Mast Cell Degranulation Assays Research Articles

Study on the Sensitization and Antigenic Epitopes of Tropomyosin from Antarctic Krill (Euphausia superba).

Antarctic krill (Euphausia superba), a shrimp-like marine crustacean, has become a beneficial source of high-quality animal protein. Meanwhile, a special focus has been placed on its potential sensitization issue. In this study, a 35 kDa protein was purified and identified to be Antarctic krill tropomyosin (AkTM) by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). The purified TM showed a strong IgE-binding capacity to shrimp/crab-allergic patients' sera, indicating that TM is the primary allergen in Antarctic krill. Simulated gastrointestinal digestion revealed that the digestion stability of TM to pepsin was higher than that to trypsin. The strong degranulation triggered by TM in RBL-2H3 cells suggested that AkTM has a strong sensitization capacity. The TM-sensitized BALB/c mice displayed severe anaphylactic symptoms; high levels of TM-specific IgE, sIgG1, and histamine; and increased IL-4, indicating that AkTM could provoke IgE-mediated allergic reactions. Bioinformatics prediction, indirect competition ELISA, and mast cell degranulation assay were used to map the antigenic epitopes of AkTM. Finally, nine peptides of T43-58, T88-101, T111-125, T133-143, T144-155, T183-197, T223-236, T249-261, and T263-281 were identified as the linear epitopes of AkTM. The findings may help us develop efficient food processing techniques to reduce krill allergy and gain a deeper comprehension of the allergenicity of krill allergens.

Molecular Cloning and Characterization of a Novel Antimicrobial Peptide from the Skin of Kaloula pulchra.

Bacterial resistance to all currently available conventional antibiotics has caused a global public health crisis and led to an imperative search for new agents. Antimicrobial peptides (AMPs) are essential components of host innate immune defense against microbial invasions. The objective of this study was to report a novel AMP, brevinin-2KP, from the skin of the black Kaloula pulchra frog and describe its structural and biological characterization. The physical and chemical parameters of brevinin-2KP were predicted with the ExPASy Bioinformatics Resource Portal. The assembled sequences were aligned with ClustalW, and the phylogenetic tree was constructed using Mega. Circular dichroism (CD) experiments were carried out to identify the secondary structure and the stability of peptide in different solvent environments. The cytotoxicity of brevinin-2KP was evaluated by the MTT test. To determine antibacterial activity of brevinin- 2KP, a standard two-fold broth dilution method was used. SEM was carried out to observe the morphological change in the bacterial treated by brevinin-2KP. The live/dead bacterial viability was measured with a LIVE/DEAD® BacLight kit. Histamine release and mast cell degranulation assays were performed. The precursor of brevinin-2KP contains 72 amino acid residues, including a conserved signal peptide, acidic propeptide with KR residues, and mature peptide with a sequence of GVITDALKGAAKTVAAELLKKAHCKLTNSC. Phylogenetic analysis based on the amino acid sequences of 34 brevinin-2 peptides from 30 anuran species demonstrates that K. pulchra is genetically closely related to the genus Hylarana. The CD spectra analysis indicates that brevinin-2KP adopts random coil in the water and an organized α-helical conformation in SDS solution. Further, this secondary structure is stable under high salt and high-temperature conditions. Brevinin-2KP is weakly active towards the tested Gram-positive and Gram-negative bacteria as well as fungi due to its membranolytic action. Moreover, brevinin-2KP inhibits the proliferation of several mammal cells with IC50 values ranging from 3.27 to 59.75 μM. In addition, brevinin-2KP promotes degranulation and histamine release of mast cells, indicating that it is involved in the inflammatory response. This is the first report on AMP identified from the skin of K. pulchra. Brevinin-2KP adopts a typical amphipathic α-helix conformation in membrane mimic environment and shows antimicrobial and antitumor activities by potential membranolytic mechanism. In addition, brevinin-2KP can promote degranulation and histamine release of mast cells. Brevinin-2KP is expected to become a good drug temple molecule.

Protease inhibitor from Libidibia ferrea seeds attenuates inflammatory and nociceptive responses in mice

Ethnopharmacological relevanceLibidibia ferrea (Mart. ex. Tul.) L.P. Queiroz is a Brazilian native tree locally known as jucá and pau-ferro, and it has been used in folk medicine for relieving, asthma, bronchitis, sore throat, rheumatism, enterocolitis and fever. The anti-inflammatory properties of L. ferrea were confirmed for its stem, fruit, leaves, bark and seeds extracts, however little is known about the natural compounds that may be associated with that response. Aim of this studyIn a normal physiological condition, many enzymes play an important role in catalyzing biological functions. Among them, proteases are of great interest. Although they take part of many biological systems, as the inflammatory process, when deregulated, proteases may cause system malfunctions, such as under- or overproduction of cytokines, or immune cells activation. Thus, protease inhibitors prevent these immune responses by regulating proteases. The objective of this study was to evaluate the anti-inflammatory and anti-nociceptive response of a protease inhibitor purified from L. ferrea seeds (LfTI). Materials and methodsIn vitro (5, 50 and 250 μg/mL of LfTI) and in vivo (0.6, 3 e 15 mg/kg of LfTI) assays were performed. Male Swiss mice weighing 18–25 g were used for cell harvesting and for the in vivo assays. The anti-inflammatory activity was analyzed in vitro by macrophage cytotoxicity, hydrogen peroxide (H2O2) production, and cell adhesion assays; and in vivo by leukocyte recruitment, nitric oxide (NO) production, vascular permeability, paw edema and mast cell degranulation assays. The anti-nociceptive activity was evaluated through abdominal writhing test induced by acetic acid and formalin sensitization. ResultsOur results showed that, in vitro, LfTI is not cytotoxic. Also, LfTI (50 μg/mL) inhibited macrophage H2O2 production (48.2%), and adhesion (48.4%). LfTI (0.6, 3 e 15 mg/kg) decreased polymorphonuclear cell recruitment dose-dependently, and it inhibited NO production (53%), vascular permeability (40.7%) and paw edema at 3 mg/kg at different time, but it did not inhibit mast cell degranulation. Besides, LfTI did not inhibit either the number of writhing or the licking time in the formalin test in the second phase (inflammatory). However, LfTI (3 mg/kg) inhibited licking time at the first phase (neurogenic) in the formalin sensitization (46.1%). ConclusionsOur results show that LfTI has anti-inflammatory and antinociceptive (neurogenic pain) effects, and these effects might be associated with the inhibition of inflammatory proteases and/or protease-activated receptors activation hindering.

IgE-Induced Mast Cell Activation Is Suppressed by Dihydromyricetin through the Inhibition of NF-κB Signaling Pathway.

Mast cells play a crucial role in the pathogenesis of type 1 allergic reactions by binding to IgE and allergen complexes and initiating the degranulation process, releasing pro-inflammatory mediators. Recently, research has focused on finding a stable and effective anti-allergy compound to prevent or treat anaphylaxis. Dihydromyricetin (DHM) is a flavonoid compound with several pharmacological properties, including free radical scavenging, antithrombotic, anticancer, and anti-inflammatory activities. In this study, we investigated the anti-allergic inflammatory effects and the underlying molecular mechanism of DHM in the DNP-IgE-sensitized human mast cell line, KU812. The cytokine levels and mast cell degranulation assays were determined by enzyme-linked immunosorbent assay (ELISA). The possible mechanism of the DHM-mediated anti-allergic signaling pathway was analyzed by western blotting. It was found that treatment with DHM suppressed the levels of inflammatory cytokines TNF-α and IL-6 in DNP-IgE-sensitized KU812 cells. The anti-allergic inflammatory properties of DHM were mediated by inhibition of NF-κB activation. In addition, DHM suppressed the phosphorylation of signal transducer and activator of transcription 5 (STAT5) and mast cell-derived tryptase production. Our study shows that DHM could mitigate mast cell activation in allergic diseases.

An Ex vivo Mast Cell Degranulation Assay using Crude Peritoneal Exudate Cells and Natural Antigen Stimulation.

Mast cell stabilizers are an essential part of allergy medication. Passive systemic anaphylaxis (PSA) is an animal assay widely used for investigating the effect of a pharmacological agent of interest on mast cells in vivo. As the anaphylactic symptoms are primarily attributed to exocytosis of the granules from mast cells, it is conceived that the agent to cause amelioration of the symptoms has a mast cell stabilizing activity. Despite the fact, it is prudent to confirm the activity by directly demonstrating the decline in the functional activity of mast cells following its treatment. In vitro degranulation assays using an immortalized mast cell line or cultured primary mast cells are routinely employed to that end. The results from the in vitro and in vivo assays may not always be akin to each other; however, as treatment conditions (e.g., treatment dose, time, surrounding environments) for the in vitro assays are often distinct from those for the in vivo assay such as PSA. In pursuit of an in vitro (or ex vivo) assay to reflect more closely the effect of a pharmacological agent on mast cells in vivo, we devised the ex vivo mast cell degranulation assay in which crude peritoneal exudate cells (PECs) isolated from the mice, treated with the agent and administered anti-dinitrophenol (DNP) IgE, were incubated directly with DNP on a carrier protein. It turned out that the assay was not only useful in validating the mast cell stabilizing activity of a pharmacological agent indicated by the in vivo assay but also practical and highly reproducible.

MRGPRX2 mediates immediate-type pseudo-allergic reactions induced by iodine-containing iohexol

BackgroundIohexol is a typical iodinated radiocontrast medium and widely used in clinical angiography. Hypersensitivity reactions induced by iohexol are common side effects known to increase the risk for patients. Iodine is the main functional group of iohexol, and it can induce delayed anaphylaxis. However, iohexol also induces immediate-type allergies, but the underlying mechanism is still not clear. MRGPRX2 is a key receptor present on mast cells, which mediates pseudo-allergic reactions induced by various drugs. MethodsWe aimed to verify the relationship between iohexol-induced anaphylactic reactions and MRGPRX2. MRGPRX2-mediated pseudo-allergic reactions induced by iohexol were investigated in vivo and in vitro using a mouse model of local and systemic anaphylaxis and mast cell degranulation assays, respectively. ResultsIohexol caused pseudo-allergic reactions in wild-type (WT) mice by activating mast cells to release histamine and cytokines. However, it did not induce a similar phenomenon in KitW-sh/W-sh (MUT) mice. Iohexol stimulated intracellular calcium ion (Ca2+) influx in MRGPRX2-HEK293, MrgprB2-HEK293, and LAD2 cells but not in NC-HEK293 cells. After knockdown of MRGPRX2 expression in LAD2 cells, the degree of iohexol-induced degranulation was reduced. In addition, after structural modification of iohexol by removal of iodine, a reduction in iohexol-induced effects, such as local and systemic anaphylaxis in mice and degranulation of LAD2 cells, could be observed. Iohexol was shown to induce immediate-type pseudo-allergic reactions via MRGPRX2, which was dependent on the presence of iodine. ConclusionsConclusively, inhibition of MRGPRX2-mediated mast cell degranulation and cytokine release is important to prevent iohexol-induced immediate-type pseudo-allergic reactions.

Paeoniflorin inhibits IgE-mediated allergic reactions by suppressing the degranulation of mast cells though binding with FcϵRI alpha subunits

Paeoniflorin (PF), a monoterpene glycoside isolated from the aqueous extract of the Chinese herb Radix Paeoniae Alba, has been used for treating various inflammatory diseases. In this study, we aimed to investigate the anti-allergic activities of PF. The anti-anaphylactic activity of PF was investigated using mast cell (MC) degranulation assay as well as Ca2+ influx in vitro and skin swelling and extravasation assays in vivo. The results showed that PF inhibited MC degranulation (histamine release from 74.5 ± 4.95 ng/ml to 58.7 ± 6.06 ng/ml) and Ca2+ influx challenged by DNP-BSA in vitro. In addition, PF reduced the degree of swelling and Evans blue exudation in mice paws. Furthermore, PF dose-dependently reduced serum inflammatory mediator release in mice sensitized with ovalbumin for 48 h by inhibiting MC degranulation. Molecular docking study revealed that PF bound better with the α subunit of FcϵRI than with the β subunit. SPR revealed that PF had a strong affinity interaction with FcϵRI α subunit and the KD value was (7.08 ± 0.97) e−6 M. Our findings revealed that PF inhibited anaphylactic responses in vivo and in vitro, and it can be considered a novel FcϵRI inhibitor for preventing MC-related allergic diseases.

Pharmacological profiling of JME-173, a novel mexiletine derivative combining dual anti-inflammatory/anti-spasmodic functions and limited action in Na+ channels

Existing evidence suggests that the local anaesthetic mexiletine can be beneficial for patients with asthma. However, caution is required since anaesthesia of the airways inhibits protective bronchodilator neuronal reflexes, limiting applications in conditions of hyperirritable airways. Here, we describe the synthesis of a new series of mexiletine analogues, which were screened for reduced activity in Na+ channels and improved smooth muscle relaxant effects, that were evaluated using the patch-clamp technique and an isolated tracheal organ bath, respectively. JME-173 (1-(4-bromo-3,5-dimethylphenoxy)propan-2-amine) was the most effective among the four mexiletine analogues investigated. JME-173 was then studied in vivo using a murine model of lung inflammation induced by cigarette smoke (CS) and in vitro using neutrophil chemotaxis and mast cell degranulation assays. Finally, the JME-173 pharmacokinetic profile was assessed using HPLC-MS/MS bioanalytical method. JME-173 directly inhibited IL-8 (CXCL8)- and FMLP-induced human neutrophil chemotaxis and allergen-induced mast cell degranulation. After oral administration 1 h before CS exposure, JME-173 (50 mg/kg) strongly reduced the increased number of macrophages and neutrophils recovered in the bronchoalveolar effluent without altering lymphocyte counts. Pharmacokinetic experiments of JME-173 (10 mg/kg, orally) showed values of maximum concentration (Cmax), maximum time (Tmax), area under the blood concentration-time curve (AUC0-t) and area under the blood concentration-time curve from 0–Inf (AUC0-inf) of 163.3 ± 38.3 ng/mL, 1.2 ± 0.3 h, 729.4 ± 118.3 ng*h/ml and 868.9 ± 117.1 ng*h/ml (means ± S.E.M.), respectively. Collectively, these findings suggest that JME-173 has the potential to be an effective oral treatment for diseases associated with bronchoconstriction and inflammation.

344 Thimerosal induces skin pseudo-allergic reaction via Mas-related G-protein coupled receptor B2

Thimerosal has been used as a preservative in a variety of products which may cause contact dermatitis. It is the second most common allergen in positive patch test reactions, though being a clinical irrelevant allergen. It is believed that thimerosal may cause contact dermatitis through delayed-type hypersensitivity response. However, recent studies have demonstrated the association between Mas-related G protein coupled receptor X2 (MRGPRX2) on mast cells and pseudo-allergic reaction. It is possible that thimerosal may cause contact dermatitis via MRGPRX2 mediated mechanism. To investigate the role of MRGPRX2/MrgprB2 in contact dermatitis induced by thimerosal, we developed a novel skin pseudo-allergic reaction mouse model, footpad swelling and extravasation assays in vivo and mast cell degranulation assay in vitro were studied. The results showed that thimerosal induced contact dermatitis in dorsal skin and footpad swelling in wild-type mice, but had no significant effect in MrgprB2-knockout mice. Thimerosal-induced dermatitis is characterized by infiltration of inflammatory cells and elevation of serum histamine and inflammatory cytokines, rather than elevation of serum IgE level. Moreover, thimerosal increased the intracellular Ca2+ concentration in HEK293 cells overexpressing MrgprB2/MRGPRX2. Downregulation of MRGPRX2 resulted in the reduced degranulation of human mast cells from LAD2 cell line. Collectively, we demonstrate that MrgprB2 mediated thimerosal-induced mast cell degranulation and pseudo-allergic reaction in mice, thimerosal also activated human mast cell via MRGPRX2, indicating that MRGPRX2 may be a key contributor to human contact dermatitis.

Cloning, Expression, and Epitope Identification of Myosin Light Chain 1: An Allergen in Mud Crab

Mud crab (Scylla paramamosain) is a commonly consumed seafood as a result of its high nutritional value; however, it is associated with food allergy. The current understanding of crab allergens remains insufficient. In the present study, an 18 kDa protein was purified from crab muscle and confirmed to be myosin light chain 1 (MLC1) by matrix-assisted laser desorption/ionization-tandem time-of-flight mass spectrometry. Total RNA was isolated and amplified to obtain a MLC1 open reading frame of 462 bp, encoding 154 amino acids. A structural analysis revealed that recombinant MLC1 (rMLC1) expressed in Escherichia coli contained α-helix and random coil. Moreover, rMLC1 displayed strong immunoactivity by dot blot and a basophil activation test. Furthermore, seven allergenic epitopes of MLC1 were predicted, and five critical epitope regions were identified by an inhibition enzyme-linked immunosorbent assay and human mast cell degranulation assay. This comprehensive research of an allergen helps to conduct component-resolved diagnoses and immunotherapies related to crab allergies.

Thimerosal induces skin pseudo-allergic reaction via Mas-related G-protein coupled receptor B2

BackgroundThimerosal has been used as a preservative in many products which may cause contact dermatitis. It is the second most common allergen in positive patch test reactions, though being a clinical irrelevant allergen. Thimerosal-induced contact dermatitis is generally considered to be a delayed-type hypersensitivity reaction, but it is difficult to explain the fact that most patients develop an allergic reaction upon first encounter with thimerosal. Recent studies have demonstrated the association between Mas-related G protein coupled receptor X2 (MRGPRX2) and pseudo-allergic reactions which occur at the first contact with stimulation. This suggests the possibility that thimerosal may cause contact dermatitis via MRGPRX2 mediated mechanism. ObjectivesTo investigate the role of Mas-related G-protein coupled receptor B2 (MrgprB2)/MRGPRX2 in contact dermatitis induced by thimerosal. MethodsThimerosal induced pseudo-allergic reactions via MrgprB2/ MRGPRX2 were investigated using a novel skin pseudo-allergic reaction mouse model, footpad swelling and extravasation assays in vivo and mast cell degranulation assay in vitro. ResultsThimerosal induced contact dermatitis in dorsal skin and footpad swelling in wild-type mice, but had no significant effect in MrgprB2-knockout mice. Thimerosal-induced dermatitis is characterized by infiltration of inflammatory cells and elevation of serum histamine and inflammatory cytokines, rather than elevation of serum IgE level. Thimerosal increased the intracellular Ca2+ concentration in HEK293 cells overexpressing MrgprB2/MRGPRX2. Downregulation of MRGPRX2 resulted in the reduced degranulation of LAD2 human mast cells. ConclusionsMrgprB2 mediates thimerosal-induced mast cell degranulation and pseudo-allergic reaction in mice. MRGPRX2 may be a key contributor to human contact dermatitis.

Reducing Allergenicity to Arginine Kinase from Mud Crab Using Site-Directed Mutagenesis and Peptide Aptamers.

The mud crab ( Scylla paramamosain) is widely consumed but can cause a severe food allergic reaction. To reduce allergenicity to arginine kinase (AK), site-directed mutagenesis was used to destroy disulfide bonds or mutate critical amino acids of conformational epitopes. Three hypoallergenic mutant AKs (mAK1, mAK2, and mAK3) were generated, with the immunoreactivity decreasing by 54.2, 40.1, and 71.4%, respectively. In comparison to recombinant AK (rAK), the structure of mAKs was clearly changed. Additionally, antisense peptides were designed on the basis of linear epitopes and pepsin-cutting sites of AK. Five peptide aptamers were screened by molecular docking and then analyzed by the immunoglobulin E inhibition enzyme-linked immunosorbent assay and human Laboratory of Allergic Diseases 2 mast cell degranulation assay. The peptide aptamers could significantly inhibit allergenicity of rAK and mAKs, and the inhibitory effect of peptide aptamer 3 was slightly better than the others. These results provide synergistic methods to reduce allergenicity to AK, which could be applied to other shellfish allergens.

Isosalvianolic acid C-induced pseudo-allergic reactions via the mast cell specific receptor MRGPRX2

Pseudo-allergic reactions occurred in patients administered drugs for the first time, seriously threaten man's survival. Due to the frequent reports of pseudo-allergic reactions to Danshen injection, in our previous study, isosalvianolic acid C in Danshen injection was found to trigger off mast cell degranulation. However, the direct involvement and the mechanisms underlying pseudo-allergic reactions have not been elucidated. In this study, the pseudo-allergic reactions induced by isosalvianolic acid C were confirmed by an ear swelling assay, a hindpaw swelling and extravasation assay in vivo and mast cell degranulation assays in vitro. We also evaluated whether the pseudo-allergic effect is related to MRGPRX2, Isosalvianolic acid C induced Ca2+ mobilization was verified as MRGPRX2-related by Ca2+ imaging using mouse peritoneal mast cells (both wild-type and MrgprB2 knockout mice), MRGPRX2-expressing HEK293 and MrgprB2-expressing HEK293 cells. MRGPRX2-related pseudo-allergic reactions induced by Isosalvianolic acid C were further confirmed by MrgprB2 knockout mice and MRGPRX2 knockdown mast cells both exhibited reduced isosalvianolic acid C-induced pseudo-allergic effects. Furthermore, both the frontal analysis and molecular docking assays showed that isosalvianolic acid C has a considerable affinity with MRGPRX2. Based on the above experiments, the western blot analyses were conducted, the results indicated that isosalvianolic acid C induced Ca2+ mobilization and degranulation via the activation of PLC-γ and IP3R, and releasing chemokines via the activation of PLC-γ, PKC and P38. This study should alarm many clinicians that medicines containing isosalvianolic acid C might induce pseudo-allergic reactions, and it may provide guidance on safe dosage of these medicines in the process of production and use.

Mast cell-mediated hypersensitivity to fluoroquinolone is MRGPRX2 dependent

Fluoroquinolones trigger anaphylaxis during clinical applications, affecting the safety of their administration. Mast cells are immune cells that act as sentinels during host defenses, mediating hypersensitivity and anaphylactic reactions. Mas-related G protein-coupled receptor X2 (MRGPRX2) is a mast cell-specific receptor that mediates cell degranulation in anaphylactic reactions. In this study, the mechanism underpinning the anaphylactic reactions caused by fluoroquinolones was investigated. Hypersensitivity was assessed through hindpaw swelling, tissue fluid leakage assays, in vivo and body temperature measurements assay in vivo, and cell calcium mobilization assays, and mast cell degranulation assays in vitro. Mast cell-deficient W-sash c-kit mutant KitW-sh/W-sh mice and MrgprB2 (the orthologous receptor of MRGPRX2 in mice) knockout mice exhibited reduced fluoroquinolone-induced anaphylactic effects. Fluoroquinolones activated mast cells in a dose-dependent manner and reduced degranulation was observed following MRGPRX2 silencing. These results reveal that fluoroquinolone-induced anaphylactic reactions are mediated by mast cells through MRGPRX2.

The discovery of quinoline-3-carboxamides as hematopoietic prostaglandin D synthase (H-PGDS) inhibitors.

With the goal of discovering more selective anti-inflammatory drugs, than COX inhibitors, to attenuate prostaglandin signaling, a fragment-based screen of hematopoietic prostaglandin D synthase was performed. The 76 crystallographic hits were sorted into similar groups, with the 3-cyano-quinoline 1a (FP IC50 = 220,000 nM, LE = 0.43) being a potent member of the 6,6-fused heterocyclic cluster. Employing SAR insights gained from structural comparisons of other H-PGDS fragment binding mode clusters, the initial hit 1a was converted into the 70-fold more potent quinoline 1d (IC50 = 3,100 nM, LE = 0.49). A systematic substitution of the amine moiety of 1d, utilizing structural information and array chemistry, with modifications to improve inhibitor stability, resulted in the identification of the 300-fold more active H-PGDS inhibitor tool compound 1bv (IC50 = 9.9 nM, LE = 0.42). This selective inhibitor exhibited good murine pharmacokinetics, dose-dependently attenuated PGD2 production in a mast cell degranulation assay and should be suitable to further explore H-PGDS biology.

Dehydroandrographolide inhibits IgE-mediated anaphylactic reactions via calcium signaling pathway

The classical mast cells degranulation pathway is mediated by FcεRI aggregation and varies in strength among subjects. Dehydroandrographolide (DA) is one of principal components of Andrographis paniculata (Burm.f.) Nees (family: Acanthaceae) and considered the main contributors of its therapeutic properties, such as anti-tumor. In this study, inhibition of IgE-mediated anaphylactic reactions and anti-inflammatory potential of DA were investigated. The anti-anaphylactic activity of DA was investigated using skin swelling and extravasation assays in vivo and mast cell degranulation assay in vitro. The release of cytokines was measured using ELISA kits. Human Phospho-Kinase Array kit and western blotting were used to explore the related molecular signaling pathways. DA inhibited IgE-mediated mast cell activation, including degranulation and release of cytokines in vitro. Moreover, DA reduced the degree of swelling and Evans blue exudation of mice paw in a dose-dependent manner by inhibiting mast cell degranulation. DA obviously reduced the concentrations of histamine, TNF-α, MCP-1, IL-8, IL-13, and IL-4 in mice serum and inhibited IgE-mediated anaphylactic reactions as a potential P-PLCγ inhibitor. Our study reveals that DA can inhibit allergic responses in vivo and in vitro, and it may be regarded as a novel P-PLCγ inhibitor for preventing mast cell-immediate and delayed allergic diseases.

Mivacurium induce mast cell activation and pseudo-allergic reactions via MAS-related G protein coupled receptor-X2

BackgroundMivacurium is a non-depolarizing muscle relaxant and widely used as a short-acting anesthetic. Pseudo-allergic reactions to mivacurium occur when it is administered during perioperative anesthesia. These reactions may present a serious threat to the patient’s life, particularly in children. MethodsMAS-related G protein coupled receptor-related pseudo-allergic reactions that were induced by mivacurium were investigated using skin swelling and extravasation assays in vivo and mast cell degranulation assay in vitro. ResultsMivacurium caused pseudo-allergic reactions in wild-type mice by inducing mast cells to release histamine. However, it did not induce a similar phenomenon in KitW-sh/W-sh mice. Furthermore, MrgprB2-knockout mice displayed no inflammatory response to mivacurium. Mivacurium induced LAD2 cell degranulation in a dose-dependent manner. Mivacurium stimulated intracellular calcium ion (Ca2+) influx in MRGPRX2-HEK293 cells but not in NC-HEK293 cells. However, mivacurium induced the release of only low levels of mediators in LAD2 cells transfected with MRGPRX2-targeted small interfering (si)RNA. Notably, cytokine release was not observed in LAD2 cells even when stimulated with high concentrations of mivacurium. ConclusionMivacurium activated MRGPRX2 and triggered mast cell degranulation, leading to anaphylactoid reactions. However, mivacurium did not induce the release of other cytokines. Therefore, the targeting of MRGPRX2 can potentially block mivacurium-induced adverse drug effects, particularly pseudo-allergic reactions.

Cisatracurium induces mast cell activation and pseudo-allergic reactions via MRGPRX2

BackgroundPseudo-allergic reactions occur when patients receive muscle relaxants during perioperative anesthesia. These reactions may result in a serious threat to the patient's life, particularly to a child's life. Cisatracurium, a relatively new NMBA, has resulted in bronchospasms and cardiovascular collapse. However, the mechanisms underlying the anaphylactoid reactions caused by cisatracurium have not been fully elucidated. MethodsIn the present study, the MRGPRX2-related pseudo-allergic reactions induced by cisatracurium were investigated using hindpaw swelling and extravasation assays in vivo and mast cell degranulation assays. ResultsCisatracurium caused anaphylactoid reactions in wild-type mice. However, cisatracurium did not induce a similar phenomenon in KitW-sh/W-sh mice. Furthermore, mast cell-related G protein-coupled receptor B2-knockout mice did not display an inflammatory response upon treatment with cisatracurium. Cisatracurium induced LAD2 cell degranulation, leading to the dose-dependent release of β-hexosaminidase, histamine and TNF-α. However, cisatracurium only induced the release of low levels of these mediator LAD2 cells transfected with MRGPRX2 siRNA. Cisatracurium also stimulated intracellular Ca2+ influx in MRGPRX2-HEK293 cells compared with that in NC-HKE293 cells. Interestingly, cytokine release was not observed in LAD2 cells even with high dose of cisatracurium. ConclusionsCisatracurium activated MRGPRX2 and triggered mast cell degranulation, leading to anaphylactoid reactions. Therefore, strategies targeting MRGPRX2 might potentially block cisatracurium-induced pseudo-allergic reactions.

Identification of Allergenic Epitopes and Critical Amino Acids of Major Allergens in Chinese Shrimp ( Penaeus chinensis) by Immunoinformatics Coupled with Competitive-Binding Strategy.

Chinese shrimp ( Penaeus chinensis) is widely cultured and consumed in Asia but is also a major food allergen locally. Although they may be the foundation for preventing and treating allergies, the allergenic epitopes of the major allergens tropomyosin (TM) and arginine kinase (AK) in Penaeus chinensis have not been identified. Here, we applied Immunoinfo-CB (immunoinformatics coupled with competitive-binding strategy) to address the point. Potential allergenic epitopes of TM and AK were predicted by multiple immunoinformatics tools, followed by validating with inhibitory dot-blot assay, indirect competition ELISA, and mast cell degranulation assay. Furthermore, critical amino acids in allergenic epitopes were also identified by Immunoinfo-CB. Our findings provide new insight into allergenic epitopes and critical amino acids of TM and AK responsible for the anaphylactic response. The Immunoinfo-CB therefore offers promises for characterization of IgE-binding epitopes that might be used as new targets for immunotherapy of food allergy.

Human mast cells release the migraine-inducing factor pituitary adenylate cyclase-activating polypeptide (PACAP).

Background Many patients with migraines suffer from allergies and vice versa, suggesting a relationship between biological mechanisms of allergy and migraine. It was proposed many years ago that mast cells may be involved in the pathophysiology of migraines. We set out to investigate the relationship between mast cell activation and known neurogenic peptides related to migraine. Methods Cultured human mast cells were assayed for the presence of neuropeptides and their receptors at the RNA and protein level. Immunohistochemistry analyses were performed on tissue resident and cultured mast cells. Mast cell degranulation assays were performed and pituitary adenylate cyclase-activating polypeptide (PACAP) activity was measured with a bioassay. Results We found that cultured and tissue resident human mast cells contain PACAP in cytoplasmic granules. No other neurogenic peptide known to be involved in migraine was detected, nor did mast cells express the receptors for PACAP or other neurogenic peptides. Furthermore, mast cell degranulation through classic IgE-mediated allergic mechanisms led to the release of PACAP. The PACAP released from mast cells was biologically active, as demonstrated using PACAP receptor reporter cell lines. We confirmed existing literature that mast cell degranulation can also be induced by several neurogenic peptides, which also resulted in PACAP release. Conclusion Our data provides a potential biological explanation for the association between allergy and migraine by demonstrating the release of biologically active PACAP from mast cells.