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Mas-related G protein-coupled receptor b2 (Mrgprb2) binding to its cationic endogenous and exogenous ligands induces mast cell degranulation and promotes inflammation in mice. However, the physiological roles of its human homologue MRGPRX2 remain unclear. Here we aimed to elucidate the mechanisms by which MRGPRX2 regulates vascular permeability, and generated MRGPRX2 knock-in (MRGPRX2-KI) and Mrgprb2 knockout (Mrgprb2-KO) mice. Substance P (SP) and ciprofloxacin strongly degranulated MRGPRX2-KI peritoneal mast cells (PMCs) better than WT PMCs, whereas Dermatophagoides pteronyssinus (Der p) extract and phenol-soluble modulin α3 (PSMα3) did not degranulate PMCs. SP-stimulated MRGPRX2-KI PMCs released large amounts of histamine and mast cell protease 4 (MCPT4) chymase. Der p extract, PSMα3, and MCPT4, but not histamine, induced SP release from dorsal root ganglion (DRG) cells. However, this effect of Der p extract/PSMα3 was suppressed by a transient receptor potential vanilloid 1 (TRPV1) antagonist. SP-, ciprofloxacin-, Der p extract-, PSMα3-, and MCPT4-induced vascular permeability was highest in MRGPRX2-KI mice, which depended on SP. In addition, SP-, ciprofloxacin- and PSMα3-induced MRGPRX2-dependent vascular hyperpermeability was suppressed by antihistamine and chymase inhibitor. TRPV1 antagonist also inhibited PSMα3-induced MRGPRX2-dependent vascular hyperpermeability. Both Mrgprb2-KO and MRGPRX2-KI did not influence the histamine-induced murine vascular hyperpermeability. Overall, our results suggest that neuronal SP induces MRGPRX2-dependent mast cell degranulation, releasing histamine and chymase, which promote vascular hyperpermeability directly or indirectly via DRG cell activation. Importantly, the worsening cycle (MRGPRX2 → mast cell degranulation → chymase → DRG activation → SP → MRGPRX2) seems to play an important role in human MRGPRX2-depdendent inflammation.

Oral immunotherapy (OIT) is an effective approach to controlling food allergy. Although the detailed molecular and cellular mechanisms of OIT are unknown currently, they must be understood to advance the treatment of allergic diseases in general. To elucidate the mechanisms of OIT, especially during the immunological transition from desensitization to allergy regulation, we generated a clinical OIT murine model and used it to examine immunological events of OIT. We found that in mice that completed OIT successfully, desensitized mast cells (MCs) showed functionally beneficial alterations, such as increased induction of regulatory cytokines and enhanced expansion of regulatory T cells. Importantly, these regulatory-T-cell-mediated inhibitions of allergic responses were dramatically decreased in mice lacking OIT-induced desensitized MC. Collectively, these findings show that the desensitization process modulates the activation of MCs, leading directly to enhanced induction of regulatory-T-cell expansion and promotion of clinical allergic unresponsiveness. Our results suggest that efficiently inducing regulatory MCs is a novel strategy for the treatment of allergic disease.

The penetration of allergens through the epithelial layer is the initial step in the development of allergic conjunctivitis. Although pollinosis patients manifest symptoms within minutes after pollen exposure, the mechanisms of the rapid transport of the allergens remain unclear. In the present study, we found that the instillation of pollen shells rapidly induces a large number of goblet cell–associated antigen passages (GAPs) in the conjunctiva. Antigen acquisition by stromal cells, including macrophages and CD11b+ dendritic cells, correlated with surface GAP formation. Furthermore, a substantial amount of antigen was transported to the stroma during the first 10 minutes of pollen exposure, which was sufficient for the full induction of an allergic conjunctivitis mouse model. This inducible, rapid GAP formation and antigen acquisition were suppressed by topical lidocaine or trigeminal nerve ablation, indicating that the sensory nervous system plays an essential role. Interestingly, pollen shell–stimulated GAP formation was not suppressed by topical atropine, suggesting that the conjunctival GAPs and intestinal GAPs are differentially regulated. These results identify pollen shell–induced GAP as a therapeutic target for allergic conjunctivitis.

BackgroundRecently, we have developed a method to identify IgE cross-reactive allergens. However, the mechanism by which IgE cross-reactive allergens cause food allergy is not yet fully understood how. In this study, we aimed to understand the underlying pathogenesis by identifying food allergens that cross-react with house dust mite allergens in a murine model.Material and methodsAllergenic protein microarray analysis was conducted using serum from mice intraperitoneally injected with Dermatophagoides pteronyssinus (Der p) extract plus alum or alum alone as controls. Der p, Dermatophagoides farinae (Der f), coho salmon extract-sensitized and control mice were analyzed. Serum levels of IgE against Der p, Der f, coho salmon extract, protein fractions of coho salmon extract separated by ammonium sulfate precipitation and anion exchange chromatography, and recombinant coho salmon tropomyosin or actin were measured by an enzyme-linked immunosorbent assay. A murine model of cutaneous anaphylaxis or oral allergy syndrome (OAS) was established in Der p extract-sensitized mice stimulated with coho salmon extract, tropomyosin, or actin.ResultsProtein microarray analysis showed that coho salmon-derived proteins were highly bound to serum IgE in Der p extract-sensitized mice. Serum IgE from Der p or Der f extract-sensitized mice was bound to coho salmon extract, whereas serum IgE from coho salmon extract-sensitized mice was bound to Der p or Der f extract. Analysis of the murine model showed that cutaneous anaphylaxis and oral allergic reaction were evident in Der p extract-sensitized mice stimulated by coho salmon extract. Serum IgE from Der p or Der f extract-sensitized mice was bound strongly to protein fractions separated by anion exchange chromatography of coho salmon proteins precipitated with 50% ammonium sulfate, which massively contained the approximately 38 kDa protein. We found that serum IgE from Der p extract-sensitized mice was bound to recombinant coho salmon tropomyosin. Der p extract-sensitized mice exhibited cutaneous anaphylaxis in response to coho salmon tropomyosin.ConclusionOur results showed IgE cross-reactivity of tropomyosin between Dermatophagoides and coho salmon which illustrates salmon allergy following sensitization with the house dust mite Dermatophagoides. Our method for identifying IgE cross-reactive allergens will help understand the underlying mechanisms of food allergies.

Mas-related G protein-coupled receptor b2 (Mrgprb2) binding to its cationic endogenous and exogenous ligands induces mast cell degranulation and promotes inflammation in mice. However, the physiological roles of its human homologue MRGPRX2 remain unclear. Here we aimed to elucidate the mechanisms by which MRGPRX2 regulates vascular permeability, and generated MRGPRX2 knock-in (MRGPRX2-KI) and Mrgprb2 knockout (Mrgprb2-KO) mice. Substance P (SP) and ciprofloxacin strongly degranulated MRGPRX2-KI peritoneal mast cells (PMCs) better than WT PMCs, whereas Dermatophagoides pteronyssinus (Der p) extract and phenol-soluble modulin α3 (PSMα3) did not degranulate PMCs. SP-stimulated MRGPRX2-KI PMCs released large amounts of histamine and mast cell protease 4 (MCPT4) chymase. Der p extract, PSMα3, and MCPT4, but not histamine, induced SP release from dorsal root ganglion (DRG) cells. However, this effect of Der p extract/PSMα3 was suppressed by a transient receptor potential vanilloid 1 (TRPV1) antagonist. SP-, ciprofloxacin-, Der p extract-, PSMα3-, and MCPT4-induced vascular permeability was highest in MRGPRX2-KI mice, which depended on SP. In addition, SP-, ciprofloxacin- and PSMα3-induced MRGPRX2-dependent vascular hyperpermeability was suppressed by antihistamine and chymase inhibitor. TRPV1 antagonist also inhibited PSMα3-induced MRGPRX2-dependent vascular hyperpermeability. Both Mrgprb2-KO and MRGPRX2-KI did not influence the histamine-induced murine vascular hyperpermeability. Overall, our results suggest that neuronal SP induces MRGPRX2-dependent mast cell degranulation, releasing histamine and chymase, which promote vascular hyperpermeability directly or indirectly via DRG cell activation. Importantly, the worsening cycle (MRGPRX2 → mast cell degranulation → chymase → DRG activation → SP → MRGPRX2) seems to play an important role in human MRGPRX2-depdendent inflammation. [ABSTRACT FROM AUTHOR]

BackgroundPatients with food allergy often suffer from atopic dermatitis, in which Staphylococcus aureus colonization is frequently observed. Staphylococcus aureus δ-toxin activates mast cells and promotes T helper 2 type skin inflammation in the tape-stripped murine skin. However, the physiological effects of δ-toxin present on the steady-state skin remain unknown. We aimed to investigate whether δ-toxin present on the steady-state skin impacts the development of food allergy.Material and methodsThe non-tape-stripped skins of wild-type, KitW-sh/W-sh, or ST2-deficient mice were treated with ovalbumin (OVA) with or without δ-toxin before intragastric administration of OVA. The frequency of diarrhea, numbers of jejunum or skin mast cells, and serum levels of OVA-specific IgE were measured. Conventional dendritic cell 2 (cDC2) in skin and lymph nodes (LN) were analyzed. The cytokine levels in the skin tissues or culture supernatants of δ-toxin-stimulated murine keratinocytes were measured. Anti-IL-1α antibody-pretreated mice were analyzed.ResultsStimulation with δ-toxin induced the release of IL-1α, but not IL-33, in murine keratinocytes. Epicutaneous treatment with OVA and δ-toxin induced the local production of IL-1α. This treatment induced the translocation of OVA-loaded cDC2 from skin to draining LN and OVA-specific IgE production, independently of mast cells and ST2. This resulted in OVA-administered food allergic responses. In these models, pretreatment with anti-IL-1α antibody inhibited the cDC2 activation and OVA-specific IgE production, thereby dampening food allergic responses.ConclusionEven without tape stripping, δ-toxin present on skin enhances epicutaneous sensitization to food allergen in an IL-1α-dependent manner, thereby promoting the development of food allergy.

Oral allergy syndrome (OAS) is an IgE-mediated immediate food allergy that is localized to the oral mucosa. Pollen food allergy syndrome (PFAS), a pollinosis-associated OAS, is caused by cross-reactivity between food and pollen allergens. However, we need to more precisely understand the underlying pathogenesis of OAS/PFAS. In the present study, we developed a method to comprehensively identify cross-reactive allergens by using murine model of OAS and protein microarray technology. We focused on lip angioedema, which is one of the most common symptoms of OAS, and confirmed that mast cells reside in the tissues inside the lower lip of the mice. Interestingly, when the food allergen ovalbumin (OVA) was injected inside the lower lip of mice with high levels of OVA-specific IgE followed by an intravenous injection of the Evans blue dye, we found immediate dye extravasation in the skin of the neck in a mast cell-dependent manner. In addition, the degree of mast cell degranulation in the oral cavity, reflecting the severity of oral allergic responses, can be estimated by measuring the amount of extravasated dye in the skin. Therefore, we used this model of OAS to examine IgE cross-reactive allergens in vivo. Protein microarray analysis showed that serum IgE from mice intraperitoneally sensitized with ragweed pollen, one of the major pollens causing pollinosis, bound highly to protein extracts from several edible plants including black peppercorn and fennel. We confirmed that the levels of black pepper-specific IgE and fennel-specific IgE were significantly higher in the serum from ragweed pollen-sensitized mice than in the serum from non-sensitized control mice. Importantly, analysis of murine model of OAS showed that the injection of black pepper or fennel extract induced apparent oral allergic responses in ragweed pollen-sensitized mice. These results indicate IgE cross-reactivity of ragweed pollen with black pepper and fennel. In conclusion, we developed mouse model of OAS to identify IgE cross-reactive pollen and food allergens, which will help understand the pathogenesis of OAS/PFAS.

Abstract Background Catheter ablation (CA) for atrial fibrillation (AF) is widely performed. However, the indication for CA in patients with asymptomatic persistent AF is still controversial. Methods Among 259 consecutive patients who were hospitalized for initial CA of AF, a total of 45 patients who had asymptomatic persistent AF were retrospectively analyzed. Quality of life (QOL) before and 1 year after CA was evaluated, and changes in the cardiac function over 5 years after CA were also examined. QOL was assessed using the AF QOL questionnaire (AFQLQ) developed by the Japanese Heart Rhythm Society. In addition, cardiac function was assessed by measuring the plasma B‐type natriuretic peptide (BNP) level, left ventricular ejection fraction (LVEF), left atrial diameter (LAD) with transthoracic echocardiogram, and left atrial (LA) volume with computed tomography (CT). Results The AFQLQ significantly improved after CA in terms of “symptom frequency” and “activity limits and mental anxiety.” The plasma BNP level, LVEF, and LAD significantly improved in the first 3 months after the first CA, with no significant changes thereafter (from 149.0 pg/dL [95% confidence intervals {CI}, 114.5‐183.5 pg/dL] to 49.8 pg/dL [95% CI, 26.5‐70.1], P

Abstract A 77‐year‐old woman with symptomatic paroxysmal atrial fibrillation (PAF) underwent pulmonary vein isolation (PVI), but subsequently experienced recurrence. In the second session, unidirectional left atrium (LA)‐left superior pulmonary vein (LSPV) conduction was revealed to exist at the carina of the LSPV. Left pulmonary vein (LPV) pacing performed in a cycle between 300 and 260 ms revealed rate‐dependent pulmonary vein (PV)‐LA conduction, and the location was estimated to be in the roof of the LSPV. PV isolation was achieved after ablation of two gaps. Consideration of the presence of rate‐dependent gaps may be useful to confirm bidirectional block lines after ablation.

Background. Severely calcified coronary artery stenting remains a challenge due to stent thrombosis, target vessel failure, and higher mortality. Moreover, optimal vessel preparation for calcified plaque with a crack formation pattern has not been established yet. We aimed to identify the effect of crack formation in calcified plaque in the coronary artery on the lumen area after stenting. Materials and Methods. We evaluated 50 consecutive patients undergoing drug-eluting stent implantation for severely calcified lesions by using optical frequency domain imaging (OFDI) (54 lesions); we analyzed OFDI image slices every 3 mm and evaluated the segments of 242 images in those who had the arc of calcium more than 180°. Crack formation in calcified plaque was classified into three types: type 0, no cracks; type 1, no dissection between calcified plaque and vessel wall; and type 2, any dissection between calcified plaque and vessel wall. Results. Type 2 had a significantly higher area expansion ratio between preballooning and poststenting (type 0, 196% (interquartile range (IQR), 163–244); type 1, 210% (IQR, 174–244); type 2, 237% (IQR, 203–294)). Conclusions. The dissection between calcified plaque and vessel wall was a significant factor affecting lumen area expansion after stenting.

Abstract Sepsis is a serious clinical problem. Negative regulation of innate immunity is associated with sepsis progression, but the underlying mechanisms remains unclear. Here we show that the receptor CD300f promotes disease progression in sepsis. CD300f −/− mice were protected from death after cecal ligation and puncture (CLP), a murine model of septic peritonitis. CD300f was highly expressed in mast cells and recruited neutrophils in the peritoneal cavity. Analysis of mice (e.g., mast cell-deficient mice) receiving transplants of wild-type or CD300f −/− mast cells or neutrophils indicated that CD300f deficiency did not influence intrinsic migratory abilities of neutrophils, but enhanced neutrophil chemoattractant production (from mast cells and neutrophils) in the peritoneal cavity of CLP-operated mice, leading to robust accumulation of neutrophils which efficiently eliminated Escherichia coli. Ceramide-CD300f interaction suppressed the release of neutrophil chemoattractants from Escherichia coli-stimulated mast cells and neutrophils. Administration of the reagents that disrupted the ceramide-CD300f interaction prevented CLP-induced sepsis by stimulating neutrophil recruitment, whereas that of ceramide-containing vesicles aggravated sepsis. Extracellular concentrations of ceramides increased in the peritoneal cavity after CLP, suggesting a possible role of extracellular ceramides, CD300f ligands, in the negative-feedback suppression of innate immune responses. Thus, CD300f is an attractive target for the treatment of sepsis.