[A MULTICENTER CLINICAL SURVEY ABOUT THE PREVALENCE OF JAPANESE CYPRESS POLLINOSIS AND THE EFFICACY OF SUBLINGUAL IMMUNOTHERAPY WITH JAPANESE CEDAR POLLEN EXTRACT DURING JAPANESE CYPRESS POLLEN DISPERSAL PERIOD].

BACKGROUND: Little is known whether sublingual immunotherapy using Japanese cedar pollen extract (cedar SLIT) is effective for not only Japanese cedar pollinosis but also Japanese cypress pollinosis. We investigated the prevalence rate of Japanese cypress pollinosis, efficacy of cedar SLIT on cypress pollinosis and patients' wish to receive cypress SLIT. METHODS: We investigated a multi-center (31 institutions), cross-sectional survey using a self-administrated questionnaire with four questions for patients received cedar SLIT aged from 5 to 69 years old. RESULTS: 2523 subjects were enrolled for analysis. 83.4% of them had pollinosis symptoms during cypress season before cedar SLIT. In such patients, 37.4% experienced lessened efficacy of cedar SLIT during cypress season. Both the prevalence of cypress pollinosis and the lessened efficacy of cedar SLIT on cypress pollinosis were significantly seen in western Japan as compared to eastern Japan. 76.1% of the subject having cypress pollinosis before SLIT wished to receive cypress SLIT if it is available. CONCLUSION: A lessened efficacy of cedar SLIT during cypress season was broadly seen in Japan, and further showed a regional difference. Together with the finding of high wish by patients, these results suggest a development of cypress SLIT is desirable.

HLA-DQ and RBFOX1 as susceptibility genes for an outbreak of hydrolyzed wheat allergy.

BACKGROUND: Food allergy is a growing health problem worldwide because of its increasing prevalence, life-threatening potential, and shortage of effective preventive treatments. In an outbreak of wheat allergy in Japan, thousands of patients had allergic reactions to wheat after using soap containing hydrolyzed wheat protein (HWP). OBJECTIVES: The aim of the present study was to investigate genetic variation that can contribute to susceptibility to HWP allergy. METHODS: We conducted a genome-wide association study of HWP allergy in 452 cases and 2700 control subjects using 6.6 million genotyped or imputed single nucleotide polymorphisms. Replication was assessed by genotyping single nucleotide polymorphisms in independent samples comprising 45 patients with HWP allergy and 326 control subjects. RESULTS: Through the genome-wide association study, we identified significant associations with the class II HLA region on 6p21 (P = 2.16 × 10-24 for rs9271588 and P = 2.96 × 10-24 for HLA-DQα1 amino acid position 34) and with the RBFOX1 locus at 16p13 (rs74575857, P = 8.4 × 10-9). The associations were also confirmed in the replication data set. Both amino acid polymorphisms (HLA-DQß1 amino acid positions 13 and 26) located in the P4 binding pockets on the HLA-DQ molecule achieved the genome-wide significance level (P < 5.0 × 10-8). CONCLUSIONS: Our data provide the first demonstration of genetic risk for HWP allergy and show that this genetic risk is mainly represented by multiple combinations of HLA variants.

Optimal step-down approach for pediatric asthma controlled by salmeterol/fluticasone: A randomized, controlled trial (OSCAR study).

BACKGROUND: Several guidelines, including the Japanese Pediatric Guideline for the Treatment and Management of Asthma (JPGL), recommend salmeterol/fluticasone combination therapy (SFC) as step 3 to 4 treatment for moderate to severe asthma. However, the optimal step-down approach to SFC remains unclear. In the current study, we examined step-down approaches in asthmatic children whose symptoms had been stabilized by SFC 100/200 µg/day. METHODS: This randomized, multicenter, open-label, parallel-group study was conducted over 12 weeks. For step-down therapy, subjects aged 5-15 years were randomly assigned to an SFC group (25/50 µg b.i.d.) or an FP group (100 µg b.i.d.), and treated for 12 weeks. Childhood Asthma Control Test (C-ACT) scores, lung function, and exhaled nitric oxide (FeNO) levels were monitored. RESULTS: Of 131 enrolled subjects, 128 completed the study and were included in the analysis. Decreases in % peak expiratory flow rate and % forced expiratory flow at 50% of vital capacity (V50) were observed in the FP group at each time point. There was a significant difference between the two groups for the change in %V50 from its previous value at each time point. There were no significant changes in FeNO levels (range 15-20 ppb) or C-ACT scores (∼26 points) within or between groups. CONCLUSIONS: A high level of asthma control was maintained with both approaches. The use of SFC step-down resulted in somewhat better respiratory function, with no worsening of airway inflammation. However, halving the dose of SFC and switching to FP alone are both optimal step-down approaches.

Differential activation of eosinophils by bacteria associated with asthma.

BACKGROUND: It has been suggested that there is a complex interaction between microbiota and various human diseases. Some bacteria have been reported to be involved in the inception and progression of asthma, and others in the protection against asthma. We know very little about the mechanisms by which bacteria do harm or good with regard to asthma. This study investigated whether bacteria exert differential effects on the functions of eosinophils, major effector cells in airway inflammation in asthma. METHODS: Eosinophils were purified from healthy adult volunteers by Percoll density gradient centrifugation and negative immunomagnetic bead selection using anti-CD16 microbeads. Three kinds of heat-killed bacteria that have been implicated in asthma, namely Staphylococcus aureus (SA), Haemophilus influenzae (HI) and a Prevotella sp. (PS), were tested for their effects on the secretion of eosinophil-derived neurotoxin (EDN), the generation of superoxides and the production of cytokines/chemokines. RESULTS: SA, but not HI or PS, induced significant EDN release in a dose-dependent manner. Superoxide generation was significantly enhanced by each of the bacterial species, but most strongly by SA, which induced significantly greater TNF-α production by eosinophils than either HI or PS. Conversely, interleukin 10, an anti-inflammatory cytokine, was more strongly induced by HI and PS than by SA. CONCLUSIONS: Bacteria exert differential effects on eosinophils. Based on these results, SA may be involved in the exacerbation of, and HI and PS in the inhibition of, eosinophilic inflammation in asthma.

Higher allergenicity of high molecular weight hydrolysed wheat protein in cosmetics for percutaneous sensitization.

BACKGROUND: Wheat protein derivatives are used in a variety of products worldwide. Gluten is commercially used 'as is' or with modifications such as hydrolysis, which is carried out to overcome its insolubility. Several cases of contact urticaria following exposure to hydrolysed wheat protein (HWP) in cosmetics or of anaphylaxis caused by deamidated gluten in food or non-food products have been described. OBJECTIVES: To evaluate the types of HWP that have higher allergenicity for percutaneous sensitization. METHODS: We enrolled 7 patients with wheat-dependent exercise-induced anaphylaxis who had been sensitized to HWP primarily through the percutaneous and/or the rhinoconjunctival route by using facial soap containing HWP. Reaction to wheat proteins was confirmed by IgE immunoblotting and basophil CD203c expression with six HWP variants. RESULTS: The IgE of all the patients reacted to HWPs composed of large polypeptide aggregates. High molecular weight (MW) HWPs were also found to induce significant enhancement of basophil CD203c expression. CONCLUSIONS: HWPs composed of large polypeptide aggregates possibly induce sensitization to a greater degree than lower-MW HWPs. Basophil surface CD203c expression is useful for evaluating the allergenicity of HWPs.

Case report of a child who may have developed anaphylaxis after ingesting raw horse meat by cross-reactivity of horse and cat pelt.

Pork-cat syndrome can occur in children younger than 10 years. A history of contact with animals since infancy and history of severe atopic dermatitis, which can promote epicutaneous sensitization to animal serum albumin, may be helpful in diagnosis.

1,25-Dihydroxyvitamin D3 upregulates functional C-x-C chemokine receptor type 4 expression in human eosinophils.

BACKGROUND: Epidemiological studies suggest that vitamin D may be protective against the inception and exacerbation of allergic diseases. However, the direct effect of vitamin D on eosinophils, the major effector cells in allergic inflammation, is not known. It has been reported that C-X-C chemokine receptor type 4 (CXCR4) in eosinophils is induced in non-Th2 cytokine milieu or in response to glucocorticoids, recruiting the cell to noninflammatory sites. OBJECTIVES: To test whether 1,25-dihydroxyvitamin D(3) [1,25-(OH)(2)D(3) or calcitriol], the active metabolite of vitamin D, acts directly on eosinophils to induce upregulation of CXCR4. METHODS: Peripheral blood eosinophils from normal volunteers were isolated by CD16 immunomagnetic beads. Vitamin D receptor (VDR) expression was detected by RT-PCR. Eosinophils were cultured with 1,25-(OH)(2)D(3) and the survival and expression of CXCR4 on eosinophils were measured by flowcytometry. Eosinophil migration by CXCL-12/SDF-1 in the presence of 1,25-(OH)(2)D(3) was also analyzed. RESULTS: Eosinophils expressed VDR. 1,25-(OH)(2)D(3) prolonged eosinophil survival and upregulated eosinophil surface expression of CXCR4 in a concentration-dependent manner. Interleukin (IL)-5 significantly reduced CXCR4 expression and migration induced by the ligand CXCL-12/SDF-1. 1,25-(OH)(2)D(3) reversed the negative effects of IL-5 on the CXCR4-CXCL12 pathway. CONCLUSION: 1,25-(OH)(2)D(3) regulates CXCR4 expression in eosinophils. The mechanism may be involved in eosinophil recruitment to noninflammatory sites where the ligand of CXCR4 is constitutively expressed.

Inhibition of eosinophil activation mediated by a Toll-like receptor 7 ligand with a combination of procaterol and budesonide.

BACKGROUND: Viral respiratory tract infections play an important role in the inception and exacerbation of asthma. Eosinophils, major effector cells in asthma, often accumulate in the airways during viral infections and are possibly activated by respiratory RNA viruses through Toll-like receptor (TLR) 7. We investigated the effect of a ß(2)-agonist, i.e. procaterol, and a corticosteroid, i.e. budesonide, that are commonly used for viral-induced asthma, on TLR7 ligand-induced activation of eosinophils in vitro. METHODS: Purified peripheral blood eosinophils were incubated with procaterol and/or budesonide and stimulated with a TLR7 ligand, i.e. R-837. Expression of CD11b was analyzed by flow cytometry. Superoxide generation was measured via the cytochrome C reduction method. IL-8 in the supernatants was assayed by ELISA. RESULTS: Although procaterol or budesonide alone did not inhibit R-837-induced CD11b expression, combinations of the 2 drugs significantly inhibited CD11b. Likewise, the combinations significantly inhibited O(2)(-) generation at low concentrations. Budesonide significantly inhibited R-837-induced IL-8 production in a concentration-dependent manner, and procaterol potentiated inhibition by budesonide although single-agent procaterol had no effect. CONCLUSION: A combination of procaterol and budesonide inhibits the TLR7-mediated effector function of eosinophils, indicating their possible anti-inflammatory effect for virus-induced asthma.

Differential activation of eosinophils by 'probiotic' Bifidobacterium bifidum and 'pathogenic' Clostridium difficile.

BACKGROUND: Recent studies suggest that probiotics alleviate pathophysiological processes of allergic diseases and inflammatory bowel diseases, whereas 'non-probiotic' microflora has negative effects. However, the underlying mechanisms are not well known, especially in relation to eosinophils, the major effector cells of these inflammatory diseases. OBJECTIVE: To investigate the effects of probiotic Bifidobacterium bifidum (BB) on human eosinophil functions compared with pathogenic Clostridium difficile (CD). METHODS: Peripheral human eosinophils were cultured with heat-killed BB or CD. FISH-labeled CD and BB were incubated with eosinophils visualized by confocal laser scanning microscopy. Superoxide generation and degranulation of eosinophils were measured with the cytochrome c reduction method and the eosinophil-derived neurotoxin (EDN) release assay, respectively. RESULTS: Confocal microscopy revealed that Cy3-labeled CD and BB were apparently ingested by eosinophils. Both bacteria induced minimal superoxide generation. However, CD elicited significantly higher EDN release than BB. GM-CSF significantly enhanced EDN release by CD but not by BB. Bacterial-induced EDN release was calcium dependent. CONCLUSION: The beneficial effect of probiotic BB might be explained, at least in part, by its ability to decrease EDN release from eosinophils compared with 'pathogenic' CD.

Basophil Activation Test Based on CD203c Expression in the Diagnosis of Fish Allergy.

PURPOSE: The basophil activation test (BAT) has been reported to be useful for the diagnosis of various food allergies, such as allergy to peanut, but not to fish. This study aimed to evaluate the diagnostic performance of the BAT for fish allergy. METHODS: We performed a retrospective review of patients with fish allergy who underwent the BAT using a panel of fish extracts (15 kinds) to examine the differential reactivity to several species of fish. The BAT score for each extract was expressed as the ratio of CD203chigh% with the extract to that with anti-IgE antibody. Clinical reactivity to each fish was confirmed by positive oral food challenge or a typical history of fish-induced immediate allergy symptoms. Receiver-operating-characteristic (ROC) analysis was performed to evaluate the diagnostic performance. RESULTS: Fifty-one patients with fish allergy were analyzed. Using extracts of 15 species of fish, the BAT was performed a total of 184 times on the patients. Clinical allergy to each species of fish was confirmed in 90 (48.9%) of those tests. ROC analysis yielded high areas under the curve for the BAT scores for the 5 most common fish species (0.72-0.88). The diagnostic accuracy ranged from 0.74 to 0.86. Using a tentative cutoff value of 0.3 deduced from the ROC analyses of the 5 fish species, the accuracy for other fish allergic reactions was generally high (0.6-1.0), except the fish tested in a small number of patients. CONCLUSIONS: The BAT score based on CD203c expression may be useful for fish allergy diagnosis, especially since a large variety of fish can be tested by the BAT using fish extracts prepared by a simple method.

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.

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.

[Factors related to recurrent wheezing after hospitalization with RSV infection with the children who were aged three years old or younger: a questionnaire survey].

Respiratory syncytial virus (RSV) infections in infancy have been related to the subsequent recurrent wheezing and asthma. However, there are a few reports about the relationship between RSV infection and subsequent wheezing in Japan. We sought to determine the contributing factors for wheezing illness after RSV infection in 99 Japanese patients with RSV-associated hospitalizations by questionnaire and follow up survey. Fifty eight patients, who were aged three years old or younger on admission and could be followed up more than one year, were analyzed. The mean duration from discharge to last survey were 703.6+/-105.5 days (432-950 days), the mean age on admission were 9.4+/-8.8 months (0-30 months). Wheezing episodes after discharge were reported in 29 of the subjects (50.0%). Univariable and multivariable analysis identified that the subsequent wheezing after RSV infection were related with the history of wheezing before admission and attending a daycare. The patient's age on admission, the patient's atopic profile, history of continuous nocturnal cough before admission, gestational ages, birth weight, length of hospital stay, perinatal abnormality, environmental tobacco smoke, parental history of allergy and asthma, presence of sibling and sibling history of allergy and asthma were not associated with subsequent wheezing. These results suggest that some host factors susceptible to wheezing and chance of infection due to attending a daycare may be related to recurrent wheezing possibly onset of bronchial asthma, after RSV infection.

Analysis of the distribution of rice allergens in brown rice grains and of the allergenicity of products containing rice bran.

To understand the allergenicity of rice bran, the distribution of rice allergens in brown rice grains was analysed and the allergenicity of cosmetics and health foods containing rice bran determined. RAG2 and a 19-kDa globulin were localized in polished rice, while a 52-kDa globulin was localized in rice bran. The 52-kDa globulin was also identified as the most likely causative allergen of rice bran allergy. Several products containing intact rice bran were found to contain the 52-kDa globulin. Our study provides the first data regarding cosmetics and health foods containing potential rice bran allergens. Western blot analysis using a rice-bran-allergic patient's plasma showed that 52-kDa globulin was detected as an IgE-binding protein of rice bran and some rice bran-containing cosmetics and health foods. Our results indicate that patients with rice bran allergy need to be careful about using products containing intact rice bran as a constituent.

Desolvation behavior of indinavir sulfate ethanol and follow-up by terahertz spectroscopy.

Active pharmaceutical ingredients are composed of single-component or multicomponent crystals. Multicomponent crystals include salts, co-crystals, and solvates. Indinavir sulfate is the ethanol solvate form of indinavir that is known to deliquesce through moisture absorption. However, the detailed behavior of solvent molecules in the crystal has not been investigated. In this study, we studied the desolvation mechanism of indinavir sulfate ethanol and investigated the behavior of solvent molecules in the solid from. Indinavir sulfate ethanol contained 1.7 molecules of ethanol, 0.7 of which desolvated at room temperature. They were originally two ethanol solvent molecules; one molecule of ethanol desolvated at room temperature, and the conformation of the remaining ethanol and t-butyl groups changed in conjunction with the removal of one ethanol molecule. Desolvation could hardly be detected by powder X-ray diffraction; however, it was detected using terahertz spectroscopy. Terahertz measurement of desolvation showed a high correlation with thermogravimetry data, suggesting that desolvation could be observed non-destructively using terahertz spectroscopy. We concluded that indinavir sulfate 1 ethanol deliquesced at 60% relative humidity, and it turned into an amorphous solid after drying.

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-alpha and GRO-alpha, 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.

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 FcepsilonRIalpha 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.

Blocking antibody is generated in allergic rhinitis patients during specific immunotherapy using standardized Japanese cedar pollen extract.

BACKGROUND: Japanese cedar pollen is by far the most important cause of allergic rhinitis in Japan. In this study, we assessed the induction of blocking antibody during specific immunotherapy (SIT) using a recently standardized allergen extract from Japanese cedar pollen. METHODS: Basophils from nonallergic subjects were passively sensitized with serum samples prepared from pollinosis patients before and after SIT; all patients showed good clinical efficacy. The cells were then stimulated with the standardized allergen, and histamine release was measured. In most experiments, the basophil stimulation buffer contained 1% serum. RESULTS: Pollinosis patients' sera obtained both before and after SIT showed essentially similar sensitizing capacity for basophils. Basophil degranulation in response to a relatively low concentration of pollen extract was effectively suppressed by addition of post-SIT serum samples, indicating the presence of blocking antibody. The blocking antibody was IgG, and its potency varied widely among the donor patients. CONCLUSIONS: The standardized allergen extract from Japanese cedar pollen is useful not only for clinical application in SIT, but also for testing for induction of blocking antibody during SIT.