Decreased Basophil Activation against House Dust Mite after Japanese Cedar Pollen Subcutaneous Immunotherapy: A Retrospective Study.

BACKGROUND: Allergen-specific immunotherapy (AIT), an established treatment for allergic diseases, prevents the development of other allergic manifestations. Although the mechanisms remain unclear, AIT has been shown to reduce basophil activation (BA) against nontarget allergens. OBJECTIVES: The aim of this study was to assess immunological changes in Dermatophagoides farinae (Der f) after Japanese cedar pollen (JCP)-based subcutaneous immunotherapy (SCIT) monotherapy. METHOD: The data of 16 patients (age: 6-37 years) with JCP-induced allergic rhinitis who were sensitive to Der f (serum Der f-specific immunoglobulin E [IgE] level >0.34 kUA/L) and received JCP-based SCIT for 5 years were reviewed retrospectively. BA by Der f and JCP extracts and serum-specific IgE and immunoglobulin G4 (IgG4) levels against these allergens were evaluated before and after completing 5 years of JCP-based SCIT monotherapy. RESULTS: The areas under the dose-response curves of BA by Der f and JCP extracts were significantly reduced (p = 0.02 and p = 0.002, respectively). JCP-specific IgE levels decreased and JCP-specific IgG4 levels increased significantly (p < 0.001 for both), whereas Der f-specific IgE and IgG4 levels did not change significantly. CONCLUSIONS: JCP-based SCIT monotherapy reduced Der f-specific BA. These findings suggest that JCP-based SCIT has the potential to modulate immune response toward nontarget allergens.

Evaluation of oral immunotherapy efficacy and safety by maintenance dose dependency: A multicenter randomized study.

BACKGROUND: Generally, oral immunotherapy (OIT) aims for daily administration. Recently, the efficacy of treatment with OIT at a low dose has been reported. However, the optimal dose and the evaluation of dose-dependent OIT outcome have not been described. METHODS: A multicenter, parallel, open-labeled, prospective, non-placebo controlled, randomized study enrolled 101 Japanese patients for treatment with OIT. We hypothesized that target dose OIT would induce short-term unresponsiveness (StU) earlier than reduced dose OIT. StU was defined as no response to 6200 mg whole egg, 3400 mg milk, and 2600 mg wheat protein, as evaluated by oral food challenge after 2-week ingestion cessation. To compare the two doses of OIT efficacy, the maximum ingestion doses during the maintenance phase of OIT were divided into 100%-dose or 25%-dose groups against their target StU dose, respectively. A total of 51 patients were assigned to the 100%-dose group [hen's egg (HE) = 26, cow's milk (CM) = 13, wheat = 12] and 50 to the 25%-dose group (HE = 25, CM = 13, wheat = 12). Primary outcome was established by comparing StU at 1 year. Secondary outcome was StU at 2 years and established by comparing allergic symptoms and immunological changes. RESULTS: The year 1 StU rates (%) for the 100%- and 25%-dose groups were 26.9 vs. 20.0 (HE), 7.7 vs. 15.4 (CM), and 50.0 vs. 16.7 (wheat), respectively. The year 2 StU rates were 30.8 vs. 36.0 (HE), 7.7 vs. 23.1 (CM), and 58.3 vs. 58.3 (wheat), respectively. There were no statistically significant differences in StU between years 1 and 2. The total allergic symptom rate in the 25%-dose group was lower than that in the 100%-dose group for egg, milk, and wheat. Antigen-specific IgE levels for egg-white, milk, and wheat decreased at 12 months. CONCLUSIONS: Reduced maintenance dose of egg OIT showed similar therapeutic efficacy to the target dose. However, we were not able to clearly demonstrate the efficacy, particularly for milk and wheat. Reducing the maintenance dose for eggs, milk, and wheat may effectively lower the symptoms associated with their consumption compared to the target OIT dose. Furthermore, aggressive reduction of the maintenance dose might be important for milk and wheat, compared to the 25%-dose OIT. TRIAL REGISTRATION: UMIN000009373, Multicenter Oral Immunotherapy for Hen's Egg, Cow's Milk, and Wheat-Allergic Children at Outpatient Clinic.

Tolerability of partially and extensively hydrolysed milk formulas in children with cow's milk allergy.

BACKGROUND AND OBJECTIVES: The safety and tolerability of hydrolysed cow's milk protein-based formulas, particularly partially hydrolysed formulas (pHFs), in children with cow's milk allergy (CMA) remain poorly understood. We evaluated the tolerability of hydrolysed cow's milk-based formulas in children with CMA. METHODS AND STUDY DESIGN: A three-period double-blind crossover evaluation compared the allergic tolerance against three dietary cow's milk-based formulas: extensively hydrolysed cow's milk formula (eHF), pHF, and regular cow's milk formula (rCMF). The primary outcome was the rate of tolerance against a maximum of 20.0 mL of formula. RESULTS: Controlled food challenges were performed in 25 children (18 boys; 7 girls) with a median age of 4.25 years (range: 1-9 years) diagnosed with CMA. The median cow's milk-specific immunoglobulin E level was 31.9 UA/mL (range: 1.16-735 UA/mL). The tolerance rate ratios for rCMF were lower than those for pHF (2 vs 16; p<0.01) and eHF (2 vs 22; p<0.01). The allergic symptom scores induced by intake of pHF and eHF were significantly lower than those of rCMF (p=0.01 and p<0.01, respectively), and the pHF and eHF scores were not significantly different. CONCLUSIONS: Compared to rCMF, the partially and extensively hydrolysed whey and casein formulas evaluated in this study were better tolerated and therefore safer for children with CMA. Although further confirmation from additional centres is needed, our findings suggest the use of pHF in patients with mild CMA. Some children with CMA react to hydrolysed formulas; therefore, food challenge tests in these children should be undertaken with caution.

Oral Immunotherapy Using Partially Hydrolyzed Formula for Cow's Milk Protein Allergy: A Randomized, Controlled Trial.

BACKGROUND: Partially hydrolyzed cow's milk protein-based formula (pHF) possesses low allergenicity. Here, we investigate the safety and efficacy of oral immunotherapy using pHF for children with cow's milk protein allergy (CMPA). OBJECTIVES: A randomized, double-blind, controlled single-center trial was conducted to evaluate the efficacy and safety of pHF oral immunotherapy in children with CMPA. METHODS: Participants were randomized into double-blind pHF-pHF and extensively hydrolyzed cow's milk protein-based formula (eHF)-pHF groups. During this phase, the pHF-pHF group received pHF and the eHF-pHF group received eHF. During the open phase, all participants received pHF. The primary end point was a change in thresholds between baseline and the end of the first phase. Secondary end points were changes in thresholds between baseline and the end of the second phase, and casein-specific immunoglobulin (Ig)E, IgG4, and basophil activation. RESULTS: Twenty-five children, aged 1-9 years, were randomized into pHF-pHF and eHF-pHF groups. The threshold between baseline and the end of the first phase was significantly elevated in the pHF-pHF group (p = 0.048), but not in the eHF-pHF group. The threshold between other phases did not change significantly in either group. There were significant decreases in casein-specific IgE antibody levels between baseline and the second phase in the eHF-pHF group (p = 0.014). No participants suffered systemic allergic reactions requiring adrenaline or systemic corticosteroids after receiving the formulas. CONCLUSIONS: The results of this trial suggest that, in children with CMPA, tolerance to cow's milk might be safely enhanced by intake of pHF, relative to that of eHF.

Long-term safety of subcutaneous immunotherapy with TO-204 in Japanese patients with house dust mite-induced allergic rhinitis and allergic bronchial asthma: Multicenter, open label clinical trial.

BACKGROUND: To evaluate the long-term safety of subcutaneous immunotherapy with TO-204, a standardized house dust mite (HDM) allergen extracts, we conducted a multicenter, open label clinical trial. METHODS: Japanese patients aged 5-65 years were eligible for the study, if they had HDM-induced allergic rhinitis (AR), allergic bronchial asthma (BA), or both. TO-204 was administered in a dose titration scheme, and the maintenance dose was determined according to the predefined criteria. The treatment period was 52 weeks, and patients who were willing to continue the treatment received TO-204 beyond 52 weeks. This clinical trial is registered at the Japan Pharmaceutical Information Center (Japic CTI-121900). RESULTS: Between July 2012 and May 2015, 44 patients (28 with AR and 16 with allergic BA) were enrolled into the study. All patients were included in the analysis. The duration of treatment ranged from 23 to 142 weeks and the median maintenance dose was 200 Japanese allergy units (JAU). Adverse events occurred in 22 patients (50%). The most common adverse event was local reactions related to the injection sites. Four patients experienced anaphylactic reactions when they were treated with the dose of 500 JAU. Two patients experienced anaphylactic shock with the doses of 1000 JAU at onset. These 6 patients could continue the study with dose reduction. CONCLUSIONS: Safety profile of TO-204 was acceptable in Japanese patients with HDM-induced AR or allergic BA. Higher doses should be administered carefully, because the risk of anaphylaxis increased at doses of 500 or 1000 JAU.

[AN EVALUATION OF SPONTANEOUS HISTAMINE RELEASE AND THE LOW RESPONDERS IN A BASOPHIL HISTAMINE RELEASE TEST].

BACKGROUND: We evaluated the clinical significance of the spontaneous histamine release ratio (SHR/T) and low responders in the automated basophil histamine release test (AllerportⓇ HRT). METHODS: This study analyzed the outcomes of 101 oral food challenges (OFC) with egg, milk or wheat (challenge-positive: n=79) in relation to the SHR/T. The traditional HRT low responders (n=27) were separated into two groups:"LOW"responders (n=10), who showed a ≥10% concentration-dependent maximum histamine release in response to the anti-human IgE stimulation, and"NON"responders who did not fulfill the criteria (n=17). RESULTS: Among the 34 patients with ≥20% SHR/T, 32 patients (94%) had a positive OFC with a low threshold dose which provoked severe symptoms. Among the"LOW"responders, four cases showed ≥10% allergen-specific maximum histamine release. On the other hand, concentration-dependent histamine release was not seen in the"NON"responders, suggesting the basophil function was not detected in this subgroup. CONCLUSION: The present study suggested that SHR/T could be an indicator of basophil activation and hypersensitivity in vivo. We also suggested that significant basophil functions might be detected among the "LOW"responders, but not among the"NON"responders.

Evaluation of allergenic potential for rice seed protein components utilizing a rice proteome database and an allergen database in combination with IgE-binding of recombinant proteins.

Among 131 rice endosperm proteins previously identified by MS-based proteomics, most of the proteins showed low or almost no sequence similarity to known allergens in databases, whereas nine proteins did it significantly. The sequence of two proteins showed high overall identity with Hsp70-like hazel tree pollen allergen (Cor a 10) and barley α-amylase (Hor v 16), respectively, whereas the others showed low identity (28-58%) with lemon germin-like protein (Cit l 1), corn zein (Zea m 50 K), wheat chitinase-like xylanase inhibitor (Tri a XI), and kinase-like pollen allergen of Russian thistle (Sal k 1). Immuno-dot blot analysis showed that recombinant proteins for these rice seed homologs were positive in the IgE-binding, but not necessarily similarity dependent, from some allergic patients. These results suggest that utilization of proteome and sequence databases in combination with IgE-binding analysis was effective to screen and evaluate allergenic potential of rice seed protein components.

Multicolor flow-cytometric analysis of milk allergen-specific T-helper type 2 cells revealed coexpression of interleukin-4 with Foxp3.

BACKGROUND: Allergen-specific T-helper type 2 (TH2) cells play an important role in the development of allergic inflammation; however, investigations of the properties of allergen-specific T cells have been challenging in humans. Despite clear evidence that forkhead box p3 (Foxp3) is expressed in conventional effector T cells, its function has remained unknown. OBJECTIVE: To characterize allergen-specific TH2 cells in milk allergy, with particular focus on the expression of Foxp3. METHODS: Twenty-one children with milk allergy and 11 children without milk allergy were studied. Peripheral blood mononuclear cells from subjects were stimulated with milk allergen for 6 hours and analyzed using multicolor flow cytometry to identify CD154(+) allergen-specific T-helper cells. Simultaneously, the expression of intracellular cytokines and Foxp3 was analyzed. RESULTS: The milk allergy group had significantly larger numbers of milk allergen-specific interleukin (IL)-4- and IL-5-producing CD4(+) T cells than the control group. Subjects in the milk allergy group had significantly more CD154(+)CD4(+) IL-10-producing cells and CD154(+)Foxp3(+)CD4(+) cells than those in the control group. In addition, the number of milk allergen-specific CD154(+)Foxp3(+)CD4(+) cells strongly correlated with that of CD154(+)IL4(+)CD4(+) cells. Bcl-2 expression in CD154(+)IL-4(+)Foxp3(+) T-helper cells was significantly lower compared with that in total CD4 cells. CONCLUSION: Increased numbers of IL-4-producing allergen-specific T-helper cells were found in patients with milk allergy. In addition, Foxp3 was coexpressed with IL-4 in allergen-specific TH2 cells from patients. This coexpression was associated with lower Bcl-2 levels and could contribute to the phenotype and function of TH2 cells.

Japanese cedar pollen-based subcutaneous immunotherapy decreases tomato fruit-specific basophil activation.

BACKGROUND: Some patients with Japanese cedar pollen (JCP)-induced allergic rhinitis develop pollen-food allergy syndrome (PFAS) as a reaction to tomato fruit. Pollen allergen-specific subcutaneous immunotherapy (SCIT) is reportedly beneficial for some associated food allergies; however, the reported changes in food allergen-specific immunoglobulin (Ig)E and IgG4 levels are inconsistent. Here, we investigated immunologic reactivity to tomato fruit after JCP-based SCIT. METHODS: Twenty-three children (aged 6-17 years) with JCP-induced allergic rhinitis and sensitized to tomato (serum tomato fruit-specific IgE level >0.34 UA/ml) received JCP-based SCIT. Basophil activation by tomato and JCP extracts and serum-specific IgE and IgG4 levels against these allergens were determined before and after 4 or 5 months of maintenance SCIT. Basophil activation was assessed by monitoring CD203c upregulation on flow cytometry. RESULTS: JCP-based SCIT significantly reduced the basophil activation caused by tomato fruit (p = 0.03) and JCP (p < 0.001) extracts. JCP-specific IgG4 levels markedly increased after SCIT (p < 0.001), whereas tomato fruit-specific IgG4 levels did not. After SCIT, no significant changes were observed in specific IgE levels for tomato fruit (p = 0.11) or JCP (p = 0.19). CONCLUSIONS: Tomato fruit-specific basophil activation decreases after JCP-based SCIT, suggesting that it is efficacious in relieving and preventing the symptoms of PFAS in patients with JCP-induced allergic rhinitis.

Hen's Egg Allergy.

Egg allergy is one of the most frequent food allergies in infants and young children. The prevalence of egg allergy is estimated to be between 1.8 and 2% in children younger than 5 years of age. The reactions are mainly mediated by IgE and partially by non-IgE or are a mix of both types. Egg white contains more than 20 different proteins and glycoproteins. Ovomucoid (Gal d 1), ovalbumin (Gal d 2), conalbumin (ovotransferrin) (Gal d 3) and lysozyme (Gal d 4) have been identified as major allergens in hen's egg. Alpha-livetin (Gal d 5) is thought to be a main egg yolk allergen responsible for bird-egg syndrome. The diagnosis of egg allergy is based on history taking, antigen-specific IgE measurements, such as the skin prick test, in vitro antigen-specific blood IgE tests and histamine release tests, and oral food challenges. The measurements of specific IgE to ovomucoid and its linear epitopes are more useful in the diagnosis of heated egg allergy and in the prediction of prognosis. Currently, the management of egg allergy is essentially minimal elimination based on the correct identification of the causative allergen. Although oral immunotherapy is promising as a tolerance induction protocol, several questions and concerns still remain, predominantly regarding safety.

[Utility of the allerport® HRT in the diagnosis of hen's egg allergy: a pediatric multicenter challenge study].

Purpose This study aimed to evaluate the utility of the Allerport® HRT in the diagnosis of hen's egg allergy. Method We enrolled 51 subjects who underwent the Allerport® HRT before an oral food challenge (OFC) consisting of heated egg. Blood samples were collected within three months prior to OFC to measure histamine release (HR) and specific IgE antibody titers. We examined whether the Allerport® HRT was useful as a means of diagnosing hen's egg allergy and predicting severity of induced symptoms. Of 51 subjects, three whose OFC results were not valid**1** and 13 who were classified as "low responders" to the Allerport® HRT (%HR due to anti-IgE below 20%) were excluded. Of the remaining 35 subjects (average age: 4 years), 23 showed positive reactions to the OFC. Quantities of histamine released in spontaneous HR and egg white (EW)- and ovomucoid (OVM)-induced HR were significantly higher in patients with positive reactions. In our receiver operating characteristic analysis, the area under the curve for %HR against 6 ng/ml of EW was 0.9601 and that against 3 ng/ml of OVM was 0.9022. The cutoff value was 15.0% for EW and 3.7% for OVM. The sensitivity was 95.7% for EW and 91.3% for OVM, and the specificity was 83.3% for EW and 58.3% for OVM. %HR correlated well with the severity of induced symptoms in the OFC. Conclusion Allerport® HRT is useful for the diagnosis of hen's egg allergy, and may also aid in predicting the severity of induced symptoms.

Japanese pediatric guideline for the treatment and management of bronchial asthma 2012.

A new version of the Japanese pediatric guideline for the treatment and management of bronchial asthma was published in Japanese at the end of 2011. The guideline sets the pragmatic goal for clinicians treating childhood asthma as maintaining a "well-controlled level" for an extended period in which the child patient can lead a trouble-free daily life, not forgetting the ultimate goal of obtaining remission and/or cure. Important factors in the attainment of the pragmatic goal are: (i) appropriate use of anti-inflammatory drugs; (ii) elimination of environmental risk factors; and (iii) educational and enlightening activities for the patient and caregivers regarding adequate asthma management in daily life. The well-controlled level refers to a symptom-free state in which no transient coughs, wheezing, dyspnea or other symptoms associated with bronchial asthma are present, even for a short period of time. As was the case in the previous versions of the guideline, asthmatic children younger than 2 years of age are defined as infantile asthma patients. Special attention is paid to these patients in the new guideline: they often have rapid exacerbation and easily present chronic asthmatic conditions after the disease is established.

Japanese Guideline for Food Allergy 2014.

A food allergy is defined as "a phenomenon in which adverse reactions are caused through antigen-specific immunological mechanisms after exposure to given food." Various symptoms of food allergy occur in many organs. Food allergies are classified roughly into 4 clinical types: (1) neonatal and infantile gastrointestinal allergy, (2) infantile atopic dermatitis associated with food allergy, (3) immediate-type food allergy (urticaria, anaphylaxis, etc.), and (4) food dependent exercise-induced anaphylaxis and oral allergy syndrome (i.e., specific forms of immediate food allergy). The therapy for food allergies includes treatment of and prophylactic measures against hypersensitivity such as anaphylaxis. A fundamental prophylactic measure is the elimination diet. However, elimination diets should be used only if necessary because of the patient-related burden. For this purpose, it is very important that causative foods be accurately identified. There are a number of means available to identify causative foods, including the history taking, a skin prick test, detection of antigen-specific IgE antibodies in the blood, the basophil histamine release test, the elimination diet test, and the oral challenge test, etc. Of these, the oral challenge test is the most reliable. However, it should be conducted under the supervision of experienced physicians because it may cause adverse reactions, such as anaphylaxis.

Allergen analysis of sea urchin roe using sera from five patients.

BACKGROUND: Sea urchin roe can cause anaphylactic reactions the first time they are consumed; therefore, careful clinical attention should be paid to their effects. However, no previous study has examined the allergens in sea urchin roe using sera from more than one patient. We attempted to identify sea urchin allergens using sera from 5 patients with sea urchin allergies. METHODS: We enrolled 5 patients with relevant medical histories, positive results on a skin prick test and/or a food challenge test, and high levels of sea urchin-specific IgE in an enzyme-linked immunosorbent assay. We performed SDS-PAGE, immunoblotting, immunoblot inhibition, and N-terminal amino acid sequence detection. RESULTS: Ten protein bands ranging from 18 to 170 kDa were detected in more than 2 patients' sera. In immunoblotting, the protein band for the 170-kDa major yolk protein was recognized by 4 of the 5 sera. Furthermore, the reaction between IgE and the protein band for egg cortical vesicle protein (18 kDa) was inhibited by the addition of salmon roe extract. CONCLUSION: Major yolk protein was confirmed to be one of the main allergens in sea urchin roe. In addition, egg cortical vesicle protein (18 kDa) was demonstrated to be an important protein for cross-reactivity with salmon roe.

Generation of transgenic rice with reduced content of major and novel high molecular weight allergens.

BACKGROUND: Rice seed proteins contain antigens that provoke allergic responses in some individuals with food allergy, particularly in those with cereal allergy, and these antigens can elicit clinical symptoms such as eczema and dermatitis. We previously generated transgenic rice with reduced accumulation of the three major allergens, which dramatically reduced the level of IgE binding from patients' sera. However, the transgenic rice still possesses allergenic reactivity. Recently, two globulin-like proteins were identified as candidates of novel high molecular weight (HMW) IgE-binding proteins that cause rice allergy. RESULTS: We identified a glucosidase family encoded by four genes as novel HMW rice allergens based on IgE antibody reactivity from individuals with allergy to rice. To further reduce allergenicity, we generated transgenic rice with reduced accumulation of these HMW allergens. We crossed the rice with reduced HMW allergens and with reduced major allergens, and all major and HMW allergens were substantially reduced in the progeny of the crossed rice. Allergen suppression did not significantly alter accumulation patterns of seed storage proteins and protein folding enzymes. The sera of a portion of patients showed low IgE-binding to the crossed line, suggesting that the crossed line is effective for a portion of patients who are allergic to proteins other than major allergens. CONCLUSIONS: The transgenic rice with reduced levels of all major and HMW allergens is thought to be an option for a portion of allergy patients with hypersensitive responses to various kinds of rice allergens.

Japanese Guideline for Food Allergy 2014.

A food allergy is defined as "a phenomenon in which adverse reactions are caused through antigen-specific immunological mechanisms after exposure to given food." Various symptoms of food allergy occur in many organs. Food allergies are classified roughly into 4 clinical types: (1) neonatal and infantile gastrointestinal allergy, (2) infantile atopic dermatitis associated with food allergy, (3) immediate-type food allergy (urticaria, anaphylaxis, etc.), and (4) food dependent exercise-induced anaphylaxis and oral allergy syndrome (i.e., specific forms of immediate food allergy). The therapy for food allergies includes treatment of and prophylactic measures against hypersensitivity such as anaphylaxis. A fundamental prophylactic measure is the elimination diet. However, elimination diets should be used only if necessary because of the patient-related burden. For this purpose, it is very important that causative foods be accurately identified. There are a number of means available to identify causative foods, including the history taking, a skin prick test, detection of antigen-specific IgE antibodies in the blood, the basophil histamine release test, the elimination diet test, and the oral challenge test, etc. Of these, the oral challenge test is the most reliable. However, it should be conducted under the supervision of experienced physicians because it may cause adverse reactions, such as anaphylaxis.

Acute eosinophilic pneumonia occurring in a dedicator of cytokinesis 8 (DOCK8) deficient patient.

Dedicator of cytokinesis 8 (DOCK8) deficiency is an autosomal recessive type of combined immunodeficiency with elevated IgE. In this report, we describe a Japanese girl of non-consanguineous family suffering from acute eosinophilic pneumonia (AEP) as a presenting feature of DOCK8 deficiency. Although AEP was self-limiting, consecutively experienced recurrent respiratory infections, severe atopic dermatitis, and vulnerability to viral infections, prompted us to evaluate the possibility of DOCK8 deficiency. Immunological assessments demonstrated decreased IgM, increased IgE, T lymphocytepenia, especially in CD4 T cells, decreased PHA blastogenesis, and decreased CD27(+) CD19(+) memory B cells. Western blotting revealed the absence of DOCK8 protein. Investigation of genomic DNA by multiplex ligation-dependent probe amplification (MLPA) revealed a heterozygous large deletion of 77 kb spanning from intron 5 to exon 22. DOCK8 cDNA sequencing revealed a nonsense mutation at position 740 (E740X). As far as we know, this is the first Japanese case of DOCK8 deficiency.