Mizuho Nagao

Allergen-Induced Basophil CD203c Expression as a Biomarker for Rush Immunotherapy in Patients with Japanese Cedar Pollinosis

Background: Rush immunotherapy (RIT) can confer rapid clinical benefit on patients with allergic rhinitis or asthma. However, biomarkers representing mechanisms for the efficacy of RIT are still to be established. CD203c is a basophil activation marker known to be upregulated by cross-linking of the FcΕRIα receptor and may serve as a useful marker. Objective: We sought to investigate the changes in allergen-induced CD203c expression in patients with Japanese cedar pollen (JCP) pollinosis who received RIT. Methods: Nine patients treated with RIT were enrolled in the study. Whole blood was incubated with various concentrations of JCP extract. CD203c expression on basophils was quantitated by means of flow cytometry. JCP-specific IgG4 levels in sera were measured with ELISA. Basophil histamine release, CAP-RAST to JCP (JCP-IgE) and total IgE were also examined. The biomarkers listed above were evaluated before and sequentially after RIT. Symptom and quality of life scores were obtained during pre- and posttreatment pollen seasons. Results: All patients showed significant improvement in symptom and quality of life scores after RIT. Serum JCP-specific IgG4 titers were significantly elevated at 1 month and remained at high levels 12 months after the treatment. Stimulation with JCP extract induced enhancement of basophil CD203c expression in a concentration-dependent manner except for 2 subjects in whom no increase in CD203c by an anti-IgE antibody was observed (nonresponders). Significant reductions in the responses were observed in 4 subjects after RIT (reduction in CD203c expression, RCE) whereas no changes were seen in 3 subjects (non-RCE). RCE subjects were older than non-RCE counterparts, with mean ages of 20 and 12 years, respectively. No significant changes in JCP-specific IgE and total IgE levels were seen before and after RIT. Conclusion: Allergen-induced CD203c expression in basophils may represent, at least in part, the cellular mechanism for the therapeutic responses to RIT for JCP pollinosis. However, further larger-scale studies to confirm the utility of the test are necessary.

Antigen-Induced Expression of CD203c on Basophils Predicts IgE-mediated Wheat Allergy

BACKGROUND For in vitro diagnosis of wheat allergy, specific IgE to wheat is known to be a poor predictive marker. Oral food challenge, the gold standard for the diagnosis, is accompanied by a risk of severe induced reactions. Reliable in vitro tests are needed to be developed for safe indication for oral challenge. OBJECTIVE We examined the utility of a basophil activation marker, CD203c, for the diagnosis of IgE-mediated wheat allergy. METHODS Fifty-eight children with suspected wheat allergy with positive CAP-FEIA to wheat were enrolled. On 70 occasions, the clinical distinction between patients with wheat allergy (WA) and patients tolerant to wheat (TW) was made by means of an oral food challenge test or recent history of immediate allergic reactions or tolerance after ingestion of wheat. Twelve replicate evaluations were performed in 9 patients over more than a 6-month interval. Thirty two patients on 43 occasions were diagnosed with WA and 27 were confirmed to be TW. One patient had both diagnoses 18 months apart. Peripheral blood was incubated with fractionated wheat extracts, purified native omega-5 gliadin (nOG5) and recombinant omega-5 gliadin (rOG5). Expression of CD203c on basophils was then analyzed by flow cytometry using a commercial kit. RESULTS All wheat proteins induced concentration-dependent enhancement of CD203c expression in WA, but did not in TW. The analysis of receiver operating characteristics (ROC) showed that nOG5-induced CD203c(high)% values provided the best power for discriminating between WA and TW, with a sensitivity of 85.0% and specificity of 77.0% at the cut-off level of 14.4%. AUC for CD203c with nOG5 were significantly higher than that for conventional CAP-FEIA, 0.89 and 0.73, respectively (p < 0.01). CONCLUSIONS Measurement of nOG-induced enhancement of CD203c on basophils is useful for the diagnosis of immediate wheat allergy in children.

Serum measurement of thymus and activation-regulated chemokine/CCL17 in children with atopic dermatitis: elevated normal levels in infancy and age-specific analysis in atopic dermatitis

Elevated blood levels of thymus and activation‐regulated chemokine (TARC)/CCL17 have been observed in atopic dermatitis (AD) and may serve as a new biomarker for AD. However, the normal levels, especially in children, have not been well determined. We sought to establish an efficient enzyme‐linked immunosorbent assay (ELISA) with a wide range of detection that would be suitable for measurement of serum TARC/CCL17 and to determine the normal ranges of this chemokine in different age groups and its diagnostic usefulness for AD. A sensitive specific ELISA for TARC/CCL17, which we previously reported, was modified to accommodate the wide range of TARC/CCL17 values often found in sera. Twenty‐seven children with AD under 6 yr of age and 25 age‐matched normal non‐atopic controls, and 18 patients with AD and 27 controls who were 6 yr and older were enrolled. The severity of AD was evaluated using the SCORAD index. The serum levels of TARC/CCL17 were measured with the ELISA, and the serum levels of IP‐10/CXCL10 were also measured. With the novel ELISA system, the assayable range of TARC/CCL17 was 14–8000 pg/ml, and the coefficient of variation at various concentrations ranged from 2.3% to 5.0%. The serum levels of TARC/CCL17 in normal individuals were significantly higher in young children, especially in the age group of 0–1 yr. The cut‐off values of TARC/CCL17 for the diagnosis of AD were 1431 pg/ml for 0–1 yr group, 803 pg/ml for 2–5 yr group and 510 pg/ml for the 6 yr and older group, with high sensitivity and specificity of 0.83 and 0.93, 0.83 and 0.92, 0.85 and 0.96, respectively. The magnitude of the decrease in the SCORAD index after treatment with topical steroids correlated significantly with the decrease in serum TARC/CCL17. There was no difference in the serum levels of IP‐10/CXCL10 between AD and the controls. The TARC/CCL17:IP‐10/CXCL10 ratio tended to be higher in the control children aged 0–1 yr than in those aged 2–5 yr. The serum level of TARC/CCL17 reflects the severity and therapeutic response in AD. The high normal levels in infants should be taken into account when assaying TARC/CCL17.

Eosinophils Promote Epithelial to Mesenchymal Transition of Bronchial Epithelial Cells

Eosinophilic inflammation and remodeling of the airways including subepithelial fibrosis and myofibroblast hyperplasia are characteristic pathological findings of bronchial asthma. Epithelial to mesenchymal transition (EMT) plays a critical role in airway remodelling. In this study, we hypothesized that infiltrating eosinophils promote airway remodelling in bronchial asthma. To demonstrate this hypothesis we evaluated the effect of eosinophils on EMT by in vitro and in vivo studies. EMT was assessed in mice that received intra-tracheal instillation of mouse bone marrow derived eosinophils and in human bronchial epithelial cells co-cultured with eosinophils freshly purified from healthy individuals or with eosinophilic leukemia cell lines. Intra-tracheal instillation of eosinophils was associated with enhanced bronchial inflammation and fibrosis and increased lung concentration of growth factors. Mice instilled with eosinophils pre-treated with transforming growth factor(TGF)-β1 siRNA had decreased bronchial wall fibrosis compared to controls. EMT was induced in bronchial epithelial cells co-cultured with human eosinophils and it was associated with increased expression of TGF-β1 and Smad3 phosphorylation in the bronchial epithelial cells. Treatment with anti-TGF-β1 antibody blocked EMT in bronchial epithelial cells. Eosinophils induced EMT in bronchial epithelial cells, suggesting their contribution to the pathogenesis of airway remodelling.

Neutrophil Proteases Activate Eosinophil Function in vitro

Background: Recent evidence suggests that both neutrophilic and eosinophilic inflammation persist in the airways of patients with severe asthma. Mechanisms for interaction between neutrophils and eosinophils are still to be understood. Since eosinophils express protease-activated receptor 2, neutrophil-derived serine proteases may activate eosinophils. Objective: We investigated the effect of neutrophil serine proteases on eosinophil effector functions. Methods: Peripheral blood eosinophils were stimulated with elastase, cathepsin G and proteinase 3. Superoxide generation was quantitated with the cytochrome C reduction method. A panel of cytokines and chemokines in the culture supernatants were measured with a multiplex beads array system. Effects of an elastase inhibitor, sivelestat, and a serine protease inhibitor, PMSF, on the protease-induced reactions were also tested. Results: Neutrophil proteases significantly induced superoxide production from eosinophils. Elastase was the most potent among them. Sivelestat and PMSF inhibited the reaction. The proteases induced production of IL-6, IL-8, TNF-α and GRO-α, that have a possible connection with neutrophilic inflammation. Conclusion: Neutrophil proteases activate eosinophils to produce superoxide, proinflammatory cytokines and neutrophilotactic chemokines and may further aggravate airway inflammation in patients with severe asthma.

Biomarkers for Allergen Immunotherapy in Cedar Pollinosis

To initiate, monitor, and complete effective immunotherapy, biomarkers to predict and visualize the immune responses are needed. First, we need to identify the right candidate for immunotherapy. Secondly, the immune responses induced by immunotherapy should be monitored. For the first objective, analysis of polymorphisms of candidate genes may be helpful, but still be in development. Regarding biomarkers for immune responsese, there are numerous reports that evaluate immunotherapy-induced immune changes such as suppression of effector cells, deviation to Th1 cytokine production, and induction of regulatory T cells. No standardized methods, however, have been established. Among them, a functional assay of blocking IgG activity, the IgE-facilitated allergen binding assay, may be useful. We quantitated induced expression of an activation marker, CD203c, on basophils and found that the assay efficiently predicts sensitivity to particular allergen and severity of the allergen-induced symptoms. In patients who received rush immunotherapy for Japanese cedar pollinosis, reduction in CD203c expression after the therapy was observed, suggesting the utility of the test for monitoring immunotherapy.

Highly increased levels of IgE antibodies to vaccine components in children with influenza vaccine–associated anaphylaxis

BACKGROUND Influenza vaccines produced in embryonated eggs might pose a risk to patients with egg allergy. However, patients experiencing influenza vaccine-associated anaphylaxis (IVA) do not always have egg allergy. In the 2011-2012 season, an unusually high incidence of IVA was reported in Japan. OBJECTIVE We sought to identify the cause of the increase in anaphylactic events in 2011-2012 in Japan. METHODS We collected blood specimens from patients with IVA from all areas of Japan. We analyzed 19 patients with confirmed IVA and 25 age-matched control subjects, including 10 with egg allergy who had no adverse events after corresponding vaccination. ELISA was used to measure specific IgE levels to the trivalent vaccines of several manufacturers and hemagglutinin proteins derived from both egg and cell cultures. Antigen-induced basophil activation was evaluated by measuring CD203c expression by means of flow cytometry. Vaccine excipients were also examined for effects on CD203c expression. RESULTS None of the patients with IVA had severe egg allergy. Levels of specific IgE antibodies to influenza vaccine antigens, whole-vaccine products from different manufacturers, and hemagglutinin proteins (A H1, H3, and B) derived from both egg and cell cultures were significantly increased in patients with IVA compared with those in control subjects. Influenza vaccine-induced CD203c expression in basophils was also highly enhanced in patients with IVA but not in control subjects. Because IVA was most frequent in patients who received 2-phenoxyethanol (2-PE)-containing vaccine, the effect of this preservative on basophil activation was examined, and the activation was slightly enhanced by 2-PE but not thimerosal. CONCLUSIONS The 2011-2012 IVA spike in Japan was caused by specific IgE antibodies to influenza vaccine components. Excipients could not be implicated, except for a modest effect of 2-PE.

Montelukast suppresses epithelial to mesenchymal transition of bronchial epithelial cells induced by eosinophils.

Epithelial to mesenchymal transition (EMT) is a mechanism by which eosinophils can induce airway remodeling. Montelukast, an antagonist of the cysteinyl leukotriene receptor, can suppress airway remodeling in asthma. The purpose of this study was to evaluate whether montelukast can ameliorate airway remodeling by blocking EMT induced by eosinophils. EMT induced was assessed using a co-culture system of human bronchial epithelial cells and human eosinophils or the eosinophilic leukemia cell lines, Eol-1. Montelukast inhibited co-culture associated morphological changes of BEAS-2b cells, decreased the expression of vimentin and collagen I, and increased the expression of E-cadherin. Montelukast mitigated the rise of TGF-β1 production and Smad3 phosphorylation. Co-culture of human eosinophils with BEAS-2B cells significantly enhanced the production of CysLTs compared with BEAS-2B cells or eosinophils alone. The increase of CysLTs was abolished by montelukast pre-treatment. Montelukast had similar effects when co-culture system of Eol-1 and BEAS-2B was used. This study showed that montelukast suppresses eosinophils-induced EMT of airway epithelial cells. This finding may explain the mechanism of montelukast-mediated amelioration of airway remodeling in bronchial asthma.

Differential response in allergen-specific IgE, IgGs, and IgA levels for predicting outcome of oral immunotherapy.

Oral immunotherapy (OIT) induces desensitization and/or tolerance in patients with persistent food allergy, but the biomarkers of clinical outcomes remain obscure. Although OIT‐induced changes in serum allergen‐specific IgE and IgG4 levels have been investigated, the response of other allergen‐specific IgG subclasses and IgA during OIT remains obscure.

Bitter Sweet: A Child Case of Erythritol-Induced Anaphylaxis

The prevalence of anaphylaxis induced by food has increased in recent years. Identifying the offending food in each case of anaphylaxis is critical for prevention of recurrence, but this is sometimes difficult since the causes are not only well-known allergens such as peanuts but sometimes rare, unexpected allergens such as oligosaccharides.1 Here, we report an 11-year-old boy who experienced anaphylaxis upon ingesting an erythritol-containing food. Erythritol is a sugar alcohol that is commonly used as a sweetener in the modern diet. An 11-year-old boy was admitted to our hospital because of anaphylaxis. At the age of 7, he began to experience episodes of urticaria and wheezing after ingesting foods. He had bronchial asthma and allergic conjunctivitis, but no family history of allergies. Based on his history and positive tests for IgE specific for wheat and peanuts at the initial examination, his physician instructed him to avoid eating foods containing wheat and peanuts. Despite elimination of those foods, he was hospitalized because of anaphylaxis after ingesting a diet sauce when he was 10. At the age of 11, he again experienced severe wheezing and generalized urticaria after eating a health-food made from grapefruit. The main raw material in that health food was grapefruit, but he never experienced symptoms after eating fresh grapefruit. We searched for a common ingredient in the diet sauce and the health food, and suspected erythritol as the possible cause of his anaphylaxis. We performed the following examinations to identify the offending allergen. First, a double-blind placebo-controlled food challenge (DBPCFC) was performed using a sweetener containing erythritol and small amounts of acesulfame potassium and sucralose. We used granulated sugar (sucrose fructose glucose) as a control by adjusting the doses to have a similar degree of sweetness. He developed cutaneous pruritus followed by wheezing, dyspnea and generalized urticaria immediately after ingestion of the erythritol-containing sweetener, but not after ingestion of granulated sugar. He was treated with inhalation of oxygen and nebulized salbutamol, intramuscular epinephrine, and intravenous chlorpheniramine and methylpredonisolone. Second, a skin-prick test was performed using two commercially available erythritol-containing sweeteners. Both sweeteners induced wheals having half the diameter of that induced by histamine. No reactions were observed in two healthy adult controls. We then performed a scratch test using pure erythritol solutions at 2 mg mL, 20 mg mL and 200 mg mL in distilled water. The patient showed positive reactions to erythritol in a concentration-dependent manner. The healthy controls showed no reactions. To further confirm the allergic reaction to erythritol in vitro, we performed a basophil activation test (BAT) for CD203 c expression. A commercial kit (Allergenicity kit, Beckman Coulter, Fullerton, CA, USA) was used for quantification of basophil CD203c expression as previously described2 with erythritol, PalsweetR (an aspartame erythritolcontaining sweetener), sucrose and granulated sugar (sucrose fructose glucose) at the indicated concentrations. Erythritol and PalsweetR induced markedly enhanced expression of CD203c on basophils from the patient, not those from two healthy adult controls (Fig. 1A and B). Neither the patient nor the controls showed any response to sucrose or granulated sugar (Fig. 1C and D). The results indicate that erythritol directly induced basophil activation in the patient. We diagnosed the patient’s reaction as erythritol allergy and instructed the patient to avoid erythritolcontaining foods. There have been some reports of allergic reactions to aspartame3,4 and other food additives.5 Erythritol allergy, especially in children, is very rare. To the best of our knowledge, only adult cases have been reported, 6,7 making the present patient the first reported pediatric case. In the reports of adult patients, the diagnosis was based on the clinical symptoms and skin tests with no confirmatory FC. We confirmed the diagnosis by DBPCFC, with additional evidence from such as BAT and skin tests. Erythritol is a natural sugar alcohol that is a product of fermentation of glucose. It is contained in natural foods such as fruits (e.g., pears and grapes) and mushrooms, and in fermented foods such as soy sauce and bean paste. The use of erythritol as a sweetener in Japan started in 1990 and Japanese consumption is currently around 6000 tons per year. When ingested, it is absorbed by the small intestine and then transferred to the blood. Erythritol is not metabolized, and more than 90% of ingested erythritol is excreted in the urine, thereby making it almost calorie-free. Although erythritol is regarded as highly safe and not carious, the present case suggests that it could be harmful if a patient develops hypersensitivity to the molecule. However, we were unable to identify a specific IgE antibody to erythritol. We did not detect IgE binding to erythritol in an ELISA system. We suspected that the negative result was due to the technical difficulty of immobilizing a sufficient amount of erythritol to the assay plate. We also did not find erythritolinduced basophil histamine release in passive sensitization experiments using serum from the patients and the methods reported elsewhere.8 These negaAllergology International. 2013;62:269-271