FcepsilonRI Research Articles

Network Pharmacology Identifies the Mechanisms of Sang-Xing-Zhi-Ke-Fang against Pharyngitis.

Background Sang-Xing-Zhi-Ke-Fang (SXZKF) demonstrates good therapeutic effect against pharyngitis. Nevertheless, the pharmacological mechanism underlying its effectiveness is still unclear. Objective To investigate the underlying mechanisms of SXZKF against pharyngitis using network pharmacology method. Methods Bioactive ingredients of SXZKF were collected and screened using published literature and two public databases. Using four public databases, the overlapping genes between these bioactive compound-related and pharyngitis-related genes were identified by Venn diagram. Protein-protein interaction (PPI) was obtained using “Search Tool for the Retrieval of Interacting Genes (STRING)” database. “Database for Annotation, Visualization, and Integrated Discovery ver. 6.8 (DAVID 6.8)” was used to perform Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis to explore the molecular mechanisms of SXZKF against pharyngitis. Finally, Cytoscape 3.7.2 software was used to construct and visualize the networks. Result A total of 102 bioactive compounds were identified. Among them, 886 compounds-related and 6258 pharyngitis-related genes were identified, including 387 overlapping genes. Sixty-three core targets were obtained, including ALB, PPARγ, MAPK3, EGF, and PTGS2. Signaling pathways closely related to mechanisms of SXZKF for pharyngitis were identified, including serotonergic synapse, VEGF signaling pathway, Fc epsilon RI signaling pathway, Ras signaling pathway, MAPK signaling pathway, and influenza A. Conclusion This is the first identification of in-depth study of SXZKF against pharyngitis using network pharmacology. This new evidence could be informative in providing new support on the clinical effects of SXZKF on pharyngitis and for the development of personalized medicine for pharyngitis.

Identification of Active Compounds of Mahuang Fuzi Xixin Decoction and Their Mechanisms of Action by LC-MS/MS and Network Pharmacology.

The decoction is an important dosage form of traditional Chinese medicine (TCM) administration. The Mahuang Fuzi Xixin decoction (MFXD) is widely used to treat allergic rhinitis (AR) in China. However, its active compounds and therapeutic mechanisms are unclear. The aim of this study was to establish an integrative method to identify the bioactive compounds and reveal the mechanisms of action of MFXD. LC-MS/MS was used to identify the compounds in MFXD, followed by screening for oral bioavailability. TCMSP, BindingDB, STRING, DAVID, and KEGG databases and algorithms were used to gather information. Cytoscape was used to visualize the networks. Twenty-four bioactive compounds were identified, and thirty-seven predicted targets of these compounds were associated with AR. DAVID analysis suggested that these compounds exert their therapeutic effects by modulating the Fc epsilon RI, B-cell receptor, Toll-like receptor, TNF, NF-κB, and T-cell receptor signaling pathways. The PI3K/AKT and cAMP signaling pathways were also implicated. Ten of the identified compounds, quercetin, pseudoephedrine, ephedrine, β-asarone, methylephedrine, α-linolenic acid, cathine, ferulic acid, nardosinone, and higenamine, seemed to account for most of the beneficial effects of MFXD in AR. This study showed that LC-MS/MS followed by network pharmacology analysis is useful to elucidate the complex mechanisms of action of TCM formulas.

Bioinformatics Analysis Identifies Potential Key Genes of Peripheral Blood Mononuclear Cell in Idiopathic Pulmonary Fibrosis

Idiopathic pulmonary fibrosis (IPF) is a chronic progressive fibrotic interstitial pneumonia with progressive worsening of dyspnea and lung function. The etiology of IPF is unknown, and the pathogenesis remains unclear. Our study aimed to investigate the key genes of the peripheral blood mononuclear cell in IPF by bioinformatics analysis. Our study used the online Gene Expression Omnibus (GEO) microarray expression profiling dataset GSE28042 to identify differentially expressed genes (DEGs) between IPF patients and healthy controls. We performed the Gene Ontology (GO) and pathway enrichment analyses of genes for annotation, visualization, and integrated discovery. The STRING database constructed Protein-protein interaction (PPI) network analysis, and hub genes were identified by the CytoHubba plugin. Moreover, we used the receiver operating characteristic (ROC) curve to assess the diagnostic value of the hub genes. In total, 28 upregulated and 44 downregulated genes were identified in the differential expression analysis. The protein-protein interaction network (PPI) was established with 69 nodes and 68 edges. The top 10 hub genes were JUN, FOS, STAT3, SOCS3, JUNB, DUSP1, IL4, FCER1A, MS4A2, and CPA3. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways enriched for the important module containing hub genes contained Fc epsilon RI signaling pathway, TNF signaling pathway, Jak-STAT signaling pathway, and MAPK signaling pathway. Additionally, the identified hub genes show high functional similarity and diagnostic value in IPF. Our study used bioinformatics analysis to provide new insight into the mechanisms underlying IPF. However, more experiments are needed to explore the relationships between the top 10 hub genes and IPF in the future.

Rapid desensitization of humanized mice with anti-human FcεRIα monoclonal antibodies

Anaphylaxis is classically mediated by allergen cross-linking of IgE bound to the α chain of FcεRI, the mast cell/basophil high affinity IgE receptor. Allergen cross-linking of the IgE/FcεRI complex activates these cells, inducing release of disease-causing mediators, cytokines, and enzymes. We previously demonstrated that IgE-mediated anaphylaxis could be safely prevented in wild-type BALB/c mice by rapid desensitization with anti-mouse FcεRIα mAb. This study sought to use humanized mice to extend these results to humans. We actively immunized huFcεRIα/F709 mice, which express human (hu) instead of mouse FcεRIα and a mutant IL-4 receptor that lacks inhibitory function. We passively immunized huFcεRIα mice, as well as human cord blood-reconstituted reNSGS mice, which are immune-deficient, produce mast cell-stimulating human cytokines, and develop numerous human mast cells. For desensitization, we used anti-huFcεRIα mAbs that bind FcεRIα regardless of its association with IgE (noncompeting mAbs), and/or mAbs that compete with IgE for huFcεRIα binding (competing mAbs). Anaphylaxis was induced by intravenous injection of antigen or anti-huIgE mAb. Anti-huFcεRIα mAb rapid desensitization was safer and more effective than allergen rapid desensitization and suppressed anaphylaxis more rapidly than omalizumab or ligelizumab. Rapid desensitization of naïve, IgE-sensitized huFcεRIα mice and huFcεRIα/F709 mice that were egg-allergic with anti-FcεRIα mAbs safely removed >98% of IgE from peritoneal mast cells and completely suppressed IgE-mediated anaphylaxis. Rapid desensitization of reNSGS mice with anti-FcεRIα mAbs also safely removed ∼98% of mast cell IgE and prevented IgE-mediated anaphylaxis. Rapid desensitization with anti-FcεRIα mAbs may be a safe, effective, and practical way to prevent IgE-mediated anaphylaxis.

Characterization of a 4 lncRNAs-based prognostic risk scoring system in adults with acute myeloid leukemia

PurposeThe study aims to develop a prognostic scoring system based on prognostic lncRNAs for acute myeloid leukemia (AML). MethodsBased on lncRNA expression profiles downloaded from The Cancer Genome Atlas (TCGA), differentially expressed long noncoding RNAs (DELs) between good prognosis and bad prognosis samples were screened, from which prognosis-related lncRNAs were selected using uni-variate and multi-variate Cox regression analysis. Based on the expression profiles of these signature prognosis-related lncRNAs, a risk scoring system was developed and applied to a training set and validated on a testing set. With sample-matched mRNAs of the signature lncRNAs, lncRNA-mRNA networks were built, followed by function analysis for the mRNAs in these networks. ResultTotal 66 DELs were identified between good prognosis and bad prognosis samples. Among these DELs, LINC01003, CTD-2234N14, RP1-137K24, and RP11-834C111 were found to be independent predictors of prognosis. A risk scoring system based on the expressions of the 4 signature lncRNAs was developed. Kaplan-Meier survival analysis found that the risk score system could classify patients into high-risk and low-risk groups with significantly different survival outcomes. Function analysis showed that the mRNAs in these lncRNA-mRNA networks were significantly linked to mTOR signaling pathway, apoptosis, Fc epsilon RI signaling pathway, B cell receptor signaling pathway, natural killer cell mediated cytotoxicity, and T cell receptor signaling pathway. ConclusionThis study suggested a promising 4 prognostic lncRNAs-based risk scoring system in AML. These 4 lncRNAs may play roles in regulating prognosis partly via mTOR signaling pathway, apoptosis, and some immune-related pathways.

Multi targeted cell membrane chromatography: A comprehensive method for screening the anaphylactoid components from complex samples

Anaphylactoid reactions induced by drugs are serious and can be life-threatening, and screening the anaphylactoid ingredients especially in complex samples is challenging. Here, a multi targeted cell membrane chromatography method, based on the Mas-related G protein coupled receptor X2, Fc epsilon RI and H1 receptors, online coupled with LC-MS system provides a comprehensive solution for screening the anaphylactoid components from complex samples. The validation including selectivity and suitability of this system has been evaluated, and it shows promising results. With optimized conditions, this method has been utilized to screen the anaphylactoid ingredients from Shenmai Injection. Ginsenoside Rb1, ginsenoside Rb2, ginsenoside Rc, ginsenoside Rd and 20(S)-ginsenoside Rg3 were identified as anaphylactoid components. The anaphylactoid effects of these five components were further verified by the in vitro sensitization assay, showing promising effects on some or all sensitization cells. In conclusion, the multi targeted cell membrane chromatography online coupled with LC-MS system developed throughout this study could be used to fully screen anaphylactoid components in complex samples. Moreover, it also provides new insights for drug quality control.

GATA2 and PU.1 Collaborate To Activate the Expression of the Mouse Ms4a2 Gene, Encoding FcεRIβ, through Distinct Mechanisms.

GATA factors GATA1 and GATA2 and ETS factor PU.1 are known to function antagonistically during hematopoietic development. In mouse mast cells, however, these factors are coexpressed and activate the expression of the Ms4a2 gene encoding the β chain of the high-affinity IgE receptor (FcεRI). The present study showed that these factors cooperatively regulate Ms4a2 gene expression through distinct mechanisms. Although GATA2 and PU.1 contributed almost equally to Ms4a2 gene expression, gene ablation experiments revealed that simultaneous knockdown of both factors showed neither a synergistic nor an additive effect. A chromatin immunoprecipitation analysis showed that they shared DNA binding to the +10.4-kbp region downstream of the Ms4a2 gene with chromatin looping factor LDB1, whereas the proximal -60-bp region was exclusively bound by GATA2 in a mast cell-specific manner. Ablation of PU.1 significantly reduced the level of GATA2 binding to both the +10.4-kbp and -60-bp regions. Surprisingly, the deletion of the +10.4-kbp region by genome editing completely abolished the Ms4a2 gene expression as well as the cell surface expression of FcεRI. These results suggest that PU.1 and LDB1 play central roles in the formation of active chromatin structure whereas GATA2 directly activates the Ms4a2 promoter.

Insights into the Allergenic Potential of the Edible Yellow Mealworm (Tenebrio molitor).

The edible yellow mealworm (Tenebrio molitor), contains an extremely diverse panel of soluble proteins, including proteins with structural functions such as muscle proteins, as well as proteins involved in metabolic functions such as enzymes. Most of these proteins display a more or less pronounced allergenic character toward previously sensitized people, especially people allergic to shrimps and other shellfish. A mass spectrometry approach following the separation of a mealworm protein, extracted by sodiumdodecyl sulfate-polyacrylamide gel electrophoresis, allowed us to identify up to 106 distinct protein fractions including molecules with structural and functional functions, susceptible to developing an allergenic potential due to the possibility of immunoglobulin E-binding cross-reactions with their counterparts occurring in shellfish. In this respect, most of the sera from people allergic to shrimps reacted with the mealworm protein extract in Western blot experiments. Moreover, the potential mealworm allergens triggered the in vitro degranulation of rat leukemic basophils transfected with the human high-affinity IgE receptor (FcεRI), upon sensitization by the IgE-containing sera from people allergic to shrimps and other shellfish foods. Owing to the large repertoire of IgE-binding cross-reacting allergens the yellow mealworm shares with other phylogenetically-related groups of arthropods, it would seem prudent to inform the consumers, especially those allergic to shellfish, by appropriate labeling on edible mealworm packages about the potential risk of developing an allergic reaction.

Co-Stimulation of Purinergic P2X4 and Prostanoid EP3 Receptors Triggers Synergistic Degranulation in Murine Mast Cells.

Mast cells (MCs) recognize antigens (Ag) via IgE-bound high affinity IgE receptors (FcεRI) and trigger type I allergic reactions. FcεRI-mediated MC activation is regulated by various G protein-coupled receptor (GPCR) agonists. We recently reported that ionotropic P2X4 receptor (P2X4R) stimulation enhanced FcεRI-mediated degranulation. Since MCs are involved in Ag-independent hypersensitivity, we investigated whether co-stimulation with ATP and GPCR agonists in the absence of Ag affects MC degranulation. Prostaglandin E2 (PGE2) induced synergistic degranulation when bone marrow-derived MCs (BMMCs) were co-stimulated with ATP, while pharmacological analyses revealed that the effects of PGE2 and ATP were mediated by EP3 and P2X4R, respectively. Consistently, this response was absent in BMMCs prepared from P2X4R-deficient mice. The effects of ATP and PGE2 were reduced by PI3 kinase inhibitors but were insensitive to tyrosine kinase inhibitors which suppressed the enhanced degranulation induced by Ag and ATP. MC-dependent PGE2-triggered vascular hyperpermeability was abrogated in a P2X4R-deficient mouse ear edema model. Collectively, our results suggest that P2X4R signaling enhances EP3R-mediated MC activation via a different mechanism to that involved in enhancing Ag-induced responses. Moreover, the cooperative effects of the common inflammatory mediators ATP and PGE2 on MCs may be involved in Ag-independent hypersensitivity in vivo.

3-Benzyl-5-((2-nitrophenoxy) methyl)-dihydrofuran-2(3H)-one suppresses FcεRI-mediated mast cell degranulation via the inhibition of mTORC2-Akt signaling

Mast cells express high-affinity IgE receptor (FcεRI) on their surface, cross-linking of which leads to the immediate release of proinflammatory mediators such as histamine but also late-phase cytokine secretion, which are central to the pathogenesis of allergic diseases. Despite the growing evidences that mammalian target of rapamycin (mTOR) plays important roles in the immune system, it is still unclear how mTOR signaling regulates mast cell function. In this study, we investigated the effects of 3-benzyl-5-((2-nitrophenoxy) methyl)-dihydrofuran-2(3H)-one (3BDO) as an mTOR agonist on FcεRI-mediated allergic responses of mast cells. Our data showed that administration of 3BDO decreased β-hexosaminidase, interleukin 6 (IL-6), and tumor necrosis factor-α (TNF-α) release in murine bone marrow-derived mast cells (BMMCs) after FcεRI cross-linking, which was associated with an increase in mTOR complex 1 (mTORC1) signaling but a decrease in activation of Erk1/2, Jnk, and mTORC2-Akt. In addition, we found that a specific Akt agonist, SC79, is able to fully restore the decrease of β-hexosaminidase release in 3BDO-treated BMMCs but has no effect on IL-6 release in these cells, suggesting that 3BDO negatively regulates FcεRI-mediated degranulation and cytokine release through differential mechanisms in mast cells. The present data demonstrate that proper activation of mTORC1 is crucial for mast cell effector function, suggesting the applicability of the mTORC1 activator as a useful therapeutic agent in mast cell-related diseases.

Clinical response to omalizumab in patients with hereditary α-tryptasemia

Hereditary α-tryptasemia (HαT) is a genetic trait that results from increased TPSAB1 copy number encoding α-tryptase and is characterized by elevated basal serum tryptase (BST) levels of 8 ng/mL or higher with variably expressed clinical symptoms. 1 Lyons J.J. Hereditary alpha tryptasemia: genotyping and associated clinical features. Immunol Allergy Clin North Am. 2018; 38: 483-495 Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar In addition to multisystem issues that involve gastrointestinal, connective tissue, and neurologic systems, many individuals with HαT report symptoms associated with mast cell activation, such as chronic spontaneous urticaria and, less commonly, allergic asthma. Increasing α-tryptase gene dosage at TPSAB1 is associated with greater symptom severity 2 Lyons J.J. Yu X. Hughes J.D. et al. Elevated basal serum tryptase identifies a multisystem disorder associated with increased TPSAB1 copy number. Nat Genet. 2016; 48: 1564-1569 Crossref PubMed Scopus (195) Google Scholar and increasing amounts of αβ-tryptase heterotetramers, which have unique enzymatic properties that may contribute to these symptoms. 3 Le Q.T. Lyons J.J. Naranjo A.N. Olivera A. Lazarus R.A. Metcalfe D.D. et al. Impact of naturally forming human alpha/beta-tryptase heterotetramers in the pathogenesis of hereditary alpha-tryptasemia. http://Doi:10.1084/jem.20190701 Google Scholar Multiple individuals in our cohorts with HαT have uncontrolled chronic spontaneous urticaria or asthma symptoms, which led to an on-label indication for treatment with the monoclonal anti-IgE antibody omalizumab, which binds to free IgE and reduces the expression of FcεRI on mast cells and basophils. 4 Beck L.A. Marcotte G.V. MacGlashan D. Togias A. Saini S. Omalizumab-induced reductions in mast cell Fce psilon RI expression and function. J Allergy Clin Immunol. 2004; 114: 527-530 Abstract Full Text Full Text PDF PubMed Scopus (395) Google Scholar ,5 Broesby-Olsen S. Vestergaard H. Mortz C.G. et al. Omalizumab prevents anaphylaxis and improves symptoms in systemic mastocytosis: efficacy and safety observations. Allergy. 2018; 73: 230-238 Crossref PubMed Scopus (72) Google Scholar Despite the strong effect observed on symptoms of mast cell mediator release among patients with chronic spontaneous urticaria and systemic mastocytosis, a recent open-label cohort study found only modest benefit in patients identified as having idiopathic mast cell activation syndrome, with 6 of 14 having minimal to no response to omalizumab and 7 of 14 having no durable response. 6 Lemal R. Fouquet G. Terriou L. et al. Omalizumab therapy for mast cell-mediator symptoms in patients with ISM, CM, MMAS, and MCAS. http://doi:10.1016/j.jaip.2019.03.039 Google Scholar Given that many of the symptoms associated with HαT are often attributed to mast cell activation, we examined which of the symptoms commonly reported in these patients respond to this specific mast cell–targeted therapy and by inference suggest which symptoms may be more directly related to mast cells bearing heterotetrameric tryptase in these patients.

Exposure to silver nanoparticles primes mast cells for enhanced activation through the high-affinity IgE receptor

Mast cells are a key effector cell in type I allergic reactions. It has been shown that environmental exposures such as diesel exhaust and heavy metals exacerbate mast cell degranulation and activation. Today, the use of engineered nanomaterials (ENMs) is rapidly expanding and silver nanoparticles (AgNP) are one of the mostly widely utilized ENMs, primarily for their antimicrobial properties, and are being incorporated into many consumer and biomedical products. We assessed whether pre-exposure of bone marrow-derived mast cells (BMMCs) to 20 nm AgNPs enhanced degranulation and activation to an allergen (dinitrophenol-conjugated human serum albumin) by measuring β-hexosaminidase release, LTB4 and IL-6 production. In addition, we assessed reactive oxygen species (ROS) generation, cell oxidative stress and toxicity as well as total and individual protein tyrosine phosphorylation (p-Tyr). We found that pre-exposure of BMMCs to AgNPs results in exacerbated allergen-mediated mast cell degranulation, LTB4 production and IL-6 release. Exposure of BMMCs to AgNPs exacerbated allergen-induced ROS generation, however, this was not associated with oxidative stress nor cell death. Finally, pre-exposure to AgNPs enhanced allergen-mediated global p-Tyr as well as individual proteins including Syk, PLCγ and LAT. Our findings indicate that pre-exposure to AgNPs exacerbates mast cell allergen-mediated phosphorylation of FcεR1-linked tyrosine kinases and ROS generation resulting in amplified early and late-phase responses. These findings suggest that exposure to AgNPs has the potential to prime mast cells to allergic immune responses, which could be of particular concern to atopic populations as the use of AgNPs in consumer and biomedical products rapidly increases.

Hymenoptera Venom Allergy: How Does Venom Immunotherapy Prevent Anaphylaxis From Bee and Wasp Stings?

Hymenoptera stings may cause both local and systemic allergic reactions and even life threatening anaphylaxis. Along with pharmaceutical drugs and foods, hymenoptera venom is one of the most common causes of anaphylaxis in humans. To date, no parameter has been identified that may predict which sensitized people will have a future systemic sting reaction (SSR), however some risk factors, such as mastocytosis and age >40 years are known. Venom immunotherapy (VIT) is the most effective method of treatment for people who had SSR, which is shown to be effective even after discontinuation of the therapy. Development of peripheral tolerance is the main mechanism during immunotherapy. It is mediated by the production of blocking IgG/IgG4 antibodies that may inhibit IgE dependent reactions through both high affinity (FcεRI) and low affinity (FcεRII) IgE receptors on mast cells, basophils and B cells. The generation of antigen specific regulatory T cells produces IL-10 and suppresses Th2 immunity and the immune responses shift toward a Th1-type response. B regulatory cells are also involved in the production of IL-10 and the development of long term immune tolerance. During VIT the number of effector cells in target organs also decreases, such as mast cells, basophils, innate type 2 lymphocytes and eosinophils. Several meta-analyses and randomized controlled studies have proved that VIT is effective for preventing SSR to a sting and improves the quality of life. In this review, the risk of SSR in venom allergy and how VIT changed this risk are discussed.

Characterization of human FcεRIα chain expression and gene copy number in humanized rat basophilic leukaemia (RBL) reporter cell lines.

Several laboratories have created rat basophil leukemia (RBL) cell lines stably transfected with the human high affinity IgE receptor (FcεRIH). More recently, humanized RBL cell lines saw the introduction of reporter genes such as luciferase (RS-ATL8) and DsRed (RBL NFAT-DsRed). These reporters are more sensitive than their parental non-reporter humanized RBL cell lines. However, no studies so far have addressed the levels of FcεRIH surface expression on humanized RBL cell lines. This is a critical parameter, as it determines the ability of these cells to be efficiently sensitized with human IgE, hence it should affect the sensitivity of the cell assay–a critical parameter for any diagnostic application. Our purpose was to assess and compare the levels of expression of the transfected FcεRIH chain in humanized RBL cell lines. We compared surface levels of FcεRIαH by flow cytometry, using a fluorescently labelled monoclonal antibody (CRA-1/AER-37) and determined receptor numbers using calibration microspheres. FcεRIαH copy numbers were assessed by qPCR, and the sequence verified. Transfection with FcεRIγH cDNA was assessed for its ability to increase FcεRIαH expression in the NFAT-DsRed reporter. While both SX-38 and RS-ATL8 expressed about 500.000 receptors/cell, RBL 703–21 and NFAT-DsRed had approximately 10- to 30-fold lower FcεRIαH expression, respectively. This was neither related to FcεRIH gene copy numbers, nor to differences in steady state mRNA levels, as determined by qPCR and RT-qPCR, respectively. Instead, FcεRIαH surface expression appeared to correlate with the co-expression of FcεRIγH. Stable transfection of NFAT-DsRed cells with pBJ1 neo-huFcεRI gamma, which constitutively expresses FcεRIγH, increased FcεRIαH chain expression levels. Levels of FcεRIαH surface expression vary greatly between humanized RBL reporter cell lines. This difference will affect the sensitivity of the reporter system when used for diagnostic purposes.

CD23 provides a noninflammatory pathway for IgE-allergen complexes

Type I hypersensitivity is mediated by allergen-specific IgE, which sensitizes the high-affinity IgE receptor FcεRI on mast cells and basophils and drives allergic inflammation upon secondary allergen contact. CD23/FcεRII, the low-affinity receptor for IgE, is constitutively expressed on B cells and has been shown to regulate immune responses. Simultaneous binding of IgE to FcεRI and CD23 is blocked by reciprocal allosteric inhibition, suggesting that the 2 receptors exert distinct roles in IgE handling. We aimed to study how free IgE versus precomplexed IgE-allergen immune complexes (IgE-ICs) target the 2 IgE receptors FcεRI and CD23, and we investigated the functional implications of the 2 pathways. We performed binding and activation assays with human cells invitro and IgE pharmacokinetics and anaphylaxis experiments invivo. We demonstrate that FcεRI preferentially binds free IgE and CD23 preferentially binds IgE-ICs. We further show that those different binding properties directly translate to distinct biological functions: free IgE initiated allergic inflammation through FcεRI on allergic effector cells, while IgE-ICs were noninflammatory because of reduced FcεRI binding and enhanced CD23-dependent serum clearance. We propose that IgE-ICs are noninflammatory through reduced engagement by FcεRI but increased targeting of the CD23 pathway.

Monomer hapten and hapten-specific IgG inhibit mast cell activation evoked by multivalent hapten with different mechanisms.

Aggregation of IgE bound to high affinity IgE receptor (FcεRI) by multivalent antigen induces mast cell activation. Reportedly, disaggregation of aggregated FcεRI immediately terminated degranulation, and formation of co-ligated FcεRI and low affinity IgG receptor FcγRIIB blocked degranulation by inhibitory signal via SH2-containing inositol 5'-phosphatase 1 (SHIP1) phosphorylation. However, their molecular mechanisms to inhibit mast cell activation have been unclear in detail. Herein, we found that addition of excess monomeric hapten (TNP-alanine) to multivalent antigen (TNP-OVA)-activated rat basophilic leukemia cells and mouse bone marrow-derived mast cells induced immediate and transient Syk dephosphorylation, which was previously phosphorylated by TNP-OVA addition. Syk dephosphorylation correlated to rapidly decreased intracellular Ca2+ concentrations ([Ca2+ ]i ), terminated degranulation, and suppressed cytokine production through inhibition of Akt and ERK phosphorylation. Addition of hapten-specific IgG monoclonal antibody (anti-TNP IgG1) to activated mast cells induced translocation of SHIP1 to the plasma membrane and its phosphorylation, indicating that co-ligation of FcεRI and FcγRIIB after FcεRI aggregation can lead to SHIP1 activation. SHIP1 phosphorylation led to gradually decreased [Ca2+ ]i , weak inhibition of degranulation, and strong inhibition of cytokine production. Our findings clearly show the inhibitory mechanism of cell function in activated mast cells by operating Fc receptor crosslinking.

Bridging the Gap: Modulatory Roles of the Grb2-Family Adaptor, Gads, in Cellular and Allergic Immune Responses.

Antigen receptor signaling pathways are organized by adaptor proteins. Three adaptors, LAT, Gads, and SLP-76, form a heterotrimeric complex that mediates signaling by the T cell antigen receptor (TCR) and by the mast cell high affinity receptor for IgE (FcεRI). In both pathways, antigen recognition triggers tyrosine phosphorylation of LAT and SLP-76. The recruitment of SLP-76 to phospho-LAT is bridged by Gads, a Grb2 family adaptor composed of two SH3 domains flanking a central SH2 domain and an unstructured linker region. The LAT-Gads-SLP-76 complex is further incorporated into larger microclusters that mediate antigen receptor signaling. Gads is positively regulated by dimerization, which promotes its cooperative binding to LAT. Negative regulation occurs via phosphorylation or caspase-mediated cleavage of the linker region of Gads. FcεRI-mediated mast cell activation is profoundly impaired in LAT- Gads- or SLP-76-deficient mice. Unexpectedly, the thymic developmental phenotype of Gads-deficient mice is much milder than the phenotype of LAT- or SLP-76-deficient mice. This distinction suggests that Gads is not absolutely required for TCR signaling, but may modulate its sensitivity, or regulate a particular branch of the TCR signaling pathway; indeed, the phenotypic similarity of Gads- and Itk-deficient mice suggests a functional connection between Gads and Itk. Additional Gads binding partners include costimulatory proteins such as CD28 and CD6, adaptors such as Shc, ubiquitin regulatory proteins such as USP8 and AMSH, and kinases such as HPK1 and BCR-ABL, but the functional implications of these interactions are not yet fully understood. No interacting proteins or function have been ascribed to the evolutionarily conserved N-terminal SH3 of Gads. Here we explore the biochemical and functional properties of Gads, and its role in regulating allergy, T cell development and T-cell mediated immunity.

Glycoprofile Analysis of an Intact Glycoprotein As Inferred by NMR Spectroscopy.

Protein N-glycosylation stands out for its intrinsic and functionally related heterogeneity. Despite its biomedical interest, Glycoprofile analysis still remains a major scientific challenge. Here, we present an NMR-based strategy to delineate the N-glycan composition in intact glycoproteins and under physiological conditions. The employed methodology allowed dissecting the glycan pattern of the IgE high-affinity receptor (FcεRIα) expressed in human HEK 293 cells, identifying the presence and relative abundance of specific glycan epitopes. Chemical shifts and differences in the signal line-broadening between the native and the unfolded states were integrated to build a structural model of FcεRIα that was able to identify intramolecular interactions between high-mannose N-glycans and the protein surface. In turn, complex type N-glycans reflect a large solvent accessibility, suggesting a functional role as interaction sites for receptors. The interaction between intact FcεRIα and the lectin hGal3, also studied here, confirms this hypothesis and opens new avenues for the detection of specific N-glycan epitopes and for the studies of glycoprotein–receptor interactions mediated by N-glycans.

Heterogeneity of Human Mast Cells With Respect to MRGPRX2 Receptor Expression and Function.

Mast cells and their mediators play a role in the control of homeostasis and in the pathogenesis of several disorders. The concept of rodent mast cell heterogeneity, initially established in the mid-1960s has been extended in humans. Human mast cells isolated and purified from different anatomic sites can be activated via aggregation of cell surface high affinity IgE receptors (FcεRI) by antigens, superantigens, anti-IgE, and anti-FcεRI. MAS-related G protein-coupled receptor-X2 (MRGPRX2) is expressed at high level in human skin mast cells (MCs) (HSMCs), synovial MCs (HSyMCs), but not in lung MCs (HLMCs). MRGPX2 can be activated by neuropeptide substance P, several opioids, cationic drugs, and 48/80. Substance P (5 × 10−7 M – 5 × 10−6 M) induced histamine and tryptase release from HSMCs and to a lesser extent from HSyMCs, but not from HLMCs and human cardiac MCs (HHMCs). Morphine (10−5 M – 3 × 10−4 M) selectively induced histamine and tryptase release from HSMCs, but not from HLMCs and HHMCs. SP and morphine were incomplete secretagogues because they did not induce the de novo synthesis of arachidonic acid metabolites from human mast cells. In the same experiments anti-IgE (3 μg/ml) induced the release of histamine and tryptase and the de novo synthesis of prostaglandin D2 (PGD2) from HLMCs, HHMCs, HSyMCs, and HSMCs. By contrast, anti-IgE induced the production of leukotriene C4 (LTC4) from HLMCs, HHMCs, HSyMCs, but not from HSMCs. These results are compatible with the heterogeneous expression and function of MRGPRX2 receptor on primary human mast cells isolated from different anatomic sites.

Identification of Novel Biomarkers for Drug Hypersensitivity After Sequencing of the Promoter Area in 16 Genes of the Vitamin D Pathway and the High-Affinity IgE Receptor.

The prevalence of allergic diseases and drug hypersensitivity reactions (DHRs) during recent years is increasing. Both, allergic diseases and DHRs seem to be related to an interplay between environmental factors and genetic susceptibility. In recent years, a large effort in the elucidation of the genetic mechanisms involved in these disorders has been made, mostly based on case-control studies, and typically focusing on isolated SNPs. These studies provide a limited amount of information, which now can be greatly expanded by the complete coverage that Next Generation Sequencing techniques offer. In this study, we analyzed the promoters of sixteen genes related to the Vitamin D pathway and the high-affinity IgE receptor, including FCER1A, MS4A2, FCER1G, VDR, GC, CYP2R1, CYP27A1, CYP27B1, CYP24A1, RXRA, RXRB, RXRG, IL4, IL4R, IL13, and IL13RA1. The study group was composed of patients with allergic rhinitis plus asthma (AR+A), patients with hypersensitivity to beta-lactams (BLs), to NSAIDs including selective hypersensitivity (SH) and cross-reactivity (CR), and healthy controls without antecedents of atopy or adverse drug reactions. We identified 148 gene variations, 43 of which were novel. Multinomial analyses revealed that three SNPs corresponding to the genes FCER1G (rs36233990 and rs2070901), and GC (rs3733359), displayed significant associations and, therefore, were selected for a combined dataset study in a cohort of 2,476 individuals. The strongest association was found with the promoter FCER1G rs36233990 SNP that alters a transcription factor binding site. This SNP was over-represented among AR+A patients and among patients with IgE-mediated diseases, as compared with control individuals or with the rest of patients in this study. Classification models based on the above-mentioned SNPs were able to predict correct clinical group allocations in patients with DHRs, and patients with IgE-mediated DHRs. Our findings reveal gene promoter SNPs that are significant predictors of drug hypersensitivity, thus reinforcing the hypothesis of a genetic predisposition for these diseases.