Shoji Mihara

Fast ω-gliadin is a major allergen in wheat-dependent exercise-induced anaphylaxis

Abstract Background: Wheat-dependent exercise-induced anaphylaxis is an anaphylaxy induced by physical exercise after ingestion of wheat. An immediate-type hypersensitivity to water/salt-insoluble fraction of wheat proteins (gluten) has been considered to underlie in this disease. Objective: The aim of the study is to determine the major allergen in Japanese patients with wheat-dependent exercise-induced anaphylaxis by using a panel of purified wheat gliadins and glutenins. Methods: Water/salt-insoluble wheat proteins, α-gliadin, β-gliadin, γ-gliadin, fast ω-gliadin, slow ω-gliadin, high molecular weight glutenin and low molecular weight glutenin, were purified, and five patients with wheat-dependent exercise-induced anaphylaxis, whose diagnose had been determined by positive-challenge test, were evaluated for skin prick test, dot-blotting test and CAP–RAST inhibition test by using these purified wheat proteins. Results: The fast ω-gliadin was the most potent allergen among these water/salt-insoluble proteins when evaluated by skin prick test and dot-blotting test. Fast and slow ω-gliadin, and γ-gliadin caused dose-dependent inhibition of the serum IgE-binding to solid-phase gluten in the patients. The incubation with fast ω-gliadin of the patients serum caused dose-dependent inhibition in the IgE-binding to γ-gliadin as well as slow ω-gliadin, indicating a cross-reactivity of these proteins in IgE-binding. Conclusion: We concluded that fast ω-gliadin is a major allergen among these water/salt-insoluble proteins for wheat-dependent exercise-induced anaphylaxis in Japanese patients, and IgE against fast ω-gliadin cross-reacts to γ-gliadin and slow ω-gliadin.

Coagulation/fibrinolysis and inflammation markers are associated with disease activity in patients with chronic urticaria

To cite this article: Takahagi S, Mihara S, Iwamoto K, Morioke S, Okabe T, Kameyoshi Y, Hide M. Coagulation/fibrinolysis and inflammation markers are associated with disease activity in patients with chronic urticaria. Allergy 2010; 65: 649–656.

Increased MCL–1 Expression Is Associated with Poor Prognosis in Ovarian Carcinomas

To investigate the potential role of the BCL–2 gene family (BAX, BCL–2, MCL–1, and BCL‐XL) in ovarian cancer development and progression, mRNA expression levels of these genes were measured using semi‐quantitative PCR in epithelial ovarian tumor tissues and normal ovaries. The immunohistochemical expression of MCL–1 in ovarian tumors was also examined. The expression levels of BAX and MCL–1 mRNA were significantly higher in ovarian cancers and in adenomas than in normal ovaries (P<0.05). In contrast, the BCL–2 mRNA expression level in ovarian cancers was significantly lower than in ovarian adenomas and in normal ovaries (P<0.05). Expression of BCL‐XL mRNA was no different between normal ovaries and ovarian tumors. Log‐rank testing showed that low BAX mRNA expression and high MCL–1 mRNA expression significantly correlate with poor survival for patients with stage III ovarian carcinomas (BAX, P=0.05; MCL–1, P=0.02). Immunohistochemical analysis showed that diffuse‐positive expression of MCL–1 protein in mucinous carcinomas was significantly higher than in mucinous low malignant potential (LMP) tumors (P=0.03). In ovarian cancer cases, diffuse‐positive expression of MCL–1 protein significantly correlates with advanced clinical stage, high histologic grade, and poor survival (stage, P<0.01; grade, P=0.01; survival, P=0.01). These results suggest that increased MCL–1 expression may play an important role in replacing the functions of increased BAX and decreased BCL–2 in ovarian carcinoma cells, thereby promoting cell survival, and resulting in a poor prognosis for patients with ovarian cancer.

Inhibition of FAS/FAS ligand-mediated apoptotic cell death of lymphocytes in vitro by circulating anti-FAS ligand autoantibodies in patients with systemic lupus erythematosus

OBJECTIVE The Fas/Fas ligand (FasL) system has been assigned a pivotal role in the establishment and maintenance of peripheral tolerance, and mice having defects in the Fas/FasL system are known to develop lupus-like symptoms. However, it remains unclear whether the Fas/FasL system is involved in the pathogenesis of systemic lupus erythematosus (SLE) in humans. This study examined whether there are circulating anti-FasL autoantibodies in the peripheral blood of patients with SLE that would interfere with Fas/FasL-mediated apoptosis. METHODS Anti-FasL autoantibodies were detected by Western blot analysis using the recombinant extracellular domain of human FasL as the antigen. Apoptosis of Fas-expressing Jurkat cells, induced by recombinant soluble FasL (sFasL) in the presence of anti-FasL autoantibodies, was assessed by DNA staining with propidium iodide, followed by flow cytometric analysis. Apoptosis of Jurkat cells by cell-bound FasL was assessed by 2-color analysis, involving TUNEL staining with fluorescein isothiocyanate-dUTP and phycoerythrin-labeled anti-CD3 monoclonal antibodies. RESULTS Among the 21 patients with SLE, 7 had IgG-isotype anti-FasL autoantibodies in their circulating blood. In addition, these autoantibodies inhibited both sFasL-mediated and cell-bound FasL-mediated apoptosis of Fas-expressing Jurkat cells. Thus, it is plausible that anti-FasL autoantibodies in patients with SLE disturb the establishment and maintenance of peripheral tolerance in vivo by inhibiting the Fas/FasL-mediated elimination of autoreactive lymphocytes. CONCLUSION These results suggest that anti-FasL autoantibodies that inhibit Fas/FasL-mediated apoptosis are involved, at least in part, in immune abnormalities and may possibly be involved in the pathogenesis of SLE.

Fungal protein MGL_1304 in sweat is an allergen for atopic dermatitis patients

BACKGROUND Sweat is a major aggravating factor of atopic dermatitis (AD) and approximately 80% of patients with AD show type I hypersensitivity against sweat. OBJECTIVE To identify and characterize an antigen in sweat that induces histamine release from basophils of patients with AD. METHODS Basophil histamine-releasing activity in sweat was purified by a combination of chromatographies, and proteins were analyzed with mass spectrometry. Recombinant proteins of the sweat antigen were generated, and their biological characteristics were studied by immunoblots, histamine release tests, and neutralization assays. RESULTS We identified a fungal protein, MGL_1304, derived from Malassezia globosa (M globosa) in the purified sweat antigen. Recombinant MGL_1304 induced histamine release from basophils of most of the patients with AD, in accordance with the semi-purified sweat antigen. Moreover, recombinant MGL_1304 abolished the binding of serum IgE of patients with AD to the semi-purified sweat antigen, or vice versa in immunoblot analysis, and attenuated the sensitization of RBL-48 mast cells expressing human FcɛRI by serum IgE. Studies of truncated mutants of MGL_1304 indicated that IgE of patients with AD recognized the conformational structure of MGL_1304 rather than short peptide sequences. Western blot analysis of the whole lysate, the culture supernatant of M globosa, and the semi-purified sweat antigen showed that MGL_1304 was produced as a minor immunological antigen of M globosa with posttranslational modification, cleaved, and secreted as a 17-kDa major histamine-releasing sweat antigen. CONCLUSION MGL_1304 is a major allergen in human sweat and could cause type I allergy in patients with AD.

Increasing the dose of cetirizine may lead to better control of chronic idiopathic urticaria : an open study of 21 patients

SIR, Chronic idiopathic urticaria (CIU) is characterized by the occurrence of spontaneous pruritic weals on most days. It is common, but often disabling because of persistent clinical symptoms which negatively influence the quality of life. Antihistamines have been the mainstay of treatment and they produce a good response in most patients, but not in all. For those patients who derive only limited benefit from the initial treatment, therapeutic guidelines advocate the use of antihistamines above the licensed or manufacturers’ recommended doses. However, there is scant evidence regarding the effectiveness of dose increases of the same antihistamine in the patients who responded poorly to the first dosage of the agent. Cetirizine is a second-generation antihistamine effective in treating patients with CIU. The manufacturer’s recommended dosage is 10 mg daily and it is permissible to increase the dose up to 20 mg daily. As cetirizine inhibits histamine-induced weal and flare reactions dose-dependently, it is plausible that higher doses of the drug will be more effective in controlling urticarial symptoms. However, the clinical effect of such dose increases has not been evaluated in patients with CIU. In some previous studies, patients with CIU were initially treated with cetirizine 5 mg, and they were allowed to increase the dose to 10 or 20 mg if no benefit was obtained at the starting dose. However, neither urticarial activity at each dose nor clinical effects of the dose increase were assessed in these studies. In the present open study, we evaluated the effect of increasing the dose of cetirizine in order to control the disease activity in patients with CIU, who had derived only limited benefit from 10 mg daily of the same drug. Patients with CIU (> 1 month duration) were recruited from secondary care hospitals. Patients with physical urticaria, or urticaria caused by medications, foods or other known causes were excluded. Prior to the dose-increasing study, patients were treated with cetirizine 10 mg daily for 1 or 2 weeks as a screening period. Twenty-one patients who responded poorly to the treatment, i.e. the change of the total daily score of urticarial symptoms (see below) was less than 1, during the screening period, were enrolled in the study. Approximately one-third of patients who entered the screening period were eligible. At the beginning of the study, patients were randomly assigned to group A (11 patients) or group B (10 patients), after obtaining informed consent, and all patients were given an increased dose of cetirizine, 20 mg daily (10 mg twice daily), for 1 or 2 weeks (period 1). Thereafter, patients in group A continued the daily dosage of cetirizine 20 mg, whereas the patients in group B received the decreased dosage of 10 mg, for an additional 1 to 2 weeks (period 2). Patients were instructed to record daily urticarial activity scores throughout the study period including the screening period. The urticarial activity was assessed by using the scoring system as previously described. Namely, each of the number of weals, the duration of the weals, and the severity of itch, was scored from 0 to 3. The total daily score of the urticarial symptoms, therefore, ranged from 0 to 9. For the assessment of the clinical effect of increase ⁄decrease in the dosage, the data were analysed by the Friedman test and the Steel–Dwass test. There were no statistically significant differences between the two groups in age (42Æ5 ± 14Æ1 vs. 36Æ9 ± 16Æ7 years, mean ± SD, for groups A and B, respectively) or mean urticarial activity scores during the screening period [weal scores 1Æ09 ± 0Æ78 vs. 1Æ11 ± 0Æ64, itch scores 1Æ53 ± 0Æ89 vs.

Food-dependent exercise-induced anaphylaxis: a report of two cases and determination of wheat-γ-gliadin as the presumptive allergen

Water/salt‐insoluble wheat proteins have been identified as the most frequent allergenic foodstuffs in patients with food‐dependent exercise‐induced anaphylaxis (FDEIA) in Japan. However, the specific allergenic proteins in wheat‐dependent exercise‐induced anaphylaxis have not been well defined. Challenge testing, skin testing and a fluoroenzyme immunoassay were used for diagnosis in two patients suspected by history of having wheat‐dependent exercise‐induced anaphylaxis. Gel chromatography and IgE immunoblotting followed by N‐terminal amino acid sequencing were used to identify the allergenic wheat protein. The challenge test revealed that both patients had FDEIA. The skin tests and the immunoassay results suggested that wheat gluten was the allergen in both patients. Gel chromatography of wheat gluten revealed that the antigens had molecular weights ranging from 40 to 250 kDa. IgE immunoblotting and subsequent N‐terminal amino acid sequencing revealed that wheat‐γ‐gliadin was the antigen predominantly bound by IgE in the two patients.

Peritoneal injection of fucoidan suppresses the increase of plasma IgE induced by OVA-sensitization

We previously reported that fucoidan, a dietary fiber purified from seaweed, inhibited IgE production by B cells in vitro. In this study, we examined the effect of fucoidan on IgE production in vivo. The OVA-induced increase of plasma IgE was significantly suppressed when fucoidan was intraperitoneally, but not orally, administered prior to the first immunization with OVA. The production of IL-4 and IFN-gamma in response to OVA in spleen cells isolated from OVA-sensitized mice treated with fucoidan in vivo was lower than that from mice treated without fucoidan. Moreover, the flow cytometric analysis and ELISpot assay revealed that the administration of fucoidan suppressed a number of IgE-expressing and IgE-secreting B cells, respectively. These results indicate that fucoidan inhibits the increase of plasma IgE through the suppression of IgE-producing B cell population, and the effect of fucoidan in vivo is crucially dependent on the route and timing of its administration.

Development of pathogenic anti-DNA antibodies in patients with systemic lupus erythematosus.

The anti‐DNA response is a hallmark of systemic lupus erythematosus (SLE). The precise mechanisms leading to anti‐DNA antibody (Ab) production remain to be studied. Nonetheless, it is becoming clear that anti‐DNA Abs cause inflammatory lesions not only via deposition of circulating immune complexes (IC) consisting of anti‐DNA Ab and antigens (Ags), but also via in situ IC formation by cationic anti‐DNA Abs. It is intriguing that cationic anti‐DNA Abs are encoded by a unique germline Vκ gene, A30, which encodes an extraordinary cationic light chain, whereas somatic mutations did not induce a cationic shift of electrical charge in human lupus nephritis, suggesting that the usage of a specific germline gene may confer the cationic charge (or pathogenicity) on anti‐DNA Abs and that somatic mutations induce the affinity maturation of Abs. Whether cationic anti‐DNA Abs will develop depends at least partly on the presence or absence of the germline A30 gene, since patients who lack this gene in the germline Vκ repertoire did not develop severe lupus nephritis. Receptor editing, a mechanism for changing the affinity of the B cell Ag receptor [surface immunoglobulin (Ig) receptor] to avoid self‐reactivity actually seems defective in patients with SUE because normal B cells edited the A30 gene, whereas SLE B cells express A30 mRNA. Thus, along with the importance of somatic mutations, polymorphisms of Ig Vκ locus, and genetic predisposition, the failure of receptor editing may contribute to the development of pathogenic anti‐DNA responses in humans.—Suzuki, N., Mihara, S., Sakane, T. Development of pathogenic anti‐DNA antibodies in patients with systemic lupus erythematosus. FASEB J. 11, 1033–1038 (1997)

Serum Gliadin Monitoring Extracts Patients with False Negative Results in Challenge Tests for the Diagnosis of Wheat-Dependent Exercise-Induced Anaphylaxis*

BACKGROUND Challenge testing with wheat plus exercise and/or aspirin is a gold standard for the diagnosis of wheat-dependent exercise-induced anaphylaxis (WDEIA); however, the test may often yield false-negative results. Our previous study suggested that an increase in serum wheat gliadin levels is required to induce allergic symptoms in patients with WDEIA. Based on this knowledge, we sought to extract the patients with false negative results in the challenge tests of WDEIA. METHODS Thirty-six patients with suspected WDEIA were enrolled. First, group categorizations-Group I, challenge tests were positive; Group II, challenge tests were negative and serum gliadin were undetectable; Group III, challenge tests were negative and serum gliadin were detectable-were given according to the results of wheat plus exercise and/or aspirin challenge testing and serum gliadin levels. Second, diagnoses were made using retests and/or dietary management in Group II and III. RESULTS Positive results for wheat plus exercise and/or aspirin challenge tests gave a diagnosis of definite WDEIA in 17 of 36 patients (Group I). Of the remaining 19 challenge negative patients, serum gliadin was undetectable in ten patients (Group II). Of the ten patients (Group II), three of them were diagnosed as definite WDEIA by retesting and six of them were diagnosed as probable WDEIA using a wheat elimination diet, whereas one patient was non-WDEIA. In the rest of the nine challenge negative patients, serum gliadin was detectable (Group III). No allergic episodes with a normal diet provided a diagnosis of non-WDEIA in seven of the nine patients, whereas the remaining two patients were probable WDEIA or had another food allergy because of repeated episodes. CONCLUSIONS Our study revealed that serum gliadin monitoring during challenge testing is useful.