UNASSIGNED: Mast cell (MC) degranulation is a key process in allergic reactions and inflammatory responses. Aspartate aminotransferase 1 (AAT1)-derived endogenous sulfur dioxide (SO2) is an important regulator of MC function. However, the mechanism underlying its role in MC degranulation remains unclear. This study aimed to investigate the mechanism by which endogenous SO2 controlled MC degranulation.
UNASSIGNED: HMC-1 and Rat basophilic leukemia cell MC line (RBL-2H3) were used in the cell experiments. SO2 content was detected by in situ fluorescent probe. MC degranulation represented by the release rate of MC β-hexosaminidase was determined using a colorimetric assay. Sulfenylation of galectin-9 (Gal-9) in MCs and purified protein was detected using a biotin switch assay. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to determine the exact sulfenylation sites of Gal-9 by SO2. Animal models of passive cutaneous anaphylaxis (PCA) and hypoxia-driven pulmonary vascular remodeling were used to investigate the effect of SO2 on mast cell activation in vivo. Site-directed mutation of Gal-9 was conducted to confirm the exact site of SO2 and support the significance of SO2/Gal-9 signal axis in the regulation of MC degranulation.
UNASSIGNED: Degranulation was increased in AAT1-knockdowned MCs, and SO2 supplementation reversed the increase in MC degranulation. Furthermore, deficiency of endogenous SO2 contributed to IgE-mediated degranulation in vitro. Besides, SO2 inhibited IgE-mediated and hypoxia-driven MC degranulation in vivo. Mechanistically, LC-MS/MS analysis and site-directed mutation results showed that SO2 sulfenylated Gal-9 at cysteine 74. Sulfenylation of the 74th cysteine of Gal-9 protein was required in the SO2-inhibited MC degranulation under both physiological and pathophysiological conditions.
UNASSIGNED: These findings elucidated that SO2 inhibited MC degranulation via sulfenylating Gal-9 under both physiological and pathophysiological conditions, which might provide a novel treatment approach for MC activation-related diseases.

BACKGROUND: Human placental extract (HPE) has been documented to facilitate the healing of certain disorders including allergy. However, the effects of HPE on the functionality of mast cells, a critical cell type in allergic diseases, have not been reported.
METHODS: To investigate the effects of HPE on the regulation of allergy with respect to the biological functions of mast cells, the mast cell line C57 or HMC-1 cells were treated with HPE followed by the assessment of cell proliferation, apoptosis, activation, chemotaxis and phagocytosis. Mouse peritoneal mast cells were also investigated for their responses to induction of apoptosis by HPE in vivo. Furthermore, the effect of HPE on mast cell degranulation was confirmed using the passive cutaneous anaphylaxis (PCA) assay, an acute allergy model.
RESULTS: HPE was capable of suppressing mast cell proliferation and inducing mast cell apoptosis. Mast cell degranulation in response to compound 48/80- or anti-DNP IgE and DNP-mediated activation was suppressed. In addition, treatment with HPE compromised the production of cytokines by mast cells and cell chemotaxis. These observations were consistent with the dampened passive cutaneous anaphylaxis (PCA) assay following treatment with HPE.
CONCLUSIONS: This study revealed a suppressive effect of HPE on overall mast cell activities, suggesting a potential regulatory role of HPE on the alleviation of allergic diseases through mast cells.

Many patients with allergies have anxiety about taking anti-allergic medicines due to their side effects and increased medical expenses. Thus, developing functional foods/agricultural products for allergy prevention is strongly desired. In this study, we revealed that a Citrus flavanone, hesperetin, amplified IgE/antigen-mediated degranulation-inhibitory potency of anti-allergic catechin, (-)-epigallocatechin-3-O-(3-O-methyl) gallate (EGCG3\'\'Me), in the rat basophilic/mast cell line RBL-2H3. Hesperetin also significantly elevated the activation of acid sphingomyelinase (ASM), essential for eliciting anti-allergic effect of EGCG3\'\'Me through the cell surficial protein, 67-kDa laminin receptor (67LR). Furthermore, oral administration of the highly absorbent hesperidin, α-glucosyl hesperidin, also enhanced the inhibitory potency of EGCG3\'\'Me-rich \'Benifuuki\' green tea (Camellia sinensis L.) on passive cutaneous anaphylaxis (PCA) reaction evoked by IgE/antigen in BALB/c mice. These observations indicate that hesperetin amplifies the ability of EGCG3\'\'Me to inhibit the IgE/antigen-mediated degranulation through activating ASM signaling.

Mast cells (MCs) are an important treatment target for high-affinity IgE Fc receptor (FcεRI)-mediated allergic diseases. The plant-derived molecule 4-methylumbelliferone (4-MU) has beneficial effects in animal models of inflammation and autoimmunity diseases. The aim of this study was to examine 4-MU effects on MC activation and probe the underlying molecular mechanism(s). We sensitized rat basophilic leukemia cells (RBLs) and mouse bone marrow-derived mast cells (BMMCs) with anti-dinitrophenol (DNP) immunoglobulin (Ig)E antibodies, stimulated them with exposure to DNP-human serum albumin (HSA), and then treated stimulated cells with 4-MU. Signaling-protein expression was determined by immunoblotting. In vivo allergic responses were examined in IgE-mediated passive cutaneous anaphylaxis (PCA) and ovalbumin (OVA)-induced active systemic anaphylaxis (ASA) mouse models. 4-MU inhibited β-hexosaminidase activity and histamine release dose-dependently in FcεRI-activated RBLs and BMMCs. Additionally, 4-MU reduced cytomorphological elongation and F-actin reorganization while down-regulating IgE/Ag-induced phosphorylation of SYK, NF-κB p65, ERK1/2, p38, and JNK. Moreover, 4-MU attenuated the PCA allergic reaction (i.e., less ear thickening and dye extravasation). Similarly, we found that 4-MU decreased body temperature, serum histamine, and IL4 secretion in OVA-challenged ASA model mice. In conclusion, 4-MU had a suppressing effect on MC activation both in vitro and in vivo and thus may represent a new strategy for treating IgE-mediated allergic conditions.

4,8-Sphingadienines (SD), metabolites of glucosylceramides (GlcCer), are sometimes determined as key mediators of the biological activity of dietary GlcCer, and cis/trans geometries of 4,8-SD have been reported to affect its activity. Since regulating excessive activation of mast cells seems an important way to ameliorate allergic diseases, this study was focused on cis/trans stereoisomeric-dependent inhibitory effects of 4,8-SD on mast cell activation. Degranulation of RBL-2H3 was inhibited by treatment of 4-cis-8-trans- and 4-cis-8-cis-SD, and their intradermal administrations ameliorated ear edema in passive cutaneous anaphylaxis reaction, but 4-trans-8-trans- and 4-trans-8-cis-SD did not. Although the activation of mast cells depends on the bound IgE contents, those stereoisomers did not affect IgE contents on RBL-2H3 cells after the sensitization of anti-TNP IgE. These results indicated that 4-cis-8-trans- and 4-cis-8-cis-SD directly inhibit the activation of mast cells. In conclusion, it was assumed that 4,8-SD stereoisomers with cis double bond at C4-position shows anti-allergic activity by inhibiting downstream pathway after activation by the binding of IgE to mast cells.

The calcium-binding protein S100A4 demonstrates important regulatory roles in many biological processes including tumorigenesis and inflammatory disorders such as allergy. However, the specific mechanism of the contribution of S100A4 to allergic diseases awaits further clarification.
To address the effect of S100A4 on the regulation of mast cell activation and its impact on allergy.
Bone marrow-derived cultured mast cells (BMMCs) were derived from wild-type (WT) or S100A4-/- mice for in vitro investigation. WT and S100A4-/- mice were induced to develop a passive cutaneous anaphylaxis (PCA) model, a passive systemic anaphylaxis (PSA) model, and an ovalbumin (OVA)-mediated mouse asthma model.
Following OVA/alum-based sensitization and provocation, S100A4-/- mice demonstrated overall suppressed levels of serum anti-OVA IgE and IgG antibodies and proinflammatory cytokines in serum, bronchoalveolar lavage fluid (BALF), and lung exudates. S100A4-/- mice exhibited less severe asthma signs which included inflammatory cell infiltration in the lung tissue and BALF, and suppressed mast cell recruitment in the lungs. Reduced levels of antigen reencounter-induced splenocyte proliferation in vitro were recorded in splenocytes from OVA-sensitized and challenged mice that lacked S100A4-/-. Furthermore, deficiency in the S100A4 gene could dampen mast cell activation both in vitro and in vivo, evidenced by reduced β-hexosaminidase release and compromised PCA and PSA reaction. We also provided evidence supporting the expression of S100A4 by mast cells.
S100A4 is required for mast cell functional activation, and S100A4 may participate in the regulation of allergic responses at least partly through regulating the activation of mast cells.

Allergy is an excessive immune response to a specific antigen. Type I allergies, such as hay fever and food allergies, have increased significantly in recent years and have become a worldwide problem. We previously reported that an ascorbic acid derivative having palmitoyl and glucosyl groups, 2-O-α-d-glucopyranosyl-6-O-hexadecanoyl-l-ascorbic acid (6-sPalm-AA-2G), showed inhibitory effects on degranulation in vitro and on the passive cutaneous anaphylaxis (PCA) reaction in mice. In this study, several palmitoyl derivatives of ascorbic acid were synthesized and a structure-activity relationship study was performed to discover more potent ascorbic acid derivatives with degranulation inhibitory activity. 6-Deoxy-2-O-methyl-6-(N-hexadecanoyl)amino-l-ascorbic acid (2-Me-6-N-Palm-AA), in which a methyl group was introduced into the hydroxyl group at the C-2 position of ascorbic acid and in which the hydroxyl group at the C-6 position was substituted with an N-palmitoyl group, exhibited much higher inhibitory activity for degranulation in vitro than did 6-sPalm-AA-2G. 2-Me-6-N-Palm-AA strongly inhibit the PCA reaction in mice at lower doses than those of 6-sPalm-AA-2G. These findings suggest that 2-Me-6-N-Palm-AA may be a promising therapeutic candidate for allergic diseases.

Japanese Cedar (JC) pollinosis is the most common seasonal allergic rhinitis in Japan. Throughout the JC pollen season, patients suffer from the allergic symptoms, resulting in a reduction of quality of life. Allergy immunotherapy (AIT) is an established treatment option for a wide range of allergens that unlike symptomatic treatments (e.g. antihistamines) may provide sustained immune tolerance. However, AIT, especially subcutaneous immunotherapy (SCIT) has a fatal anaphylaxis risk due to the use of crude allergen extracts. Consequently, development of allergen derivatives with substantially reduced anaphylactic potential is desirable. An allergen derivative that showed reduced IgE-binding and anaphylactic potential was developed through conjugation of native Cry j 1 (n Cry j 1), a major JC allergen, to the polysaccharide pullulan followed by chemical but non-covalent denaturation. The resulting Cry j 1 allergen derivative, Dn p-Cry j 1, showed reduced IgE-binding and IgE-mediated effector cell activation in vitro using an ELISA competition assay and a mast cell activation model (EXiLE). Reduced anaphylactic potential of Dn p-Cry j 1 in vivo was demonstrated using the rat passive cutaneous anaphylaxis (PCA) assay. The difference in anaphylactic potential of Dn p-Cry j 1 compared to n Cry j 1 in wild-type rats was of the same magnitude as the difference seen in the anaphylaxis reactions obtained with n Cry j 1 in wild-type rats and mast-cell deficient rats, indicating a dramatic reduction in anaphylactic potential of Dn p-Cry j 1. These results indicate that Dn p-Cry j 1 is a promising candidate for next-generation JC AIT.

Blocking the major cat allergen, Fel d 1, with mAbs was effective in preventing an acute cat allergic response.
This study sought to extend the allergen-specific antibody approach and demonstrate that a combination of mAbs targeting Bet v 1, the immunodominant and most abundant allergenic protein in birch pollen, can prevent the birch allergic response.
Bet v 1-specific mAbs, REGN5713, REGN5714, and REGN5715, were isolated using the VelocImmune platform. Surface plasmon resonance, x-ray crystallography, and cryo-electron microscopy determined binding kinetics and structural data. Inhibition of IgE-binding, basophil activation, and mast cell degranulation were assessed via blocking ELISA, flow cytometry, and the passive cutaneous anaphylaxis mouse model.
REGN5713, REGN5714, and REGN5715 bind with high affinity and noncompetitively to Bet v 1. A cocktail of all 3 antibodies, REGN5713/14/15, blocks IgE binding to Bet v 1 and inhibits Bet v 1- and birch pollen extract-induced basophil activation ex vivo and mast cell degranulation in vivo. Crystal structures of the complex of Bet v 1 with immunoglobulin antigen-binding fragments of REGN5713 or REGN5715 show distinct interaction sites on Bet v 1. Cryo-electron microscopy reveals a planar and roughly symmetrical complex formed by REGN5713/14/15 bound to Bet v 1.
These data confirm the immunodominance of Bet v 1 in birch allergy and demonstrate blockade of the birch allergic response with REGN5713/14/15. Structural analyses show simultaneous binding of REGN5713, REGN5714, and REGN5715 with substantial areas of Bet v 1 exposed, suggesting that targeting specific epitopes is sufficient to block the allergic response.

UNASSIGNED: Huoxiangzhengqi oral liquid (HXZQ-OL), a traditional Chinese medicine formula, has antibacterial, anti-inflammation and gastrointestinal motility regulation effects.
UNASSIGNED: The study investigates the anti-allergic activity and underlying mechanism of HXZQ-OL.
UNASSIGNED: IgE/Ag-mediated RBL-2H3 cells were used to evaluate the anti-allergic activity of HXZQ-OL (43.97, 439.7 and 4397 μg/mL) in vitro. The release of cytokines and eicosanoids were quantified using ELISA. RT-qPCR was used to measure the gene expression of cytokines. The level of intracellular Ca2+ was measured with Fluo 3/AM. Immunoblotting analysis was performed to investigate the mechanism of HXZQ-OL. In the passive cutaneous anaphylaxis (PCA), BALB/c mice (5 mice/group) were orally administrated with HXZQ-OL (263.8, 527.6 and 1055 mg/kg/d) or dexamethasone (5 mg/kg/d, positive control) for seven consecutive days.
UNASSIGNED: HXZQ-OL not only inhibited degranulation of mast cells (IC50, 123 μg/mL), but also inhibited the generation and secretion of IL-4 (IC50, 171.4 μg/mL), TNF-α (IC50, 88.4 μg/mL), LTC4 (IC50, 52.9 μg/mL) and PGD2 (IC50, 195.8 μg/mL). Moreover, HXZQ-OL suppressed the expression of IL-4 and TNF-α mRNA, as well as the phosphorylation of Fyn, Lyn and multiple downstream signalling proteins including MAPK and PI3K/NF-κB pathways. In addition, HXZQ-OL (527.5 mg/kg) attenuated the IgE-mediated PCA with 55% suppression of Evans blue exudation in mice.
UNASSIGNED: HXZQ-OL attenuated the activation of mast cell and PCA. Therefore, HXZQ-OL might be used as an alternative treatment for allergic diseases.