Release Of Allergic Mediators Research Articles

Microtubule nucleation in mouse bone marrow-derived mast cells is regulated by the concerted action of GIT1/βPIX proteins and calcium.

Ag-mediated activation of mast cells initiates signaling events leading to Ca(2+) response, release of allergic mediators from cytoplasmic granules, and synthesis of cytokines and chemokines. Although microtubule rearrangement during activation has been described, the molecular mechanisms that control their remodeling are largely unknown. Microtubule nucleation is mediated by complexes that are formed by γ-tubulin and γ-tubulin complex proteins. In this study, we report that, in bone marrow-derived mast cells (BMMCs), γ-tubulin interacts with p21-activated kinase interacting exchange factor β (βPIX) and G protein-coupled receptor kinase-interacting protein (GIT)1. Microtubule regrowth experiments showed that the depletion of βPIX in BMMCs stimulated microtubule nucleation, whereas depletion of GIT1 led to the inhibition of nucleation compared with control cells. Phenotypic rescue experiments confirmed that βPIX and GIT1 represent negative and positive regulators of microtubule nucleation in BMMCs, respectively. Live-cell imaging disclosed that both proteins are associated with centrosomes. Immunoprecipitation and pull-down experiments revealed that an enhanced level of free cytosolic Ca(2+) affects γ-tubulin properties and stimulates the association of GIT1 and γ-tubulin complex proteins with γ-tubulin. Microtubule nucleation also was affected by Ca(2+) level. Moreover, in activated BMMCs, γ-tubulin formed complexes with tyrosine-phosphorylated GIT1. Further experiments showed that GIT1 and βPIX are involved in the regulation of such important physiological processes as Ag-induced chemotaxis and degranulation. Our study provides for the first time, to our knowledge, a possible mechanism for the concerted action of tyrosine kinases, GIT1/βPIX proteins, and Ca(2+) in the propagation of signals leading to the regulation of microtubule nucleation in activated mast cells.

15,16-Dihydrotanshinone I suppresses IgE-Ag stimulated mouse bone marrow-derived mast cell activation by inhibiting Syk kinase

Ethnopharmacological relevance15,16-Dihydrotanshinone I (DHT-I), isolated from the dried root of Salvia miltiorrhiza Bung, which is traditionally used to treat cardiovascular and inflammatory diseases agent in Chinese medicine. DHT-I has been reported to have a broad range of biological activities, including antibacterial activity, and has been used to treat circulatory disorders, hepatitis, inflammation, cancer, and neurodegenerative diseases. Aim of the studyThe aim of this study was to evaluate the anti-allergic inflammatory effects of DHT-I on degranulation and on the generation of eicosanoids, such as, prostaglandin D2 (PGD2) and leukotriene C4 (LTC4), in IgE/Ag-stimulated bone marrow-derived mast cells (BMMCs). Materials and methodsThe anti-allergic inflammatory activity of DHT-I was evaluated using BMMCs. The effects of DHT-I on mast cell activation were investigated by following degranulation and eicosanoid generation using ELISA and immunoblotting and immunoprecipitation techniques. ResultsDHT-I at a concentration of 20μM markedly inhibited degranulation and the generation of PGD2 and LTC4 in IgE/Ag-stimulated BMMCs (about 90% inhibitions, respectively). Analyses of FcεRI-mediated signaling pathways demonstrated that DHT-I inhibited the phosphorylations of spleen tyrosine kinase (Syk) and linker for activation of T cells (LAT), and inhibited downstream signaling process, including [Ca2+]i mobilization induced by the phosphorylation of phospholipase Cγ1 (PLCγ1), and the activations of mitogen-activated protein kinases (MAPKs) and the Akt-nuclear factor-κB (NF-κB) pathway. ConclusionsDHT-1 inhibits the release of allergic inflammatory mediators from IgE/Ag-stimulated mast cells by suppressing a FcεRI-mediated Syk-dependent signal pathway. This result suggests DHT-I offers a novel developmental basis for drugs targeting allergic inflammatory diseases.

Phaseolin: A 47.5kDa protein of red kidney bean (Phaseolus vulgaris L.) plays a pivotal role in hypersensitivity induction

Red kidney bean (Phaseolus vulgaris L.), a protein rich legume, is consumed globally due to its delicacy. This study was aimed to purify, characterize and assess allergenicity of one of its clinically relevant allergens, later identified as phaseolin. This study was carried out using clinical, in vivo and ex vivo approaches. Phaseolin, an abundant protein of red kidney bean, was purified by column chromatography and reverse-phase-HPLC techniques and characterized by peptide mass fingerprinting. The IgE immunoblotting using red kidney bean allergic patients sera showed phaseolin as a major IgE binding protein of red kidney bean. Phaseolin treated mice demonstrated enhanced levels of specific IgE and IgG1, mouse mast cell protease-1, mRNA expressions of IL-4, IL-5, IL-13 and GATA-3 in the lungs, spleen and intestine along with anaphylactic symptoms indicative of allergic responses. Further, flow cytometry analysis and immunohistochemical studies indicated increased levels of IL-4, IL-5, IL-13 and GATA-3, respectively as compared to controls. The level of Foxp3 was found suppressed in the intestine of phaseolin treated mice when compared to the control. Further, phaseolin treated mice showed positive results in type 1 skin test. Bone marrow derived mast cells (BMMCs) and rat basophilic leukemia (RBL-2H3) cells showed enhanced release of allergic mediators like β-hexosaminidase, histamine, cysteinyl leukotrienes and prostaglandin D2. Taken together, phaseolin was found to possess characteristics of a potential allergen that may lead to hypersensitivity responses in the susceptible individuals and this may be one of the major proteins responsible for allergenicity of red kidney bean.

Twenty‐first century mast cell stabilizers

Mast cell stabilizing drugs inhibit the release of allergic mediators from mast cells and are used clinically to prevent allergic reactions to common allergens. Despite the relative success of the most commonly prescribed mast cell stabilizer, disodium cromoglycate, in use for the preventative treatment of bronchial asthma, allergic conjunctivitis and vernal keratoconjunctivitis, there still remains an urgent need to design new substances that are less expensive and require less frequent dosing schedules. In this regard, recent developments towards the discovery of the next generation of mast cell stabilizing drugs has included studies on substances isolated from natural sources, biological, newly synthesized compounds and drugs licensed for other indications. The diversity of natural products evaluated range from simple phenols, alkaloids, terpenes to simple amino acids. While in some cases their precise mode of action remains unknown it has nevertheless sparked interest in the development of synthetic derivatives with improved pharmacological properties. Within the purely synthetic class of inhibitors, particular attention has been devoted to the inhibition of important signalling molecules including spleen TK and JAK3. The statin class of cholesterol-lowering drugs as well as nilotinib, a TK inhibitor, are just some examples of clinically used drugs that have been evaluated for their anti-allergic properties. Here, we examine each approach under investigation, summarize the test data generated and offer suggestions for further preclinical evaluation before their therapeutic potential can be realized.

Tako-Tsubo cardiomyopathy or allergic acute coronary syndrome: that is the question

We read with great interest the article by Fassio et al. [1] which provided a detailed review on Kounis syndrome (allergic acute coronary syndrome). The authors approached the issue both from the cardiologist’s and the allergist’s view. Authors report that initial descriptions of Kounis syndrome were focused on patients with ‘‘angiographycally’’ normal coronary arteries and acute release of allergic mediators (histamine or leukotrienes); the prognosis of these patients is good, with the left ventricular function normalizing over 3 days to several weeks [2]. However, we recently reported a case (a 70-year-old woman) of transient apical ballooning (Tako-Tsubo syndrome) [3], with mild ST-elevation in anterior leads and increased troponin-I levels [1.09 ng/ml (n.v. 0.03)], triggered by administration of antibiotics (cephalosporin) in a patient with history of asthma and allergy. Tako-Tsubo syndrome is characterized by acute onset of chest symptoms, ECG changes with elevated cardiac markers mimicking acute myocardial infarction, left ventricular wall motion abnormalities in the apical region with preserved function of base, and normal coronary arteries [4]. Those affected are typically older women presenting after a stressful trigger, either emotional or physical. According to authors’ opinion, could there be a link between Kounis syndrome’s variant with ‘‘angiographycally’’ normal coronary arteries and Tako-Tsubo syndrome triggered by administration of drugs in patient with history of asthma and allergy?

Effect of KOB03, a polyherbal medicine, on ovalbumin-induced allergic rhinitis in guinea pigs

BackgroundKOB03 is a polyherbal medicine that originated from the oriental prescription for the treatment of chronic allergic diseases such as rhinitis and asthma. This study aims to evaluate the effect of KOB03 on ovalbumin (OVA)-induced allergic rhinitis (AR) in guinea pigs.MethodsHartley guinea pigs were sensitized to OVA by intraperitoneal injection on days 0, 7, and 14 and challenged with intranasal exposure to OVA three times for 7 days after the last sensitization. KOB03 at doses of 200 and 500 mg/kg were orally administrated to guinea pigs once daily during challenge. The serum levels of histamine, OVA-specific immunoglobulin (Ig) E, eosinophil cationic protein (ECP) and cytokines (TNF-α, IL-4 and IFN-γ) in OVA sensitization/challenge-induced AR guinea pigs were measured. We also observed histological changes in nasal tissues of AR guinea pigs by staining with H&E, Periodic acid-Schiff, and toluidine blue.ResultsThe administration of KOB03 at a dose of 500 mg/kg significantly decreased the serum levels of histamine (P = 0.001), OVA-specific IgE (P = 0.0017), ECP (P = 0.008), and TNF-α (P = 0.0003) in OVA-sensitized/challenged guinea pigs compared with controls. KOB03 significantly decreased the serum levels of a Th2 cytokine, IL-4 (P = 0.017), while significantly increasing the levels of a Th1 cytokine, IFN-γ (P = 0.0006) in OVA-sensitized/challenged guinea pigs compared with controls. In addition, KOB03 suppressed the epithelial destruction, goblet cell hyperplasia and eosinophilic infiltration into nasal mucosa associated with AR.ConclusionKOB03 may regulate allergic inflammation in AR by inhibiting nasal damage, the release of allergic mediators and modulating the balance of Th1/Th2 cytokines.

Sugammadex and rocuronium‐induced anaphylaxis

The study by Clarke et al., designed to examine whether sugammadex might attenuate rocuronium-induced anaphylaxis, appears to conclude prematurely that ‘sugammadex is unlikely to modify significantly the clinical course of an established allergic reaction’ [1]. There have been at least six reports (references available from author) of apparent mitigation, from five countries. Before the introduction of sugammadex, the capacity to remove an allergen from the circulation so easily did not exist, and the implications for the future of allergy management with inclusion complexes (not just for rocuronium) are significant. Within the presently accepted understanding of allergic mediator release, it is difficult to understand how sugammadex could so quickly alleviate anaphylactic symptoms [2]. However, as well as the classical pathway involving IgE and its high-affinity receptor FcεRI, we are just beginning to understand some mast cell activation pathways and effector mechanisms, for example, identification of an alternative pathway mediated by IgG, FcγRIII, macrophages and platelet activating factor (PAF), and the previously unsuspected importance of nitric oxide, endothelial nitric oxide synthase, PAF, PI3K/Akt signalling, cytokines IL-4 and IL-13, sphingosine-1-phosphate and sphingosine kinases [3-5]. In addition, there is still much to learn about sugammadex. How, for example, does the drug interact with mast cells? Degranulation is a rapid process leading to release of potent mediators that increase capillary permeability (wheal) and vasodilation (flare). Histamine liberation and increased local blood flow begins within two minutes of allergen challenge and up to 50% of wheal size cannot be explained by histamine alone [6, 7]. Any anti-anaphylactic effects of sugammadex, therefore, may not be seen if researchers were only to analyse cutaneous signs. There is a considerable amount of further research to be done before it can be so readily concluded that sugammadex’s potential for modifying an established allergic reaction to rocuronium can be discounted.

Intracellular adenosine inhibits IgE-dependent degranulation of human skin mast cells (177.19)

Abstract Adenosine is a purine nucleoside that modulates the immediate release (degranulation) of allergic mediators from mast cells. We show that adenosine at relatively high concentrations (IC50=245 µM) inhibits IgE-dependent degranulation of human skin mast cells by a non-toxic intracellular mechanism. We demonstrate that pre-treatment with the specific inhibitor of equilibrative nucleoside transporter 1 (ENT1/SLC29A1), NBMPR, dose-dependently prevented the inhibitory effect of adenosine. In contrast, blocking adenosine A2a receptor (A2aAR) signaling with the specific antagonist ZM241385 was ineffective. Thus, blocking the influx of extracellular adenosine, but not A2aAR signals, was necessary and sufficient to prevent the adenosine-mediated inhibition of degranulation. The inhibitory effect of adenosine was not due to acute toxicity since human skin mast cells cultured up to 3 days with adenosine at concentrations as high as 0.5 mM remained completely viable and activation-competent. Importantly, the inhibitory action of intracellular adenosine was specific for FcϵRI-induced signaling pathways since degranulation induced by Complement Factor 5a, Substance P, or calcium ionophore A23187 was not affected. Moreover, activation (phosphorylation) of the FcϵRI-proximal and critical signaling molecule Syk was significantly inhibited in adenosine-treated mast cells. Overall, these findings provide the first evidence of an inhibitory anti-allergic effect of intracellular adenosine.

Effects of Novel Nanomaterials on Allergic Mediator Release from Human Mast Cells and Basophils through Non-Ige Mediated Pathways

Mast cells (MC) and peripheral blood basophils (PBB) are well known for their role in the allergic response mediated through high affinity IgE receptors (FceRI). However, these cells can also be stimulated by other non-allergic secretagogues to release their inflammatory mediators. Certain fullerene derivatives (FD) have already been shown to stabilize FceRI-mediated MC/PBB responses, but it is not know if they also stabilize these cells through non-IgEmediated mechanisms. A panel of FD was synthesized and tested for their ability to inhibit non-FceRI mediated release from human MC and PBB. It was found that specifically engineered FD could significantly inhibit calcium ionophore, compound 48/80, somatostatin, and poly L-lysine induced MC degranulation and cytokine production, as well as blunt degranulation and cytokine production from N-formyl-methionine-leucine-phenylalanine (fMLP), poly L-lysine, and calcium ionophore stimulated PBB. The mechanism of inhibition was due in part to the prevention of secretagogueinduced increases in cellular reactive oxygen species (ROS) and calcium levels as well as the reduced activation of the MAPK signaling intermediates ERK1/ERK2 and LAT. Additionally, preincubation of MC with FD blunted the prostaglandin D2 (PGD2) production upon exposure to inflammatory stimuli. In both cell types, the extent of inhibition of mediator release in response to each secretagogue was dependent on the moieties/side chains attached to the carbon cage. These results further extend the utility of fullerene nanomaterials to control mediator release through non-IgE mediated pathways in MC/PBB.

An IgE Immediate Reaction to Thiocolchicoside

Hypersensitivity reactions due to muscle relaxant drugs may be related either to a nonspecific release of allergic mediators or to allergic reactions induced by the molecules themselves. Rare cases of hypersensitivity reactions have been associated to thiocolchicoside, and no case of IgE-mediated immediate reaction has actually been reported to date. We report the first documented case of immediate anaphylaxis to thiocolchicoside.

N-3 polyunsaturated fatty acids reduce allergic type mediator release by human mast cells in vitro via inhibition of reactive oxygen species

N-3 polyunsaturated fatty acids reduce allergic type mediator release by human mast cells in vitro via inhibition of reactive oxygen species

STIM1-Directed Reorganization of Microtubules in Activated Mast Cells

Activation of mast cells by aggregation of the high-affinity IgE receptors (FcεRI) initiates signaling events leading to the release of inflammatory and allergic mediators stored in cytoplasmic granules. A key role in this process play changes in concentrations of intracellular Ca(2+) controlled by store-operated Ca(2+) entry (SOCE). Although microtubules are also involved in the process leading to degranulation, the molecular mechanisms that control microtubule rearrangement during activation are largely unknown. In this study, we report that activation of bone marrow-derived mast cells (BMMCs) induced by FcεRI aggregation or treatment with pervanadate or thapsigargin results in generation of protrusions containing microtubules (microtubule protrusions). Formation of these protrusions depended on the influx of extracellular Ca(2+). Changes in cytosolic Ca(2+)concentration also affected microtubule plus-end dynamics detected by microtubule plus-end tracking protein EB1. Experiments with knockdown or reexpression of STIM1, the key regulator of SOCE, confirmed the important role of STIM1 in the formation of microtubule protrusions. Although STIM1 in activated cells formed puncta associated with microtubules in protrusions, relocation of STIM1 to a close proximity of cell membrane was independent of growing microtubules. In accordance with the inhibition of Ag-induced Ca(2+) response and decreased formation of microtubule protrusions in BMMCs with reduced STIM1, the cells also exhibited impaired chemotactic response to Ag. We propose that rearrangement of microtubules in activated mast cells depends on STIM1-induced SOCE, and that Ca(2+) plays an important role in the formation of microtubule protrusions in BMMCs.

Mountain cedar pollen induces IgE-independent mast cell degranulation, IL-4 production, and intracellular reactive oxygen species generation

Cedar pollens cause severe allergic disease throughout the world. We have previously characterized allergenic pollen glycoproteins from mountain cedar ( Juniperus ashei) that bind to allergen-specific immunoglobulin E (IgE). In the present report, we investigated an alternative pathway of mast cell activation by mountain cedar pollen extract through IgE-independent mechanisms. We show that mountain cedar pollen directly induces mast cell serotonin and IL-4 release and enhances release induced by IgE cross-linking. Concomitant with mediator release, high levels of intracellular reactive oxygen species (ROS) were generated, and both ROS and serotonin release were inhibited by anti-oxidants. These findings suggest that alternative mechanisms exist whereby pollen exposure enhances allergic inflammatory mediator release through mechanisms that involve ROS. These mechanisms have the potential for enhancing the allergenic potency of pollens.

Ocular surface disease: A case of vernal keratoconjunctivitis

Vernal keratoconjunctivitis (VKC) is a recurrent, chronic allergic inflammation of the ocular surface associated with remodeling of the cornea and conjunctiva, with a potential to adversely affect the patient's vision and quality of life. It affects mainly children and young adults. The purpose of this interventional case report is to present a typical case of limbal VKC, which was unresponsive to antiallergic treatment alone. A 5-year-old boy, who was previously unsuccessful with conventional anti-allergic therapy alone, presented with clinicopathological features pathognomonic of limbal VKC. The patient was successfully managed with a combination of palliative, anti-allergic and anti-inflammatory therapy. Prophylaxis with an anti-allergic pharmaceutical agent was initiated for long term use to prevent the release of allergic mediators. This case highlights the importance of prescribing the most appropriate pharmaceutical medication for VKC since conjunctival fibroproliferative lesions are often resistant to anti-allergy therapy. Allergic inflammatory mediators are responsible for the fibroproliferative changes of the conjunctiva and corneal lesions in VKC. Thus, it is crucial to prescribe the most appropriate pharmaceutical medication that will prevent the release of allergic inflammatory mediators, control the allergic inflammatory cascade and prevent ocular surface damage. The fine points of this case are presented with a review of applicable literature.

A New Class of Human Mast Cell and Peripheral Blood Basophil Stabilizers that Differentially Control Allergic Mediator Release

Treatments for allergic disease block the effects of mediators released from activated mast cells and blood basophils. A panel of fullerene derivatives was synthesized and tested for their ability to preempt the release of allergic mediators in vitro and in vivo. The fullerene C(70)-tetraglycolic acid significantly inhibited degranulation and cytokine production from mast cells and basophils, while C(70)-tetrainositol blocked only cytokine production in mast cells and degranulation and cytokine production in basophils. The early phase of FcepsilonRI inhibition was dependent on the blunted release of intracellular calcium stores, elevations in reactive oxygen species, and several signaling molecules. Gene microarray studies further showed the two fullerene derivatives inhibited late phase responses in very different ways. C(70)-tetraglycolic acid was able to block mast cell-driven anaphylaxis in vivo, while C(70)-tetrainositol did not. No toxicity was observed with either compound. These findings demonstrate the biological effects of fullerenes critically depends on the moieties added to the carbon cage and suggest they act on different FcepsilonRI-specific molecules in mast cells and basophils. These next generation fullerene derivatives represent a new class of compounds that interfere with FcepsilonRI signaling pathways to stabilize mast cells and basophils. Thus, fullerene-based therapies may be a new approach for treating allergic diseases.

Effects of Novel Nanomaterials on Allergic Mediator Release from Human Mast Cells through Non-IgE Mediated Pathways

Effects of Novel Nanomaterials on Allergic Mediator Release from Human Mast Cells through Non-IgE Mediated Pathways

Maternal Bisphenol A Exposure Promotes the Development of Experimental Asthma in Mouse Pups

BackgroundWe recently reported that various environmental estrogens induce mast cell degranulation and enhance IgE-mediated release of allergic mediators in vitro.ObjectivesWe hypothesized that environmental estrogens would enhance allergic sensitization as well as bronchial inflammation and responsiveness. To test this hypothesis, we exposed fetal and neonatal mice to the common environmental estrogen bisphenol A (BPA) via maternal loading and assessed the pups’ response to allergic sensitization and bronchial challenge.MethodsFemale BALB/c mice received 10 μg/mL BPA in their drinking water from 1 week before impregnation to the end of the study. Neonatal mice were given a single 5 μg intraperitoneal dose of ovalbumin (OVA) with aluminum hydroxide on postnatal day 4 and 3% OVA by nebulization for 10 min on days 13, 14, and 15. Forty-eight hours after the last nebulization, we assessed serum IgE antibodies to OVA by enzyme-linked immunosorbent assay (ELISA) and airway inflammation and hyperresponsiveness by enumerating eosinophils in bronchoalveolar lavage fluid, whole-body barometric plethysmography, and a forced oscillation technique.ResultsNeonates from BPA-exposed mothers responded to this “suboptimal” sensitization with higher serum IgE anti-OVA concentrations compared with those from unexposed mothers (p < 0.05), and eosinophilic inflammation in their airways was significantly greater. Airway responsiveness of the OVA-sensitized neonates from BPA-treated mothers was enhanced compared with those from unexposed mothers (p < 0.05).ConclusionsPerinatal exposure to BPA enhances allergic sensitization and bronchial inflammation and responsiveness in a susceptible animal model of asthma.

Roles for SH2 and SH3 domains in Lyn kinase association with activated FcεRI in RBL mast cells revealed by patterned surface analysis

In mast cells, antigen-mediated cross-linking of IgE bound to its high-affinity surface receptor, FcepsilonRI, initiates a signaling cascade that culminates in degranulation and release of allergic mediators. Antigen-patterned surfaces, in which the antigen is deposited in micron-sized features on a silicon substrate, were used to examine the spatial relationship between clustered IgE-FcepsilonRI complexes and Lyn, the signal-initiating tyrosine kinase. RBL mast cells expressing wild-type Lyn-EGFP showed co-redistribution of this protein with clustered IgE receptors on antigen-patterned surfaces, whereas Lyn-EGFP containing an inhibitory point mutation in its SH2 domain did not significantly accumulate with the patterned antigen, and Lyn-EGFP with an inhibitory point mutation in its SH3 domain exhibited reduced interactions. Our results using antigen-patterned surfaces and quantitative cross-correlation image analysis reveal that both the SH2 and SH3 domains contribute to interactions between Lyn kinase and cross-linked IgE receptors in stimulated mast cells.

Andolast Acts at Different Cellular Levels to Inhibit Immunoglobulin E Synthesis

The anti-asthmatic agent andolast is thought to inhibit the release of allergic mediators, but its mechanism of action is not fully understood. We investigated whether the compound inhibits immunoglobulin E (IgE) synthesis and tested the hypothesis that andolast affects immunoglobulin class switching. Interleukin (IL)-4 and the interaction of CD40 expressed on B cells with its ligand on T cells are necessary for IgE synthesis. Thus, peripheral blood mononuclear cells (PBMCs) from 40 asthmatic, 16 non-asthmatic allergic, and 9 normal donors were stimulated with IL-4 and/or anti-CD40 antibody. T cells from 9 additional allergic donors were activated with anti-CD3/CD28 antibodies to express IL-4 mRNA. After incubation in the absence or presence of test compounds, immunoglobulin concentrations were measured by enzyme immunoassay, and mRNA levels were analyzed by RT-PCR. Andolast significantly inhibited IgE synthesis by stimulated PBMCs from both asthma patients and combined allergic/normal donors. In mechanistic studies, andolast was found to act at different cellular levels. Firstly, it reduced by about 45 percent (p<0.05) the levels of IL-4 mRNA in T cells stimulated with anti-CD3/CD28. Secondly, andolast reduced by about 36 percent (p<0.05) the expression of epsilon germline transcripts in PBMCs stimulated with IL-4/anti-CD40. Thirdly, the effect of andolast on immunoglobulin synthesis was selective in that the production of IgG4 antibodies was not significantly inhibited. Our findings, while supporting the evidence that andolast is effective for the treatment of asthma, provide new insights into its mechanism of action.

A novel druglike spleen tyrosine kinase binder prevents anaphylactic shock when administered orally

The spleen tyrosine kinase (Syk) is recognized as a potential pharmaceutical target for the treatment of type I hypersensitivity reactions including allergic rhinitis, urticaria, asthma, and anaphylaxis because of its critical position upstream of immunoreceptor signaling complexes that regulate inflammatory responses in leukocytes. Our aim was to improve the selectivity of anti-Syk therapies by impeding the interaction of Syk with its cellular partners, instead of targeting its catalytic site. We have previously studied the inhibitory effects of the anti-Syk intracellular antibody G4G11 on Fc epsilonRI-induced release of allergic mediators. A compound collection was screened by using an antibody displacement assay to identify functional mimics of G4G11 that act as potential inhibitors of the allergic response. The effects of the selected druglike compounds on mast cell activation were evaluated in vitro and in vivo. We discovered compound 13, a small molecule that inhibits Fc epsilonRI-induced mast cell degranulation in vitro and anaphylactic shock in vivo. Importantly, compound 13 was efficient when administered orally to mice. Structural analysis, docking, and site-directed mutagenesis allowed us to identify the binding cavity of this compound, located at the interface between the 2 Src homology 2 domains and the interdomain A of Syk. We have isolated a new class of druglike compounds that modulate the interaction of Syk with some of its macromolecular substrates implicated in the degranulation pathway in mast cells.