Alpha/Beta Gliadin MM1 Is a Novel Antigen for Wheat-Dependent Exercise-Induced Anaphylaxis.

INTRODUCTION: Screening for ω-5 gliadin specific IgE antibody (sIgE) has high diagnostic utility in cases of suspected wheat-dependent exercise-induced anaphylaxis (WDEIA); however, negative cases may require confirmatory tests, such as the oral challenge test. Thus, newly identified allergens that can be used for the serological diagnosis of WDEIA are needed. This study aimed to identify additional sIgE biomarkers of WDEIA. METHODS: Forty-two patients with WDEIA (5 negative/37 positive for ω-5 gliadin sIgE) were enrolled. For comparison, 8 patients with immediate-type wheat allergy without WDEIA and 20 healthy controls without wheat allergy were also enrolled. Extracted wheat proteins were separated by 2D-PAGE. Proteins that reacted with serum IgE antibody in 2D Western blotting (2D-WB) were identified using mass spectrometry. Recombinant proteins were synthesized in Escherichia coli, and the antigenicity was tested using ELISA and the basophil activation test. RESULTS: In 2D-WB, nine proteins reacted with the serum IgE antibody from at least 60% of patients with WDEIA (n ≥ 25/42). ELISA revealed that alpha/beta gliadin MM1 exhibited the highest positive immunoreactivity in 23 of 26 patients who were positive for ω-5 gliadin sIgE (88%) and in 5 of 5 patients who were negative for ω-5 gliadin sIgE (100%). Alpha/beta gliadin MM1 exhibited significantly higher basophil activation in 14 patients with WDEIA when compared to 5 individuals without a wheat allergy. CONCLUSIONS: Alpha/beta gliadin MM1 sIgE exhibited the highest seropositivity, even among patients who were negative for ω-5 gliadin sIgE. The inclusion of alpha/beta gliadin MM1 in allergen-sIgE tests may improve the sensitivity for diagnosing WDEIA.

Nattokinase (Bac s 1), a subtilisin family serine protease, is a novel allergen contained in the traditional Japanese fermented food natto.

BACKGROUND: Immediate allergy caused by natto, a popular Japanese food prepared by fermenting soybeans with Bacillus subtilis var. natto, has been reported. Polygamma glutamic acid (PGA) in the sticky substance around natto beans has been reported to be a causative allergen of natto allergy. However, some of our patients with natto allergy were negative for PGA in the skin prick test (SPT). The sticky substance of natto beans contains a subtilisin family serine protease, nattokinase, along with PGA. In this study, we aimed to examine the antigenicity of nattokinase in natto allergy. METHODS: Eight patients, who developed symptoms after ingesting natto and positively reacted to natto (seven to the sticky substance in natto and one to the whole natto product) in their SPT, were enrolled in this study. To analyze IgE reactivity, we performed immunoblotting, ELISA, and SPT for natto (bean and sticky substance), and/or PGA, and/or nattokinase and/or cultured B. subtilis var. natto extract. RESULTS: In the SPT, four cases each were PGA-positive and PGA-negative. Immunoblotting of the sera from PGA-negative patients showed a protein band at 30 kDa, which was identified as nattokinase. Three PGA-negative cases, but not three PGA-positive cases, showed a positive reaction to nattokinase in the SPT and had a history of atopic dermatitis. The ELISA for nattokinase revealed a positive reaction of PGA-negative cases and negative reaction of PGA-positive cases in the SPT. CONCLUSIONS: We identified a subtilisin family serine protease, nattokinase, as a novel allergen in natto allergy patients unsensitized to PGA.

The Microbiome in Comedonal Contents of Inflammatory Acne Vulgaris is Composed of an Overgrowth of Cutibacterium Spp. and Other Cutaneous Microorganisms.

Background: Acne vulgaris (acne) and cutaneous resident microorganisms are considered to be closely related. However, the bacterial and fungal microbiota in the comedonal contents of inflammatory acne lesions have not yet been investigated in detail. Purpose: To clarify the relationship between cutaneous microorganisms and acne, we examined the microbiome in the comedonal contents of inflammatory acne and on the facial skin of patients with acne using 16s rRNA and ITS gene sequencing with a next-generation sequencer (NGS). Patients and Methods: Twenty-two untreated Japanese acne outpatients were examined. The comedonal contents of inflammatory acne lesions on the face were collected using a comedo extractor. Skin surface samples from facial skin were collected using the swab method. Results: The results obtained revealed that the predominant bacteria in the comedonal contents of inflammatory acne were Cutibacterium spp. (more prominent in areas with large amounts of sebum), while those on the skin surface were Staphylococcus spp. Malassezia spp., particularly Malassezia restricta, were the predominant fungi in both the comedonal contents of inflammatory acne and on the skin surface. The bacterial microbiome in comedonal contents exhibited stronger metabolic activity, including the production of enzymes related to acne, than that on the skin surface. Conclusion: These results indicate that acne is an inflammatory disease involving the overgrowth of Cutibacterium acnes and other cutaneous resident microorganisms, including Malassezia spp.

An epidermal keratinocyte homogenate induced type 2 and proinflammatory cytokine expression in cultured dermal cells.

BACKGROUND: The "itch-scratch cycle" is a clinically well-known cause of exacerbation of atopic dermatitis (AD), but the underlying molecular mechanisms remain largely unknown. OBJECTIVE: To test our hypothesis that some molecules released from damaged epidermal keratinocytes by scratching inducetype 2 responses in intact skin dermal cells, leading to exacerbation of AD. METHODS: Normal human dermal fibroblasts (NHDF) and human dermal blood microvascular endothelial cells (HMVEC-dBl) were treated with an epidermal keratinocyte homogenate (EKH). We used qPCR and ELISA, respectively, to measure the mRNA expressions and protein concentrations of various cytokines, including IL-6, IL-8, thymic stromal lymphopoietin (TSLP), and IL-33. We analyzed IL-33 protein expression in the nuclear fractions of NHDF by Western blotting. We also investigated the effects of IL-1R1 gene-silencing and several AD therapeutic drugs on EKH induction of cytokine production by the dermal cells. RESULTS: EKH significantly induced IL-6 and IL-8 in NHDF and HMVEC-dBl. EKH also induced type 2 cytokines, TSLP and IL-33, in NHDF. IL-1R1 gene-silencing in NHDF partially attenuated the induction. Dexamethasone (a corticosteroid) significantly inhibited, while ABT494 (a JAK1 inhibitor) partially inhibited, EKH's induction of cytokines in fibroblasts. In contrast, ABT494 was more effective than dexamethasone in inhibiting the cytokine induction in HMVEC-dBl. CONCLUSION: This study showed that a homogenate of epidermal keratinocytes significantly induced AD-related cytokines in cultured dermal cells, and IL-1α, an alarmin, might be involved in that induction. Combined use of corticosteroids and JAK1 inhibitors is likely to block the itch-scratch cycle by targeting different types of dermal cells.

Gene Expression Patterns in Distinct Endoscopic Findings for Eosinophilic Gastritis in Children.

BACKGROUND: Eosinophilic gastritis (EG) is clinicopathologically characterized by both marked gastric eosinophilia and clinical symptoms. The endoscopic findings in EG vary among patients, leading to clinical confusion. However, little is known about the relationship between precise endoscopic findings and the pathophysiological process responsible for EG. OBJECTIVE: We aimed to elucidate whether the gross endoscopic findings of EG can be classified into distinct gene expression profiles. METHODS: We enrolled pediatric patients who underwent gastrointestinal endoscopy for clinical symptoms suggestive of eosinophilic gastrointestinal disorder between 2011 and 2016. EG was diagnosed when gastric eosinophilia was greater than or equal to 30 eosinophils/hpf. The gene expression profiles of gastric biopsies were assessed using microarray technology. RESULTS: Patients with EG and control subjects (n = 8, each) were examined. On the microarray, 1,999 genes were differentially expressed between EG and the controls (≥2-fold difference, adjusted P value < .05), including significant upregulation of eotaxin-3 (C-C chemokine ligand 26). The endoscopic findings of patients with EG fell roughly into 2 types, namely, ulcerative and nodular lesions. Despite identifying distinct patterns of gene expression, most differentially regulated genes overlapped between the 2 endoscopic finding types. Several gene ontology terms were enriched in the substantially overlapped genes, but not in each of the distinct genes. CONCLUSIONS: Our results strongly indicate that ulcerative and nodular lesions are a single disease, EG, or a variation thereof, in spite of morphological differences. Our findings may contribute to a better understanding of the pathogenesis of EG, as well as to more accurate diagnosis of this disease.

Gene expression profiling of acute type A aortic dissection combined with in vitro assessment.

OBJECTIVES: The mechanisms underlying aortic dissection remain to be fully elucidated. We aimed to identify key molecules driving dissection through gene expression profiling achieved by microarray analysis and subsequent in vitro experiments using human aortic endothelial cells (HAECs) and aortic vascular smooth muscle cells (AoSMCs). METHODS: Total RNA, including microRNA (miRNA), was isolated from the intima-media layer of dissected ascending aorta obtained intraoperatively from acute type A aortic dissection (ATAAD) patients without familial thoracic aortic disease (n = 8) and that of non-dissected ascending aorta obtained from transplant donors (n = 9). Gene expression profiling was performed with mRNA and miRNA microarrays, and results were confirmed by quantitative polymerase chain reaction (qPCR). Target genes and miRNA were identified by gene ontology analysis and a literature search. To reproduce the in silico results, HAECs and AoSMCs were stimulated in vitro by upstream cytokines, and expression of target genes was assessed by qPCR. RESULTS: Microarray analysis revealed 1536 genes (3.6%, 1536/42 545 probes) and 41 miRNAs (3.0%, 41/1368 probes) that were differentially expressed in the ATAAD group (versus donor group). The top 15 related pathways included regulation of inflammatory response, growth factor activity and extracellular matrix. Gene ontology analysis identified JAK2 (regulation of inflammatory response), PDGFA, TGFB1, VEGFA (growth factor activity) and TIMP3, TIMP4, SERPINE1 (extracellular matrix) as the target genes and miR-21-5p, a TIMP3 repressor, as target miRNA that interacts with the target genes. Validation qPCR confirmed the altered expression of all 7 target genes and miR-21-5p in dissected aorta specimens (all genes, P < 0.05). Ingenuity pathway analysis showed TNF-α and TGF-ß to be upstream cytokines for the target genes. In vitro experiments showed these cytokines inhibit TIMP3 expression (P < 0.05) and enhance VEGFA expression (P < 0.01) in AoSMCs but not HAECs. miR-21-5p expression increases in AoSMCs under TNF-α and TGF-ß stimulation (fold change: 1.36; P = 0.011). CONCLUSIONS: Results of our novel approach, integrating in vitro assessment into gene expression profiling, implicated chronic inflammation characterized by MMP-TIMP dysregulation, increased VEGFA expression, and TGF-ß signalling in the development of dissection. Further investigation may reveal novel diagnostic biomarkers and uncover the mechanism(s) underlying ATAAD.

Platelets constitutively express IL-33 protein and modulate eosinophilic airway inflammation.

BACKGROUND: Although platelets play a key role in allergic inflammation in addition to their well-established role in hemostasis, the precise mechanisms of how platelets modulate allergic inflammation are not fully understood. IL-33 is an essential regulator of innate immune responses and allergic inflammation. OBJECTIVE: We sought to determine the expression of IL-33 protein by platelets and its functional significance in airway inflammation. METHODS: IL-33 protein in human platelets, the human megakaryocyte cell line MEG-01, and bone marrow-derived mouse megakaryocytes was detected by using Western blot analysis and fluorescent immunostaining. We examined the functional relevance of IL-33 protein in platelets by comparing platelet-intact and platelet-depleted groups in a murine model of IL-33-dependent airway eosinophilia elicited by intranasal administration of papain. We further compared the additive effect of administration of platelets derived from wild-type versus IL-33-deficient mice on the papain-induced eosinophilia. RESULTS: Platelets and their progenitor cells, megakaryocytes, constitutively expressed IL-33 protein (31 kDa). Papain-induced IL-33-dependent airway eosinophilia in mice was significantly attenuated by platelet depletion. Conversely, concomitant administration of platelets derived from wild-type mice but not IL-33-deficient mice enhanced the papain-induced airway eosinophilia. CONCLUSIONS: Our novel findings suggest that platelets might be important cellular sources of IL-33 protein in vivo and that platelet-derived IL-33 might play a role in airway inflammation. Therefore platelets might become an attractive novel therapeutic target for asthma and probably allergic inflammation.

Epicutaneous Sensitization in Patients with Atopic Dermatitis.

Atopic dermatitis (AD) is the most common chronic skin disorder among infants and young children. It is characterized by chronic relapsing eczema with itch and is caused by skin barrier dysfunction and immunological dysregulation. Scratching of the lesion site can damage the skin and increase epithelial permeability, thereby allowing large molecular weight antigens to be absorbed transcutaneously. However, a recent study demonstrated that Langerhans cells are localized close below the skin surface and extend dendrites vertically to penetrate the tight junctions (TJs) in erythematous lesions, even though the TJs were functionally intact. Therefore, epidermal barrier disruption per se is not critical for antigen uptake by Langerhans cells. In contrast, 2 critical damage-associated patterns-IL-33 and IL-1alpha-are reportedly released from damaged epithelial cells. Dendritic cells activated by IL-33 and thymic stromal lymphopoietin (TSLP) (induced by IL-1 alpha) express MHC class II and costimulatory molecules and facilitate naive T cell differentiation into IL-5- and IL-13-secreting Th2 cells. IL-33 and TSLP also activate type 2 innate lymphoid cells, induce large amounts of IL-5 and IL-13, and participate in the pathogenesis of AD. Primary prevention and proactive treatment of AD are critically important for preventing epicutaneous sensitization in AD patients. However, future studies are required to elucidate the most beneficial primary prevention strategies, including the indicated patient cohort and the timing and method of their application.

Recent advances in understanding the roles of vascular endothelial cells in allergic inflammation.

Allergic disorders commonly involve both chronic tissue inflammation and remodeling caused by immunological reactions to various antigens on tissue surfaces. Due to their anatomical location, vascular endothelial cells are the final responders to interact with various exogenous factors that come into contact with the epithelial surface, such as pathogen-associated molecular patterns (PAMPs) and antigens. Recent studies have shed light on the important roles of endothelial cells in the development and exacerbation of allergic disorders. For instance, endothelial cells have the greatest potential to produce several key molecules that are deeply involved in allergic inflammation, such as periostin and thymus and activation-regulated chemokine (TARC/CCL17). Additionally, endothelial cells were recently shown to be important functional targets for IL-33--an essential regulator of allergic inflammation. Notably, almost all endothelial cell responses and functions involved in allergic inflammation are not suppressed by corticosteroids. These corticosteroid-refractory endothelial cell responses and functions include TNF-α-associated angiogenesis, leukocyte adhesion, IL-33-mediated responses and periostin and TARC production. Therefore, these unique responses and functions of endothelial cells may be critically involved in the pathogenesis of various allergic disorders, especially their refractory processes. Here, we review recent studies, including ours, which have elucidated previously unknown pathophysiological roles of vascular endothelial cells in allergic inflammation and discuss the possibility of endothelium-targeted therapy for allergic disorders.