Results: Pre-incubation with scGOS/lcFOS, scFOS/lcFOS, or galectin-9 reduced anti-IgE-mediated basophil degranulation. scFOS/lcFOS or 5 µg/mL galectin-9 also decreased peanut-specific basophil degranulation by approximately 20%, mainly in whole blood from female patients. Inhibitory effects were not related to diminished FcεRI expression on basophils. Galectin-9 was increased in plasma after pre-incubation with scGOS/lcFOS, and both NDOs and 5 µg/mL galectin-9 increased MCP-1 production. Conclusion and clinical relevance: The prebiotic mixture scFOS/lcFOS and galectin-9 can contribute to decreased degranulation of basophils in vitro in peanut-allergic patients. The exact mechanism needs to be elucidated, but these NDOs might be useful in reducing allergic symptoms.

The Basophil Activation Test Can Be of Value for Diagnosing Immediate Allergic Reactions to Omeprazole.

BACKGROUND: Proton pump inhibitors (PPIs) are commonly used to treat gastrointestinal diseases. Incidence of hypersensitivity reactions to PPIs has risen, likely because of increased consumption. Their diagnosis is difficult, with skin tests (STs) presenting low sensitivity, making it necessary to perform drug provocation tests (DPTs). The value of in vitro tests for the diagnosis of immediate reaction to PPI is unclear. OBJECTIVE: To analyze the diagnostic value of the basophil activation test (BAT) in a group of patients diagnosed with immediate allergy to omeprazole. METHODS: The study included 42 patients with confirmed immediate allergic reactions to omeprazole confirmed by positive ST results or DPT results and 22 age- and sex-matched subjects tolerant to PPIs. BAT was performed with omeprazole, pantoprazole, and lansoprazole using CD63 and CD203c as activation markers. RESULTS: ST sensitivity was 66.7% with a specificity of 100%. BAT using CD63 with a stimulation index of more than 2 as positive revealed a sensitivity of 73.8%, a specificity of 100%, a positive predictive value of 100%, and a negative predictive value of 66.7%. BAT was positive in 57.1% of patients with negative ST result, and thus by combining ST and BAT we can correctly diagnose 85.7% of patients with immediate allergy to omeprazole. CONCLUSION: BAT represents a complementary tool for inclusion in the allergological workup for patients allergic to omeprazole. When combined with ST, it can be of value to guide the clinician as to whether to perform a DPT.

[A CASE OF A 7-YEAR-OLD GIRL WITH ANAPHYLAXIS CAUSED BY PECTIN IN ALBEDO OF CITRUS UNSHIU BUT NOT BY THE FRUIT ITSELF].

Pectin is used in several foods as an additive and a thickner. But some cases of anaphylaxis have been reported. Most of these are induced by occasional exposures; however, no cases of anaphylaxis after eating a Citrus unshiu, the albedo of which is rich in pectin, have been reported.A 7-year-old girl developed barking cough and pruritus approximately two hours after eating a frozen Citrus unshiu. She had a history of anaphylaxis induced by consuming cashew nuts. Skin testing and basophil activation tests were performed using a commercially available pectin product. Both tests were positive. In an oral food challenge test, she felt abdominal pain and nausea only after eating fruit, along with the albedo, of Citrus unshiu. We concluded that this case was induced by pectin present in the albedo of Citrus unshiu, but not by the fruit itself. We should consider that patients with cashew nut allergies have a possibility of pectin allergies as well, and that pectin in the albedo of Citrus unshiu may induce anaphylaxis.

Characterization of a Major Allergen from Mongolian Oak, Quercus mongolica, a Dominant Species of Oak in Korea.

BACKGROUND: Oaks are the most common trees in Korean forests, and Mongolian oak, Quercus mongolica, is the dominant species. However, no allergen has been characterized from Mongolian oak. In this study, we tried to characterize a major allergen from Mongolian oak. METHODS: A molecule homologous to pathogenesis-related 10 (PR-10)-like protein, Que m 1, was cloned by RT-PCR. Its recombinant protein, along with Que a 1, an allergen from white oak (Q. alba), was produced. The allergenicity and diagnostic value of recombinant Que m 1, Que a 1, and Bet v 1 proteins were compared by ELISA using sera from oak-sensitized subjects. A basophil activation test was also performed using CD63 expression as an activation marker. RESULTS: Que m 1 sequence shares 57.5-96.2% amino acid sequence identity with PR-10-like allergens from various plants. Specific IgE to recombinant Que m 1, Que a 1, and Bet v 1 were detected in 92.0, 74.0, and 38.0% of 50 serum samples from Korean tree pollinosis patients. Recombinant Que m 1 was able to inhibit IgE reactivity to Que a 1 and Bet v 1, indicating its strong cross-reactivity. The activation patterns of basophils from 5 patients were similar in terms of the CD63 expression and protein concentration of challenged Bet v 1 and Que m 1. CONCLUSIONS: A major allergen, Que m 1, was cloned, and its recombinant protein was produced from Mongolian oak, a dominant species in Korea. Recombinant Que m 1 is potentially useful for the diagnosis and treatment of tree pollinosis in Korea.

A Recombinant Fragment of Human Surfactant Protein D Suppresses Basophil Activation and T-Helper Type 2 and B-Cell Responses in Grass Pollen-induced Allergic Inflammation.

RATIONALE: Recombinant fragment of human surfactant protein D (rfhSP-D) has been shown to suppress house dust mite- and Aspergillus fumigatus-induced allergic inflammation in murine models. OBJECTIVES: We sought to elucidate the effect of rfhSP-D on high-affinity IgE receptor- and CD23-mediated, grass pollen-induced allergic inflammatory responses. METHODS: rfhSP-D, containing homotrimeric neck and lectin domains, was expressed in Escherichia coli BL21(λDE3)pLysS cells. Peripheral blood mononuclear cells and sera were obtained from individuals with grass pollen allergy (n = 27). The effect of rfhSP-D on basophil activation and histamine release was measured by flow cytometry. IgE-facilitated allergen binding and presentation were assessed by flow cytometry. T-helper cell type 2 (Th2) cytokines were measured in cell culture supernatants. The effect of rfhSP-D on IgE production by B cells when stimulated with CD40L, IL-4, and IL-21 was also determined. MEASUREMENTS AND MAIN RESULTS: rfhSP-D bound to Phleum pratense in a dose- and calcium-dependent manner. Allergen-induced basophil responsiveness and histamine release were inhibited in the presence of rfhSP-D, as measured by CD63, CD203c (P = 0.0086, P = 0.04205), and intracellularly labeled diamine oxidase (P = 0.0003, P = 0.0148). The binding of allergen-IgE complexes to B cells was reduced by 51% (P = 0.002) in the presence of rfhSP-D. This decrease was concomitant with reduction in CD23 expression on B cells (P < 0.001). rfhSP-D suppressed allergen-driven CD27-CD4+CRTh2+ T-cell proliferation (P < 0.01), IL-4, and IL-5 levels (all P < 0.01). Moreover, rfhSP-D inhibited CD40L/IL-4- and IL-21-mediated IgE production (77.12%; P = 0.02) by B cells. CONCLUSIONS: For the first time, to our knowledge, we show that rfhSP-D inhibited allergen-induced basophil responses at a single-cell level and suppressed CD23-mediated facilitated allergen presentation and Th2 cytokine production. In addition, rfhSP-D inhibited IgE synthesis by B cells, which is also a novel observation.

Basophil activation test for inhalant allergens in pediatric patients with allergic rhinitis.

OBJECTIVE: Flow cytometric quantification of in vitro basophil activation can be quite performant and reliable tool to measure IgE-dependent allergen-specific responses in allergic patients. Current study aimed to evaluate the clinical relevance of basophil activation test (BAT) for the diagnosis of pediatric grass pollen and house dust mite (HDM) allergies. METHODS: Forty-seven patients suffering from allergic rhinitis with HDM and grass pollen co-sensitization with clinical history of allergic rhinitis and/or asthma and 15 non-allergic healthy subjects were enrolled. BAT was determined by flow cytometry upon double staining with anti-IgE/anti-CD63 mAb. RESULTS: Regarding HDM with cut-off point greater than 12.5% for CD63+ basophils sensitivity and specificity of the BAT were 90% and 73%, with positive predictive value (PPV) and negative predictive value (NPV) as 0.70 and 0.91, respectively. The analysis of concordance of being either allergic or healthy in comparison to BAT results for HDM revealed a substantial concordance (κ index = 0.61, p < 0.001). Grass pollen with cut-off point greater than 11%, BAT attained a sensitivity, specificity, PPV, and NPV of 96%, 93%, 0.98, and 0.88, respectively. The analysis of concordance of being either allergic or healthy in comparison to BAT results for grass pollen revealed an almost perfect concordance (κ index = 0.87, p < 0.001). CONCLUSION: Our findings concluded that BAT is reliable technique in the diagnosis of sensitization to grass pollen and HDM. The sensitivity of BAT in pollen allergic children was found to be remarkably higher in our cohort compared to other studies.