Makiko Hiragun

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.

Germline mutation in ATR in autosomal- dominant oropharyngeal cancer syndrome

ATR (ataxia telangiectasia and Rad3 related) is an essential regulator of genome integrity. It controls and coordinates DNA-replication origin firing, replication-fork stability, cell-cycle checkpoints, and DNA repair. Previously, autosomal-recessive loss-of-function mutations in ATR have been demonstrated in Seckel syndrome, a developmental disorder. Here, however, we report on a different kind of genetic disorder that is due to functionally compromised ATR activity, which translates into an autosomal-dominant inherited disease. The condition affects 24 individuals in a five-generation pedigree and comprises oropharyngeal cancer, skin telangiectases, and mild developmental anomalies of the hair, teeth, and nails. We mapped the disorder to a ∼16.8 cM interval in chromosomal region 3q22-24, and by sequencing candidate genes, we found that ATR contained a heterozygous missense mutation (c.6431A>G [p.Gln2144Arg]) that segregated with the disease. The mutation occurs within the FAT (FRAP, ATM, and TRRAP) domain-which can activate p53-of ATR. The mutation did not lead to a reduction in ATR expression, but cultured fibroblasts showed lower p53 levels after activation of ATR with hydroxyurea than did normal control fibroblasts. Moreover, loss of heterozygosity for the ATR locus was noted in oropharyngeal-tumor tissue. Collectively, the clinicopathological and molecular findings point to a cancer syndrome and provide evidence implicating a germline mutation in ATR and susceptibility to malignancy in humans.

Prognosis of chronic spontaneous urticaria in 117 patients not controlled by a standard dose of antihistamine

Chronic spontaneous urticaria (CSU) is a common skin disorder, but its clinical course reported so far is largely variable, probably due to the heterogeneity of the clinical background of patients and pathogenesis of this disease.

Elevated serum IgE against MGL_1304 in patients with atopic dermatitis and cholinergic urticaria

BACKGROUND MGL_1304 secreted by Malassezia globosa is contained in human sweat and induces histamine release from basophils in patients with atopic dermatitis (AD) at a high positive rate. The aims of this study were to establish the enzyme-linked immunosorbent assay (ELISA) measuring specific immunoglobulins against MGL_1304 and to investigate the levels of these immunoglobulins in sera of patients with various allergic diseases. METHODS Purified MGL_1304 from human sweat (QRX) and recombinant MGL_1304 (rMGL_1304) were prepared for ELISA. To quantify the amount of MGL_1304-specific immunoglobulins, the standard serum was created by pooling sera of 20 patients with AD whose basophils released histamine in response to QRX. A monoclonal antibody which exhibited the highest neutralizing ability against QRX was established as Smith-2, and used as a capture antibody for the assay of QRX-specific IgE. A total of 156 subjects [normal controls (n = 23), AD (n = 63), cholinergic urticaria (CU) (n = 24), bronchial asthma (n = 32), and allergic rhinitis (n = 14)] were enrolled in this study. RESULTS ELISA methods to quantify the specific IgE, IgG and IgG4 against MGL_1304 in sera were successfully established. Levels of QRX-specific IgE in sera of patients with AD and CU were significantly higher than those of normal controls. Moreover, the levels of QRX-specific IgE and rMGL_1304-specific IgE in patients with AD were significantly correlated with their disease severities. CONCLUSIONS These ELISA methods to quantify the specific immunoglobulins against MGL_1304 are easy and useful means to assess allergy to MGL_1304. MGL_1304 contained in sweat is an important antigen for patients with AD and CU.

The Sensitivity and Clinical Course of Patients with Wheat-Dependent Exercise-Induced Anaphylaxis Sensitized to Hydrolyzed Wheat Protein in Facial Soap - Secondary Publication

BACKGROUND Recently, an increasing number of patients with wheat-dependent exercise-induced anaphylaxis (WDEIA) have been reported in Japan. Most of them had developed this condition during or after using hydrolyzed wheat protein (HWP)-containing soap (HWP-WDEIA). METHODS To clarify the relation between WDEIA and HWP-containing soap and their prognosis, we retrospectively studied the patients who visited Hiroshima University Hospital and were diagnosed as WDEIA from January 2010 to June 2011. We took detailed clinical histories, performed skin prick tests, serum immunoassays for antigen-specific IgE and basophil histamine release test, and followed up their clinical courses after the diagnosis. RESULTS Among 36 patients with WDEIA, 30 patients had used only one type of HWP-soap. The patients with HWP-WDEIA were mainly women and had developed facial symptoms and angioedema. They suffered from blood pressure reductions less frequently than patients with conventional WDEIA. The levels of gluten-specific IgE were higher than those of omega-5 gliadin in patients with HWP-WDEIA (P < 0.05, One-way ANOVA). All patients with HWP-WDEIA were positive against HWP in histamine release test. Among the conventional wheat antigens, glutenins induced the highest histamine release from basophils of patients with HWP-WDEIA. The sensitivities of patients against glutens and glutenins were reduced over months along with the discontinuance of HWP-soap. CONCLUSIONS The development of HWP-WDEIA is associated with the use of HWP-soap. The sensitivity to HWP that cross reacts with non-processed wheat may be reduced or possibly cured after the discontinuation of HWP-soap.

Remission rate of patients with wheat allergy sensitized to hydrolyzed wheat protein in facial soap

Hydrolyzed wheat protein (HWP) is made from gluten by acid or enzymemodification and used formany cosmetics around theworld. Recently, reports emerged about 2169 patients with wheat allergy sensitized to HWP in facial soap (HWP-wheat allergy) and consequently represented in an important social problem in Japan.1e3 Most patients had used the same soap bar named “Cha-no-ShizukuTM” containing HWP. HWPused in “Cha-no-ShizukuTM”wasGlupearl19S.Although itwasalsocontained inseveralother typesof soap bars, all of themhad alreadybeen recalled.Most of the patients sensitized to HWPwere female and their peak agewas in the 40s.3 Several severe cases led toanaphylaxis. 56%and16%of thepatientsdeveloped symptoms after exercise or oral intake of NSAIDs, respectively. Although serumwheatand gluten-specific IgE are negative and u-5 gliadin-specific IgE is positive in CAP-FEIA (CAP-fluoro enzyme immune assay) in most patients with conventional wheatdependent exercise-induced anaphylaxis (WDEIA),4 wheatand gluten-specific IgE are positive and u-5 gliadin-specific IgE is negative in many patients with HWP-wheat allergy, even in the WDEIA subtype.3,5 Moreover, patients with HWP-wheat allergy exhibit positive skin prick tests and basophil histamine release tests (HRT) against HWP, Glupearl 19S. Those positive reactions against HWP are specific to patients with HWP-wheat allergy.5 Wepreviously reporteda remissioncaseofHWP-wheatallergyafter cessation of HWP-containing soap6 and also reported that hypersensitivities againstwheat componentsweremostly reduced in those patients.5 However, substantial numbers of patients still avoid food containingwheat, and the remission rate and/or change of hypersensitivityagainstHWPofoverall patientshavenot yetbeendetermined. Here, we investigated clinical remission rate of HWP-wheat allergy and negative conversion rate of ex vivo HRT against HWP. We retrospectively studied 110 patients who were diagnosed with HWP-wheat allergy, visited Hiroshima University Hospital from January 2010 to December 2013, and who were followed until September 2014. Patients were diagnosed according to the criteria for immediate wheat allergy to the hydrolyzed wheat (Glupearl 19S) contained in “Cha-no-Shizuku” soap and some other products by the Special Committee for the Safety of Protein Hydrolysate in Cosmetics (Supplementary Table 1).7 The remission rate of HWPwheat allergy and the negative conversion rate of HRT against HWPwere analyzed by KaplaneMeier methods, and related factors were analyzed by the log-rank test. We defined “Remission” as condition free of symptoms for more than 3 months without any

Evaluation of recombinant MGL_1304 produced by Pichia pastoris for clinical application to sweat allergy

BACKGROUND We previously identified MGL_1304 secreted by Malassezia globosa as a sweat antigen for patients with atopic dermatitis (AD) and cholinergic urticaria (ChU). However, purifying native MGL_1304 from human sweat or culture supernatant of M. globosa (sup-MGL_1304) is costly and time-consuming. Moreover, recombinant MGL_1304 expressed by using Escherichia coli (TF-rMGL_1304) needs a large chaperon protein and lacks the original glycosylation of yeasts. Thus, we generated a recombinant MGL_1304 by Pichia pastoris (P-rMGL_1304) and investigated its characteristic features. METHODS Recombinant MGL_1304 proteins expressed by E. coli and P. pastoris were generated. Properties of these recombinants and native antigens were compared by western blot analysis, histamine release tests (HRT) of patients with AD and ChU, and β-hexosaminidase release tests with RBL-48 cells. P-rMGL_1304-specific IgE in sera of patients with AD were measured by sandwich ELISA. RESULTS Western blot analysis revealed that IgE of patients with AD bound to all MGL_1304 recombinants and native antigens. The histamine releasing ability of P-rMGL_1304 was 100 times higher than that of TF-rMGL_1304, and was comparable to that of sup-MGL_1304. Degranulation rates of RBL-48 cells, sensitized with sera of patients with AD in response to the stimulation of P-rMGL_1304, were comparable to those of sup-MGL_1304, whereas those of TF-rMGL_1304 were relatively weak. The levels of P-rMGL_1304-specific IgE in sera of patients with AD were correlated with their disease severities. CONCLUSIONS P-rMGL_1304 has an antigenicity comparable to the native antigen, and is more useful than TF-rMGL_1304, especially in HRT and degranulation assay of RBL-48 cells.

Diagnostic Tests for Urticaria

Clinical diagnosis is made essentially by the characteristics of skin eruptions and detailed history taking. Particular, but not general blood examinations and/or skin biopsy should be performed if clinical diagnosis suggests the necessity of these examinations.

Novel and recurrent C1 inhibitor gene mutations in nine Japanese patients with hereditary angioedema

C1 inhibitor (C1-INH) deficiency (hereditary or acquired gioedema; HAE [OMIM106100] or AAE) is characterized by curring episodes of subcutaneous or submucosal swellings, pically involving the face, limbs, tongue, bowels or upper airways ]. Laryngeal attack can cause airway obstruction which may be tal. Therefore, prompt diagnosis and treatment are essential. Two assical types of HAE, type 1 and 2, are autosomal dominant sorders due to heterozygous deficiencies of the C1-INH gene ERPING1). Type 1 shows decreased antigenic and functional vels of C1-INH and type 2 shows normal levels of antigenic -INH but low levels of functional C1-INH [2]. DNA screening of the C1-INH gene establishes the genetic termination of the C1-INH deficiency. It is especially useful for e diagnosis of sporadic cases accounting for 20–30% of patients ith HAE as a result of de novo mutations without family history of gioedema [3]. According to the C1-INH mutation database AEdb, http://hae.enzim.hu), more than 250 different mutations ve been reported [4]. The mutations have been found distributed er all exons and splice sites of the C1-INH gene. Gross mutation is sponsible for approximately 15% of the mutations detected in tients with HAE, and the remaining 85% being due to small/point utations. This database has been well established by reports ainly from Europe and North America, while the genetic formation of HAE in Asian races is uncertain. The prevalence HAE type 1 and type 2 has been estimated at 1/50,000 [2]. On the her hand, a nation-wide prevalence survey of HAE in Japan has own only 52 type 1 or type 2 patients with HAE, even though panese population is about 128 million [5], and only 23 utations have been reported in Japan so far [6]. Similarly, only 3 HAE patients have been reported from China, which is the ost populous country in the world [7]. In this study, we investigated the genetic analysis of nine panese patients with HAE in order to increase the genetic formation of HAE in Japan. HAE was diagnosed based on repeated isodes of swelling in the skin and/or mucosa and decreased vels of C1 inhibitor function and C4 in plasma. Genomic DNA was tracted from peripheral blood of the patients by using Genomic A Mini Kit (Invitrogen, Carlsbad, CA). Polymerase chain reaction CR) were carried out using 9 pairs of primers to amplify the 8 ons, referring to previously described information [8]. DNA quencing was performed using an Applied Biosystems 3100ant DNA Analyzer (Applied Biosystems, Warrington, UK). The udy was approved by the Genome Ethics Committee for perimental Research Involving Human Subjects, Hiroshima iversity Hospital, and written informed consent for participan was obtained from the patient and/or their families. se w Subjects of this study consisted of four male and five female tients with an age range from 20 to 69 years. Five patients had milial history of angioedema and the other four patients were oradic cases. Genetic analysis of the C1-INH gene in this study vealed 4 frameshift mutations, 2 nonsense mutations, one issense mutation, one splice site mutation and large deletion. these mutations, 4 mutations: p.Arg40fsX17 (c.119insA), Ala46fsX9 (c.138_207del), p.Asp256fsX22 (c.766delG) and Gln338X (c.1012C > T) were novel (Fig. 1) and 5 mutations: Lys329fsX7 (c.987delG) [6], p.Arg466His (c.1397G > A) [4], p. g492X (c.1480C > T) [4], splicing defect (c.551-2_A > G) [4] and large rearrangement (>650 kbp including entire exons), which as reported separately [8], were recurrent (Table 1). Based on the netic analysis from our study and previous reports about panese patients with HAE, the mutations were widely distributacross the C1-INH gene and the distributions of the mutations by pes in Japan do not seem to be apparently different from those ported in Europe. We have classified four individuals into type 1 HAE and one dividual into type 2 HAE by the level of C1-INH protein. We could t classify four individuals because of lack of the serum test of C1H protein level. According to previous studies, almost 100% in pe 2 HAE showed missense mutations and it almost invariably sults in HAE type II when mutations occur within the C1-INH active site located in exon 8 at arginine 444 [9]. All of four classified individuals showed nonsense or frameshift mutations hich caused premature termination expecting to lead to reduced pression of C1-INH. Actually, the mutation in patient 1, Arg492X, was identified in type 1 HAE. From these observations, e speculated that these 4 unclassified individuals were type 1 E. Only one patient was found with HAE type 2 in this study 1%), which is in accordance with another report in Japan (12.5%) ]. Previously the prevalence of type 2 has been reported around % [2], but a recent study showed the number of type 2 was 6% of tients with HAE identified in several countries of Europe [10]. erefore, there seem to be regional variation in occurrence garding HAE type 2. Among nine patients in this study, two patients received acheostomy due to severe swelling on the respiratory trunk. mamoto et al. [6] also reported that 32% patients with HAE had acheostomy or tracheal intubation in Japan. The risk of severe spiratory symptoms in Japanese patients with HAE was as quent as in western countries. Therefore, proper diagnosis of E is necessary to prevent the patients from asphyxiation. oreover DNA screening of the C1-INH gene is useful in that it veals the segregation of genetic carriers of HAE within affected

Oral administration of β-carotene or lycopene prevents atopic dermatitis-like dermatitis in HR-1 mice

Atopic dermatitis (AD) is a chronic relapsing eczematous skin disease. Certain populations of patients are resistant to standard therapies with topical steroids and/or calcineurin inhibitors, and require systemic medication, such as immunosuppressants. Recently, several reports have shed light on the anti‐allergic effects of carotenoids. Therefore, we investigated the effect of p.o. administration of β‐carotene or lycopene on AD‐like symptoms of HR‐1 hairless mice fed with a low zinc/magnesium diet. Mice were divided into four groups: (i) fed with a standard diet (Co group); (ii) low zinc/magnesium diet (HR group); (iii) low zinc/magnesium and β‐carotene diet (HR‐C group); and (iv) low zinc/magnesium and lycopene diet (HR‐L group). They were then fed these diets for 8 weeks. Severities of dermatitis were assessed by their appearance, and histopathological and hematological observations. Mice in the HR group developed AD‐like dermatitis both clinically and histologically. HR‐C and HR‐L group mice also developed xerosis and wrinkle‐like skin changes, but they were milder than those of HR group mice. Histological analysis revealed that epidermis thickening and inflammatory cell infiltration in the skin of the HR‐C and HR‐L groups were both statistically less than those of the HR group. The concentration of thymus and activation regulated chemokine in the skin of the HR‐L group and the concentration of CCL27 in the skin of the HR‐C group were significantly lower than those of the HR group, respectively. In conclusion, p.o. administration of β‐carotene or lycopene prevents AD‐like symptoms in association with a suppression of T‐helper 2 chemokines in a murine model. Ingestion of carotenoids may be beneficial for patients with AD.