Sustained silencing peanut allergy by xanthopurpurin is associated with suppression of peripheral and bone marrow IgE-producing B cell.

Introduction: Peanut allergy is an immunoglobulin E (IgE) mediated food allergy. Rubia cordifolia L. (R. cordifolia), a Chinese herbal medicine, protects against peanut-induced anaphylaxis by suppressing IgE production in vivo. This study aims to identify IgE-inhibitory compounds from the water extract of R. cordifolia and investigate the underlying mechanisms using in vitro and in vivo models. Methods: Compounds were isolated from R. cordifolia water extract and their bioactivity on IgE production was assessed using a human myeloma U266 cell line. The purified active compound, xanthopurpurin (XPP), was identified by LC-MS and NMR. Peanut-allergic C3H/HeJ mice were orally administered with or without XPP at 200µg or 400µg per mouse per day for 4 weeks. Serum peanut-specific IgE levels, symptom scores, body temperatures, and plasma histamine levels were measured at challenge. Cytokines in splenocyte cultures were determined by ELISA, and IgE + B cells were analyzed by flow cytometry. Acute and sub-chronic toxicity were evaluated. IL-4 promoter DNA methylation, RNA-Seq, and qPCR analysis were performed to determine the regulatory mechanisms of XPP. Results: XPP significantly and dose-dependently suppressed the IgE production in U266 cells. XPP significantly reduced peanut-specific IgE (>80%, p <0.01), and plasma histamine levels and protected the mice against peanut-allergic reactions in both early and late treatment experiments (p < 0.05, n=9). XPP showed a strong protective effect even 5 weeks after discontinuing the treatment. XPP significantly reduced the IL-4 level without affecting IgG or IgA and IFN-γ production. Flow cytometry data showed that XPP reduced peripheral and bone marrow IgE + B cells compared to the untreated group. XPP increased IL-4 promoter methylation. RNA-Seq and RT-PCR experiments revealed that XPP regulated the gene expression of CCND1, DUSP4, SDC1, ETS1, PTPRC, and IL6R, which are related to plasma cell IgE production. All safety testing results were in the normal range. Conclusions: XPP successfully protected peanut-allergic mice against peanut anaphylaxis by suppressing IgE production. XPP suppresses murine IgE-producing B cell numbers and inhibits IgE production and associated genes in human plasma cells. XPP may be a potential therapy for IgE-mediated food allergy.

Untargeted serum metabolomic analysis reveals a role for purinergic signaling in FPIES.

BACKGROUND: Food protein-induced enterocolitis syndrome (FPIES) is a non-IgE-mediated food allergy with a typical onset in infancy. Its symptoms are distinct from those of IgE-mediated food allergies and include severe repetitive vomiting, lethargy, and pallor. FPIES reactions are associated with TH17 cytokines and a systemic innate immune activation; however, the link between immune activation and symptoms is poorly understood. OBJECTIVE: Our aim was to use an untargeted metabolomics approach to identify novel pathways associated with FPIES reactions. METHODS: Serum samples were obtained before, during, and after oral food challenge (OFC) (10 subjects with FPIES and 10 asymptomatic subjects), and they were analyzed by untargeted metabolomics. Two-way ANOVA with false discovery rate adjustment was used for analysis of metabolites. Stomach and duodenal biopsy specimens from non-FPIES donors were stimulated with adenosine in vitro and serotonin measured by immunoassay. RESULTS: The levels of a total of 34 metabolites, including inosine and urate of the purine signaling pathway, were increased during OFCs performed on the patients with symptomatic FPIES compared with the levels found for asymptomatic subjects. Expression of the purine receptors P2RX7 and P2RY10 and the ectonucleotidase CD73 in peripheral blood was significantly reduced after OFC of the patients with FPIES. The level of the serotonin metabolite 5-hydroxyindoleacetate was significantly elevated after reaction. Adenosine stimulation of gastric and duodenal biopsy specimens from FPIES-free donors induced a significant release of serotonin, suggesting a link between purinergic pathway activation and serotonin release. CONCLUSIONS: Activation of the purinergic pathway during FPIES reactions provides a possible mechanism connecting inflammation and vomiting by triggering serotonin release from gastric and duodenal mucosa.

Anti-IgE effect of small-molecule-compound arctigenin on food allergy in association with a distinct transcriptome profile.

BACKGROUND: Excessive production of IgE plays a major role in the pathology of food allergy. In an attempt to identify anti-IgE natural products, Arctium Lappa was one of the most effective herbs among approximately 300 screened medicinal herbs. However, little is known about its anti-IgE compounds. OBJECTIVE: To identify compounds from Arctium Lappa for targeted therapy on IgE production and explore their underlying mechanisms. METHODS: Liquid-liquid extraction and column chromatographic methods were used to purify the compounds. IgE inhibitory effects were determined on IgE-producing human myeloma U266 cells, peanut-allergic murine model and PBMCs from food-allergic patients. Genes involved in IgE inhibition in PBMCs were studied by RNA sequencing. RESULTS: The main compounds isolated were identified as arctiin and arctigenin. Both compounds significantly inhibited IgE production in U266 cells, with arctigenin the most potent (IC50=5.09µg/mL). Arctigenin (at a dose of 13 mg/kg) markedly reduced peanut-specific IgE levels, blocked hypothermia and histamine release in a peanut-allergic mouse model. Arctigenin also significantly reduced IgE production and Th2 cytokines (IL-5, IL-13) by PBMCs. We found 479 differentially expressed genes in PBMCs with arctigenin treatment (p < .001 and fold-change ≥1.5), involving 24 gene ontology terms (p < .001, FDR <0.05); cell division was the most significant. Eleven genes including UBE2C and BCL6 were validated by qPCR. CONCLUSION: Arctigenin markedly inhibited IgE production in U266 cells, peanut-allergic murine model and PBMCs from allergic patients by down-regulating cell division, cell cycle-related genes and up-regulating anti-inflammatory factors.

Systems Pharmacology and In Silico Docking Analysis Uncover Association of CA2, PPARG, RXRA, and VDR with the Mechanisms Underlying the Shi Zhen Tea Formula Effect on Eczema.

Eczema is a complex chronic inflammatory skin disease impacted by environmental factors, infections, immune disorders, and deficiencies in skin barrier function. Shi Zhen Tea (SZT), derived from traditional Chinese medicine Xiao-Feng-San, has shown to be an effective integrative therapy for treating skin lesions, itching, and sleeping loss, and it facilitates reduction of topical steroid and antihistamine use in pediatric and adult patients with severe eczema. Yet, its active compounds and therapeutic mechanisms have not been elucidated. In this study, we sought to investigate the active compounds and molecular mechanisms of SZT in treating eczema using systems pharmacology and in silico docking analysis. SZT is composed of 4 medicinal herbs, Baizhu (Atractylodis macrocephalae rhizome), Jingjie (Schizonepetae herba), Kushen (Sophorae flavescentis radix), and Niubangzi (Arctii fructus). We first identified 51 active compounds from SZT and their 81 potential molecular targets by high-throughput computational analysis, from which we identified 4 major pathways including Th17 cell differentiation, metabolic pathways, pathways in cancer, and the PI3K-Akt signaling pathway. Through network analysis of the compound-target pathway, we identified hub molecular targets within these pathways including carbonic anhydrase II (CA2), peroxisome proliferator activated receptor γ (PPAR γ), retinoid X receptor α (RXRA), and vitamin D receptor (VDR). We further identified top 5 compounds including cynarine, stigmasterin, kushenol, ß-sitosterol, and (24S)-24-propylcholesta-5-ene-3ß-ol as putative key active compounds on the basis of their molecular docking scores with identified hub target proteins. Our study provides an insight into the therapeutic mechanism underlying multiscale benefits of SZT for eczema and paves the way for developing new and potentially more effective eczema therapies.

Acute FPIES reactions are associated with an IL-17 inflammatory signature.

BACKGROUND: Food protein-induced enterocolitis syndrome (FPIES) is a non-IgE-mediated food allergy characterized by profuse vomiting within hours of ingestion of the causative food. We have previously reported that FPIES is associated with systemic innate immune activation in the absence of a detectable antigen-specific antibody or T-cell response. The mechanism of specific food recognition by the immune system remains unclear. OBJECTIVE: Our aim was to identify immune mechanisms underlying FPIES reactions by proteomic and flow cytometric analysis of peripheral blood. METHODS: Children with a history of FPIES underwent supervised oral food challenge. Blood samples were taken at baseline, at symptom onset, and 4 hours after symptom onset. We analyzed samples from 23 children (11 reactors and 12 outgrown). A total of 184 protein markers were analyzed by proximity ligation assay and verified by multiplex immunoassay. Analysis of cell subset activation was performed by mass cytometry and spectral cytometry. RESULTS: Symptomatic FPIES challenge results were associated with significant elevation of levels of cytokines and chemokines, including IL-17 family markers (IL-17A, IL-22, IL-17C, and CCL20) and T-cell activation (IL-2), and innate inflammatory markers (IL-8, oncostatin M, leukemia inhibitory factor, TNF-α, IL-10, and IL-6). The level of the mucosal damage marker regenerating family member 1 alpha (REG1A) was also significantly increased. These biomarkers were not increased in asymptomatic challenges or IgE-mediated allergy. The level of phospho-STAT3 was significantly elevated in myeloid and T cells after challenge in individuals with symptoms. Mass cytometry indicated preferential activation of nonconventional T-cell populations, including γδ T cells and CD3+CD4-CD8-CD161+ cells; however, the potential sources of IL-17 in PBMCs were primarily CD4+ TH17 cells. CONCLUSIONS: These results demonstrate a unique IL-17 signature and activation of innate lymphocytes in FPIES.

Eosinophilic esophagitis as a complication of food oral immunotherapy.

PURPOSE OF REVIEW: Food oral immunotherapy (OIT) has emerged as way to mitigate serious allergic reactions including life-threatening anaphylaxis related to accidental ingestion. However, gastrointestinal-related adverse effects of OIT have been reported and are often cited as reasons for discontinuation of therapy. We summarize recent research on the prevalence of eosinophilic esophagitis (EoE) in patients undergoing OIT. RECENT FINDINGS: We examined 12 recent studies on OIT for peanut, milk, walnut, egg, and wheat, which enrolled a total of 620 patients. Gastrointestinal symptoms were common during OIT, and while generally mild, 24 (3.9%) patients from the reviewed studies reported gastrointestinal symptoms that were significant enough to prompt discontinuation of OIT. Of these, two (0.3% of the total 620 patients or 8.3% of those with gastrointestinal symptoms) patients had biopsy-confirmed EoE. One of these patients was subsequently found to also have ulcerative colitis that had been previously undiagnosed. SUMMARY: EoE is a rare but concerning side effect of OIT. More research is needed to better elucidate both the OIT-related and patient-related factors that may predispose individuals to develop EoE. The presence of comorbid conditions and/or preexisting subclinical esophageal eosinophilia may account for some of cases of EoE identified during OIT.

IgE-binding epitope mapping of tropomyosin allergen (Exo m 1) from Exopalaemon modestus, the freshwater Siberian prawn.

Exopalaemon modestus (EM) is a shrimp delicacy that could cause food allergy, the major allergen of EM is Exo m 1. The amino acid (AA) sequence, IgE-binding epitopes and allergenic peptides in gastrointestinal (GI) digests of Exo m 1, and their effects on basophil function were investigated. Exo m 1 has an AA-sequence of high similarity with other shrimp tropomyosins, while not 100% matching. The IgE-binding epitopes of Exo m 1 are epitope 1 (43-59, VHNLQKRMQQLENDLDS), epitope 2 (85-105, VAALNRRIQLLEEDLERSEER), epitope 3 (131-164, ENRSLSDEERMDALENQLKEARFLAEEADRKYDE), epitope 4 (187-201, ESKIVELEEELRVVG) and epitope 5 (243-280, ERSVQKLQKEVDRLEDELVNEKEKYKSITDELDQTFSE). Among the thirty-three peptides of Exo m 1 identified in GI digests, two were highly recognized by IgE, twenty-four moderately or weakly bound IgE, and seven had no IgE-reactivities. These IgE-binding epitopes and GI digestion induced-allergenic peptides could activate basophil degranulation, and CD63 and CD203c expression, they could be potential peptide-based immunotherapy for shrimp allergic individuals.

Non-IgE-mediated gastrointestinal food allergies in children.

Non-IgE-mediated gastrointestinal food allergic disorders (non-IgE-GI-FA) including food protein-induced enterocolitis syndrome (FPIES), food protein-induced enteropathy (FPE), and food protein-induced allergic proctocolitis (FPIAP) are relatively uncommon in infants and young children, but are likely under-diagnosed. Non-IgE-GI-FA have a favorable prognosis, with majority resolving by age 3-5 years. Diagnosis relies on the recognition of symptoms pattern in FPIAP and FPIES and biopsy in FPE. Further studies are needed for a better understanding of the pathomechanism, which will lead eventually to the development of diagnostic tests and treatments. Limited evidence supports the role of food allergens in subsets of constipation, gastroesophageal reflux disease, irritable bowel syndrome, and colic. The immunologic pathomechanism is not fully understood and empiric prolonged avoidance of food allergens should be limited to minimize nutrient deficiency and feeding disorders/food aversions in infants.

Effect of chemical modifications on allergenic potency of peanut proteins.

BACKGROUND: Modification of native peanut extracts could reduce adverse effects of peanut immunotherapy. OBJECTIVE: We sought to compare native and chemically modified crude peanut extract (CPE) and major peanut allergens Ara h 2 and Ara h 6 in a mediator-release assay based on the rat basophilic leukemia (RBL) cell line transfected with human Fcε receptor. METHODS: Native Ara h 2/6 was reduced and alkylated (RA), with or without additional glutaraldehyde treatment (RAGA). CPE was reduced and alkylated. Sera of subjects with peanut allergy (16 males; median age 7 years) were used for overnight RBL-passive sensitization. Cells were stimulated with 0.1 pg/mL to 10 µg/mL of peanut. ß-N-acetylhexosaminidase release (NHR) was used as a marker of RBL degranulation, expressed as a percentage of total degranulation caused by Triton X. RESULTS: Median peanut-specific immunoglobulin E was 233 kUA/L. Nineteen subjects were responders, NHR ≥ 10% in the mediator release assay. Responders had reduced NHR by RA and RAGA compared with the native Ara h 2/6. Modification resulted in a later onset of activation by 10- to 100-fold in concentration and a lowering of the maximum release. Modified RA-Ara h 2/6 and RAGA-Ara h 2/6 caused significantly lower maximum mediator release than native Ara h 2/6, at protein concentrations 0.1, 1, and 10 ng/mL (p < 0.001, < 0.001, and < 0.001, respectively, for RA; and < 0.001, 0.026, and 0.041, respectively, for RAGA). RA-CPE caused significantly lower maximum NHR than native CPE, at protein concentration 1 ng/mL (p < 0.001) and 10 ng/mL (p < 0.002). Responders had high rAra h 2 immunoglobulin E (mean, 61.1 kUA/L; p < 0.001) and higher NHR in mediator release assay to native Ara h 2/6 than CPE, which indicates that Ara h 2/6 were the most relevant peanut allergens in these responders. CONCLUSIONS: Chemical modification of purified native Ara h 2 and Ara h 6 reduced mediator release in an in vitro assay ∼100-fold, which indicates decreased allergenicity for further development of the alternative candidate for safe peanut immunotherapy.

Berberine and limonin suppress IgE production by human B cells and peripheral blood mononuclear cells from food-allergic patients.

BACKGROUND: Currently, there is no satisfactory treatment for IgE-mediated food allergy. Food Allergy Herbal Formula 2 (FAHF-2) and butanol-purified FAHF-2 (B-FAHF-2) have been shown to protect against peanut-induced anaphylaxis and inhibit IgE synthesis in a murine model. OBJECTIVE: To determine which herbs and compounds in FAHF-2 and B-FAHF-2 suppress IgE production. METHODS: The effect of FAHF-2 and B-FAHF-2 on IgE production was determined using a human B-cell line (U266). Individual compounds were isolated and identified using column chromatography, liquid chromatographic mass spectrometry, and nuclear magnetic resonance techniques. The potency of compounds on IgE suppression were investigated using U266 cells and verified using human peripheral blood mononuclear cells (n = 25) from peanut-allergic patients. Epsilon germline transcript expression was determined. Phosphorylated IκBα level was analyzed using the In-Cell Western assay. The mRNA expression of signal transducer and activator of transcription-3, T-box transcription factor TBX21, interferon-γ, forkhead box P3, GATA-binding protein 3, interleukin-10, and interleukin-5 also were analyzed using real-time polymerase chain reaction. RESULTS: FAHF-2 and B-FAHF-2 inhibited IgE production by U266 cells. B-FAHF-2 was 9 times more effective than FAHF-2. Two compounds that inhibited IgE production were isolated from Philodendron chinensis and identified as berberine and limonin. Berberine was more potent and inhibited IgE production by peripheral blood mononuclear cells by 80% at 0.62 µg/mL. Berberine significantly inhibited ε-germline transcript expression by peripheral blood mononuclear cells. Phosphorylated IκBα level was significantly suppressed and mRNA expressions of T-box transcription factor TBX21 and signal transducer and activator of transcription-3 were significantly increased by berberine. CONCLUSION: Berberine and limonin mediated IgE suppression. The mechanism by which berberine modulates ε-germline transcript expression might be through regulating the phosphorylated IκBα level and the expressions of signal transducer and activator of transcription-3 and T-box transcription factor TBX21. TRIAL REGISTRATION: Clinicaltrials.gov identifier NCT00602160.

Definition, etiology, and diagnosis of food protein-induced enterocolitis syndrome.

PURPOSE OF REVIEW: Food protein-induced enterocolitis syndrome (FPIES) is a poorly understood non-IgE-mediated food hypersensitivity, primarily affecting infants and toddlers. There are few data regarding pathophysiology of FPIES that suggest local intestinal imbalance between TNF-α and TGF-ß. Patients frequently present with multiple reactions, which are characterized by projectile, repetitive emesis, dehydration, lethargy, and failure to thrive. Despite the severity of presentation, the diagnosis is frequently delayed, and patients often undergo extensive and invasive evaluation prior to reaching the diagnosis. RECENT FINDINGS: Reviews published in the last year provide a general approach to diagnosis and management of FPIES and aim to increase awareness and understanding of FPIES among general pediatricians. SUMMARY: Multicenter studies are necessary to reevaluate and modify the oral food challenge criteria. Research on the pathophysiology of FPIES reactions is necessary to provide insight into the evidence-based approach to diagnosis and management of FPIES. Registries are needed to understand the phenotype, triggers, and prevalence of FPIES.

In vitro assessment of the allergenicity of novel MF59-adjuvanted pandemic H1N1 influenza vaccine produced in dog kidney cells.

A licensed inactivated MF59-adjuvanted seasonal influenza vaccine (Optaflu) produced in canine kidney cells (MDCK 33016-PF) contained no egg proteins and did not trigger degranulation in rat basophilic leukemia (RBL) cells passively sensitized with human anti-dog IgE, supporting its safe use in dog-allergic individuals. The cell-derived pandemic H1N1 influenza vaccine was also adjuvanted with the emulsion adjuvant MF59, and support for its similar safe use was sought. We sought to evaluate in vitro allergenicity of the MF59-adjuvanted cell-derived pandemic H1N1 influenza vaccine in subjects with dog allergy, with a mediator release assay. RBL-2H3 cells transfected with human Fcε receptor type 1 were sensitized with sera from adult dog-allergic subjects and stimulated with serial dilutions of pandemic H1N1 influenza vaccine and dog dander extract. ß-N-hexosaminidase release (NHR) was used as a marker of RBL degranulation.. Median dog dander-specific IgE in 30 dog-allergic subjects was 27.7 kU(A)/L (range 10.1; > 100); and in 5 dog non-allergic subjects was < 0.35 kU(A)/L (UniCAP system). Median (range) maximum NHR in dog-allergic subjects was: pandemic H1N1 influenza vaccine 1.1% (0; 4.4) and dog dander 6.9% (0.7; 37.3), P < 0.001. In conclusion, MF59-adjuvanted pandemic H1N1 influenza vaccine produced in continuous canine kidney cells did not trigger degranulation in RBL cells passively sensitized with human anti-dog IgE, supporting its safe use in dog-allergic individuals.

Significance of ovomucoid- and ovalbumin-specific IgE/IgG(4) ratios in egg allergy.

BACKGROUND: The role of specific IgG(4) antibodies in natural tolerance acquisition remains a matter of debate; the specific IgE/IgG(4) ratio might add value to the measurement of absolute amounts of IgE for assessing the ongoing status of egg reactivity. OBJECTIVE: We sought to determine the significance of IgG(4) antibodies to ovalbumin (OVA) and ovomucoid (OVM) in egg-allergic children. METHODS: One hundred seven egg-allergic children (mean age 6.9 years; range 1.6-18.6 years) were challenged to baked egg. The outcomes of the challenges were related to the level of specific IgE and IgG(4) to OVM and OVA, component IgE/IgG(4) ratios, and mediator release in a functional assay based on the rat basophil leukemia cell line. RESULTS: Baked egg-reactive children had significantly higher OVA and OVM ratios of IgE/IgG(4) and mediator release in the rat basophil leukemia-based assay than did tolerant children (P < .05 for both). The OVA- and OVM-specific IgE/IgG(4) ratios and mediator release were correlated. In the receiver operating characteristic analysis, the areas under the curve for a logistic regression model including specific IgE and IgG(4) to OVA and OVM were significantly greater compared with the areas under the curve for egg white-specific IgE and OVM-specific IgE. CONCLUSIONS: The balance between IgE and IgG(4) to OVA and OVM has functional consequences. A model that includes the interactions between IgE and IgG(4) to OVA and OVM accurately predicts reactivity to baked egg and warrants further investigation.

Strategies to prevent or reduce allergic disease.

The need for allergy prevention strategies has never been greater. Surging rates of food allergy and eczema are now adding to the already substantial burden of asthma and respiratory allergic diseases. The parallel rise in many other immune diseases suggests that the developing immune system is highly vulnerable to modern environmental changes. These strong environmental pressures may be one reason why simple allergen avoidance strategies have not been successful. Another more recent strategy to curtail the allergy epidemic has been to identify factors associated with modern lifestyle that may be causally linked with allergic disease, in an attempt to restore more favourable conditions for immune tolerance during early development. More hygienic conditions and disruption of microbial exposure have prompted strategies to restore this balance using probiotic and prebiotic supplements. Modern dietary changes linked with allergic diseases have prompted supplementation studies to assess the preventive merits of specific immunomodulatory dietary nutrients such as polyunsaturated fatty acids. Other nutrients such as antioxidants, folate, and vitamin D are also currently under investigation. Modern environmental pollutants have also been associated with adverse effects on immune development and the risk of disease. While many of these avenues have provided some promise, they have not yet translated into specific recommendations. Current evidence-based guidelines for allergy prevention remain limited to avoidance of cigarette smoke, promotion of breastfeeding and the use of hydrolysed formula when breastfeeding is not possible. Allergen avoidance strategies have been largely removed from most guidelines. It is hoped that a number of ongoing studies will help provide clearer recommendations around the use of probiotics, prebiotics, specific dietary nutrients and the role of early introduction of allergenic foods for the promotion of tolerance. Despite the current uncertainties, prevention remains the best long-term strategy to reduce the growing burden of allergic disease.

Mechanisms underlying differential food allergy response to heated egg.

BACKGROUND: Egg white proteins are usually subjected to heating, making them edible for the majority of children with egg allergy. OBJECTIVE: We sought to investigate the underlying mechanisms responsible for the reduced allergenicity displayed by heat-treated egg white allergens. METHODS: C3H/HeJ mice were orally sensitized with ovalbumin (OVA) or ovomucoid and challenged with native or heated proteins to evaluate their allergenicity. Immunoreactivity was assessed by immunoblotting using sera from children with egg allergy. In vitro gastrointestinal digestion of native and heated OVA and ovomucoid was studied by SDS-PAGE and liquid chromatography. Intestinal uptake of intact native and heated OVA and ovomucoid by human intestinal epithelial (Caco-2) cells was investigated. Rat basophil leukemia cells passively sensitized with mouse serum and human basophils passively sensitized with serum from children with egg allergy were used to assess the effector cell activation by heated, digested, and transported OVA and ovomucoid. RESULTS: Heated OVA and ovomucoid did not induce symptoms of anaphylaxis in sensitized mice when administered orally. Heating did not completely destroy IgE-binding capacity of OVA or ovomucoid but enhanced in vitro digestibility of OVA. Digestion of both OVA and ovomucoid diminished mediator release in rat basophil leukemia assay and basophil activation. Heating of allergens prevented transport across human intestinal epithelial cells in a form capable of triggering basophil activation or T-cell activation. CONCLUSION: Heat treatment reduces allergenicity of OVA and ovomucoid. This is partially a result of the enhanced gastrointestinal digestibility of heated OVA and the inability of heated OVA or ovomucoid to be absorbed in a form capable of triggering basophils.

In vitro assessment of the allergenicity of a novel influenza vaccine produced in dog kidney cells in individuals with dog allergy.

BACKGROUND: An inactivated influenza vaccine produced in canine kidney cells (MDCK 33016-PF) contains no egg proteins and may be used to immunize egg-allergic patients. Although no major dog allergens were identified in MDCK 33016-PF cells, minor dog allergens might be present and cause reactions in dog-allergic individuals. OBJECTIVE: To evaluate the allergenicity of the inactivated influenza vaccine produced in cell culture in a mediator release assay. METHODS: Rat basophil leukemia (RBL) cells transfected with human IgE receptor-1 were sensitized with sera from dog-allergic adults with positive skin prick test reactions to dog extract and detectable dog dander IgE and were stimulated with serial dilutions of vaccine and dog dander extract. N-hexosaminidase release (NHR) was used as a marker of RBL cell degranulation. Western blots were performed, and UniCAP was used to measure dog-specific IgE antibody levels. RESULTS: The median (interquartile range) level of dog dander IgE was 8.31 kU(A)/L (1.895-14.5 kU(A)/L) and of dog epithelium IgE was 3.19 kU(A)/L (0.835-6.27 kU(A)L). Median (range) maximum NHR (at the first 10-fold dilution) was 0% (0%-1.4%) to vaccine and 10.2% (0%-35.9%) to dog dander (P < .001). In an egg-allergic control subject, the maximum NHR to a vaccine cultured in chick embryo and containing egg protein was 10.2%. IgE antibodies in pooled sera did not bind to vaccine on immunoblots but produced strong binding to dog dander and epithelium extracts. Serum from an egg-allergic control subject strongly bound embryonated egg-derived vaccine. CONCLUSION: An influenza vaccine produced in continuous canine kidney cells did not trigger degranulation in RBL cells passively sensitized with human anti-dog IgE.