@article{gracioso martins_snider_popowski_schuchard_tenorio_akunuri_wee_peters_jansson_shirwaiker_et al._2023, title={Low-dose intrapulmonary drug delivery device for studies on next-generation therapeutics in mice}, volume={359}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-85161646295&partnerID=MN8TOARS}, DOI={10.1016/j.jconrel.2023.05.039}, abstractNote={Although nebulizers have been developed for delivery of small molecules in human patients, no tunable device has been purpose-built for targeted delivery of modern large molecule and temperature-sensitive therapeutics to mice. Mice are used most of all species in biomedical research and have the highest number of induced models for human-relevant diseases and transgene models. Regulatory approval of large molecule therapeutics, including antibody therapies and modified RNA highlight the need for quantifiable dose delivery in mice to model human delivery, proof-of-concept studies, efficacy, and dose-response. To this end, we developed and characterized a tunable nebulization system composed of an ultrasonic transducer equipped with a mesh nebulizer fitted with a silicone restrictor plate modification to control the nebulization rate. We have identified the elements of design that influence the most critical factors to targeted delivery to the deep lungs of BALB/c mice. By comparing an in silico model of the mouse lung with experimental data, we were able to optimize and confirm the targeted delivery of over 99% of the initial volume to the deep portions of the mouse lung. The resulting nebulizer system provides targeted lung delivery efficiency far exceeding conventional nebulizers preventing waste of expensive biologics and large molecules during proof-of-concept and pre-clinical experiments involving mice. (Word Count =207).}, journal={Journal of Controlled Release}, author={Gracioso Martins, A.M. and Snider, D.B. and Popowski, K.D. and Schuchard, K.G. and Tenorio, M. and Akunuri, S. and Wee, J. and Peters, K.J. and Jansson, A. and Shirwaiker, R. and et al.}, year={2023}, pages={287–301} }
 @article{hedgespeth_snider_bitting_cruse_2023, title={The exon-skipping oligonucleotide, KitStop, depletes tissue-resident mast cells in vivo to ameliorate anaphylaxis}, volume={14}, ISSN={["1664-3224"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-85148252370&partnerID=MN8TOARS}, DOI={10.3389/fimmu.2023.1006741}, abstractNote={Introduction Anaphylaxis represents the most extreme and life-threatening form of allergic disease and is considered a medical emergency requiring immediate intervention. Additionally, some people with mastocytosis experience recurrent episodes of anaphylaxis during normal daily activities without exposure to known triggers. While acute therapy consists primarily of epinephrine and supportive care, chronic therapy relies mostly on desensitization and immunotherapy against the offending allergen, which is a time-consuming and sometimes unsuccessful process. These treatments also necessitate identification of the triggering allergen which is not always possible, and thus highlighting a need for alternative treatments for mast cell-mediated diseases. Methods The exon-skipping oligonucleotide KitStop was administered to mice intradermally, intraperitoneally, or systemically at a dose of 12.5 mg/kg. Local mast cell numbers were enumerated via peritoneal lavage or skin histology, and passive systemic anaphylaxis was induced to evaluate KitStop’s global systemic effect. A complete blood count and biochemistry panel were performed to assess the risk of acute toxicity following KitStop administration. Results Here, we report the use of an exon-skipping oligonucleotide, which we have previously termed KitStop, to safely reduce the severity and duration of the anaphylactic response via mast cell depopulation in tissues. KitStop administration results in the integration of a premature stop codon within the mRNA transcript of the KIT receptor—a receptor tyrosine kinase found primarily on mast cells and whose gain-of-function mutation can lead to systemic mastocytosis. Following either local or systemic KitStop treatment, mice had significantly reduced mast cell numbers in the skin and peritoneum. In addition, KitStop-treated mice experienced a significantly diminished anaphylactic response using a model of passive systemic anaphylaxis when compared with control mice. Discussion KitStop treatment results in a significant reduction in systemic mast cell responses, thus offering the potential to serve as a powerful additional treatment modality for patients that suffer from anaphylaxis.}, journal={FRONTIERS IN IMMUNOLOGY}, author={Hedgespeth, Barry A. A. and Snider, Douglas B. and Bitting, Katie J. and Cruse, Glenn}, year={2023}, month={Jan} }
 @article{bitting_hedgespeth_ehrhardt-humbert_arthur_schubert_bradding_tilley_cruse_2022, title={Identification of redundancy between human Fc epsilon RI beta and MS4A6A proteins points toward additional complex mechanisms for Fc epsilon RI trafficking and signaling}, volume={78}, ISSN={["1398-9995"]}, url={https://doi.org/10.1111/all.15595}, DOI={10.1111/all.15595}, abstractNote={Allergic diseases are triggered by signaling through the high‐affinity IgE receptor, FcεRI. In both mast cells (MCs) and basophils, FcεRI is a tetrameric receptor complex comprising a ligand‐binding α subunit (FcεRIα), a tetraspan β subunit (FcεRIβ, MS4A2) responsible for trafficking and signal amplification, and a signal transducing dimer of single transmembrane γ subunits (FcεRIγ). However, FcεRI also exists as presumed trimeric complexes that lack FcεRIβ and are expressed on several cell types outside the MC and basophil lineages. Despite known differences between humans and mice in the presence of the trimeric FcεRI complex, questions remain as to how it traffics and whether it signals in the absence of FcεRIβ. We have previously reported that targeting FcεRIβ with exon‐skipping oligonucleotides eliminates IgE‐mediated degranulation in mouse MCs, but equivalent targeting in human MCs was not effective at reducing degranulation.}, number={5}, journal={ALLERGY}, author={Bitting, Katie and Hedgespeth, Barry and Ehrhardt-Humbert, Lauren C. and Arthur, Greer K. and Schubert, Alicia G. and Bradding, Peter and Tilley, Stephen L. and Cruse, Glenn}, year={2022}, month={Dec} }
 @article{cortes_brodsky_chen_pridgen_odle_snider_cruse_putikova_masuda_doyle_et al._2022, title={Immunologic and pathologic characterization of a novel swine biomedical research model for eosinophilic esophagitis}, volume={3}, ISSN={["2673-6101"]}, DOI={10.3389/falgy.2022.1029184}, abstractNote={Eosinophilic esophagitis (EoE) is a chronic allergy-mediated condition with an increasing incidence in both children and adults. Despite EoE's strong impact on human health and welfare, there is a large unmet need for treatments with only one recently FDA-approved medication for EoE. The goal of this study was to establish swine as a relevant large animal model for translational biomedical research in EoE with the potential to facilitate development of therapeutics. We recently showed that after intraperitoneal sensitization and oral challenge with the food allergen hen egg white protein (HEWP), swine develop esophageal eosinophilia—a hallmark of human EoE. Herein, we used a similar sensitization and challenge treatment and evaluated immunological and pathological markers associated with human EoE. Our data demonstrate that the incorporated sensitization and challenge treatment induces (i) a systemic T-helper 2 and IgE response, (ii) a local expression of eotaxin-1 and other allergy-related immune markers, (iii) esophageal eosinophilia (>15 eosinophils/0.24 mm2), and (iv) esophageal endoscopic findings including linear furrows and white exudates. Thereby, we demonstrate that our sensitization and oral challenge protocol not only induces the underlying immune markers but also the micro- and macro-pathological hallmarks of human EoE. This swine model for EoE represents a novel relevant large animal model that can drive translational biomedical research to develop urgently needed treatment strategies for EoE.}, journal={FRONTIERS IN ALLERGY}, author={Cortes, Lizette M. and Brodsky, David and Chen, Celine and Pridgen, Tiffany and Odle, Jack and Snider, Douglas B. and Cruse, Glenn and Putikova, Arina and Masuda, Mia Y. and Doyle, Alfred D. and et al.}, year={2022}, month={Nov} }
 @article{arthur_cruse_2022, title={Regulation of Trafficking and Signaling of the High Affinity IgE Receptor by Fc epsilon RI beta and the Potential Impact of Fc epsilon RI beta Splicing in Allergic Inflammation}, volume={23}, ISSN={["1422-0067"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-85122521799&partnerID=MN8TOARS}, DOI={10.3390/ijms23020788}, abstractNote={Mast cells are tissue-resident immune cells that function in both innate and adaptive immunity through the release of both preformed granule-stored mediators, and newly generated proinflammatory mediators that contribute to the generation of both the early and late phases of the allergic inflammatory response. Although mast cells can be activated by a vast array of mediators to contribute to homeostasis and pathophysiology in diverse settings and contexts, in this review, we will focus on the canonical setting of IgE-mediated activation and allergic inflammation. IgE-dependent activation of mast cells occurs through the high affinity IgE receptor, FcεRI, which is a multimeric receptor complex that, once crosslinked by antigen, triggers a cascade of signaling to generate a robust response in mast cells. Here, we discuss FcεRI structure and function, and describe established and emerging roles of the β subunit of FcεRI (FcεRIβ) in regulating mast cell function and FcεRI trafficking and signaling. We discuss current approaches to target IgE and FcεRI signaling and emerging approaches that could target FcεRIβ specifically. We examine how alternative splicing of FcεRIβ alters protein function and how manipulation of splicing could be employed as a therapeutic approach. Targeting FcεRI directly and/or IgE binding to FcεRI are promising approaches to therapeutics for allergic inflammation. The characteristic role of FcεRIβ in both trafficking and signaling of the FcεRI receptor complex, the specificity to IgE-mediated activation pathways, and the preferential expression in mast cells and basophils, makes FcεRIβ an excellent, but challenging, candidate for therapeutic strategies in allergy and asthma, if targeting can be realized.}, number={2}, journal={INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES}, author={Arthur, Greer K. and Cruse, Glenn}, year={2022}, month={Jan} }
 @article{snider_arthur_falduto_olivera_ehrhardt-humbert_smith_smith_metcalfe_cruse_2022, title={Targeting KIT by frameshifting mRNA transcripts as a therapeutic strategy for aggressive mast cell neoplasms}, volume={30}, ISSN={["1525-0024"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-85114708401&partnerID=MN8TOARS}, DOI={10.1016/j.ymthe.2021.08.009}, abstractNote={Activating mutations in c-KIT are associated with the mast cell (MC) clonal disorders cutaneous mastocytosis and systemic mastocytosis and its variants, including aggressive systemic mastocytosis, MC leukemia, and MC sarcoma. Currently, therapies inhibiting KIT signaling are a leading strategy to treat MC proliferative disorders. However, these approaches may have off-target effects, and in some patients, complete remission or improved survival time cannot be achieved. These limitations led us to develop an approach using chemically stable exon skipping oligonucleotides (ESOs) that induce exon skipping of precursor (pre-)mRNA to alter gene splicing and introduce a frameshift into mature KIT mRNA transcripts. The result of this alternate approach results in marked downregulation of KIT expression, diminished KIT signaling, inhibition of MC proliferation, and rapid induction of apoptosis in neoplastic HMC-1.2 MCs. We demonstrate that in vivo administration of KIT targeting ESOs significantly inhibits tumor growth and systemic organ infiltration using both an allograft mastocytosis model and a humanized xenograft MC tumor model. We propose that our innovative approach, which employs well-tolerated, chemically stable oligonucleotides to target KIT expression through unconventional pathways, has potential as a KIT-targeted therapeutic alone, or in combination with agents that target KIT signaling, in the treatment of KIT-associated malignancies.}, number={1}, journal={MOLECULAR THERAPY}, author={Snider, Douglas B. and Arthur, Greer K. and Falduto, Guido H. and Olivera, Ana and Ehrhardt-Humbert, Lauren C. and Smith, Emmaline and Smith, Cierra and Metcalfe, Dean D. and Cruse, Glenn}, year={2022}, month={Jan}, pages={295–310} }
 @article{ozpinar_frey_arthur_mora-navarro_biehl_snider_cruse_freytes_2021, title={Dermal Extracellular Matrix-Derived Hydrogels as an In Vitro Substrate to Study Mast Cell Maturation}, volume={27}, ISSN={["1937-335X"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-85110277858&partnerID=MN8TOARS}, DOI={10.1089/ten.tea.2020.0142}, abstractNote={Mast cells (MCs) are pro-inflammatory tissue-resident immune cells that play a key role in inflammation. MCs circulate in peripheral blood as progenitors and undergo terminal differentiation in the tissue microenvironment where they can remain for many years. This in situ maturation results in tissue- and species-specific MC phenotypes, culminating in significant variability in response to environmental stimuli. There are many challenges associated with studying mature tissue-derived MCs, particularly in humans. In cases where cultured MCs are able to differentiate in two-dimensional in vitro cultures, there remains an inability for full maturation. Extracellular matrix (ECM) scaffolds provide for a more physiologically relevant environment for cells in vitro and have been shown to modulate the response of other immune cells such as T cells, monocytes, and macrophages. To improve current in vitro testing platforms of MCs and to assess future use of ECM scaffolds for MC regulation, we studied the in vitro response of human MCs cultured on decellularized porcine dermis hydrogels (dermis extracellular matrix hydrogel [dECM-H]). This study investigated the effect of dECM-H on cellular metabolic activity, cell viability, and receptor expression compared to collagen type I hydrogel (Collagen-H). Human MCs showed different metabolic activity when cultured in the dECM-H and also upregulated immunoglobulin E (IgE) receptors associated with MC maturation/activation compared to collagen type I. These results suggest an overall benefit in the long-term culture of human MCs in the dECM-H compared to Collagen-H providing important steps toward a model that is more representative of in vivo conditions. Graphical abstract [Formula: see text] Impact statement Mast cells (MCs) are difficult to culture in vitro as current culture conditions and substrates fail to promote similar phenotypic features observed in vivo. Extracellular matrix (ECM)-based biomaterials offer three-dimensional, tissue-specific environments that more closely resemble in vivo conditions. Our study explores the use of dermal ECM hydrogels for MC culture and shows significant upregulation of metabolic activity, cell viability, and gene expression of markers associated with MC maturation or activation compared to collagen type I-hydrogel and tissue culture plastic controls at 7 days. These results are among the first to describe MC behavior in response to ECM hydrogels.}, number={15-16}, journal={TISSUE ENGINEERING PART A}, author={Ozpinar, Emily W. and Frey, Ariana L. and Arthur, Greer K. and Mora-Navarro, Camilo and Biehl, Andreea and Snider, Douglas B. and Cruse, Glenn and Freytes, Donald O.}, year={2021}, month={Aug}, pages={1008–1022} }
 @article{kong_bennett_jania_chason_german_adouli_budney_oby_heusden_lazarowski_et al._2021, title={Identification of an ATP/P2X7/mast cell pathway mediating ozone-induced bronchial hyperresponsiveness}, volume={6}, ISSN={["2379-3708"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-85118886921&partnerID=MN8TOARS}, DOI={10.1172/jci.insight.140207}, abstractNote={Ozone is a highly reactive environmental pollutant with well-recognized adverse effects on lung health. Bronchial hyperresponsiveness (BHR) is one consequence of ozone exposure, particularly for individuals with underlying lung disease. Our data demonstrated that ozone induced substantial ATP release from human airway epithelia in vitro and into the airways of mice in vivo and that ATP served as a potent inducer of mast cell degranulation and BHR, acting through P2X7 receptors on mast cells. Both mast cell–deficient and P2X7 receptor–deficient (P2X7–/–) mice demonstrated markedly attenuated BHR to ozone. Reconstitution of mast cell–deficient mice with WT mast cells and P2X7–/– mast cells restored ozone-induced BHR. Despite equal numbers of mast cells in reconstituted mouse lungs, mice reconstituted with P2X7–/– mast cells demonstrated significantly less robust BHR than mice reconstituted with WT mast cells. These results support a model where P2X7 on mast cells and other cell types contribute to ozone-induced BHR.}, number={21}, journal={JCI INSIGHT}, author={Kong, Xiaomei and Bennett, William C. and Jania, Corey M. and Chason, Kelly D. and German, Zachary and Adouli, Jennifer and Budney, Samuel D. and Oby, Brandon T. and Heusden, Catharina and Lazarowski, Eduardo R. and et al.}, year={2021}, month={Nov} }
 @article{ozpinar_frey_cruse_freytes_2021, title={Mast Cell-Biomaterial Interactions and Tissue Repair}, volume={27}, ISSN={["1937-3376"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-85122126010&partnerID=MN8TOARS}, DOI={10.1089/ten.teb.2020.0275}, abstractNote={Tissue engineers often use biomaterials to provide structural support along with mechanical and chemical signals to modulate the wound healing process. Biomaterials that are implanted into the body interact with a heterogeneous and dynamic inflammatory environment that is present at the site of injury. Whether synthetically-derived, naturally-derived, or a combination of both, it is important to assess biomaterials for their ability to modulate inflammation in order to understand their potential clinical use. One important, but under-explored cell in the context of biomaterials is the mast cell (MC). MCs are granulocytic leukocytes that engage in a variety of events in both the innate and adaptive immune systems. Though highly recognized for their roles in allergic reactions, MCs play an important role in wound healing by recognizing antigens through pattern recognition receptors and the high-affinity immunoglobulin E (IgE) receptor, FcεRI, and releasing granules that affect cell recruitment, fibrosis, extracellular matrix deposition, angiogenesis, and vasculogenesis. MCs also mediate the foreign body response, contributing to the incorporation or rejection of implants. Studies of MC-biomaterial interactions can aid in the elucidation of MC roles during the host tissue response. This review is designed for those in the tissue engineering and biomaterials fields who are interested in exploring the role MCs may play in wound-biomaterial interactions and wound healing. With this review, we hope to inspire more research in the MC-biomaterial space in order to accelerate the design and construction of optimized implants.}, number={6}, journal={TISSUE ENGINEERING PART B-REVIEWS}, author={Ozpinar, Emily W. and Frey, Ariana L. and Cruse, Glenn and Freytes, Donald O.}, year={2021}, month={Dec}, pages={590–603} }
 @article{chee_nandi_nellenbach_mihalko_snider_morrill_bond_sproul_sollinger_cruse_et al._2020, title={Nanosilver composite pNIPAm microgels for the development of antimicrobial platelet-like particles}, volume={108}, ISSN={["1552-4981"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-85080073268&partnerID=MN8TOARS}, DOI={10.1002/jbm.b.34592}, abstractNote={Platelets crucially facilitate wound healing but can become depleted in traumatic injury or chronic wounds. Previously, our group developed injectable platelet-like particles (PLPs) comprised of highly deformable, ultralow crosslinked pNIPAm microgels (ULCs) coupled to fibrin binding antibodies to treat post-trauma bleeding. PLP fibrin-binding facilitates homing to sites of injury, promotes clot formation, and, due to high particle deformability, induces clot retraction. Clot retraction augments healing by increasing clot stability, enhancing clot stiffness, and promoting cell migration into the wound bed. Because post-traumatic healing is often complicated by infection, the objective of these studies was to develop antimicrobial nanosilver microgel composite PLPs to augment hemostasis, fight infection, and promote healing post-trauma. A key goal was to maintain particle deformability following silver incorporation to preserve PLP-mediated clot retraction. Clot retraction, antimicrobial activity, hemostasis after trauma, and healing after injury were evaluated via confocal microscopy, colony-forming unit assays, a murine liver trauma model, and a murine full-thickness injury model in the absence or presence of infection, respectively. We found that nanosilver incorporation does not affect base PLP performance while bestowing significant antimicrobial activity and enhancing infected wound healing outcomes. Therefore, Ag-PLPs have great promise for treating hemorrhage and improving healing following trauma.}, number={6}, journal={JOURNAL OF BIOMEDICAL MATERIALS RESEARCH PART B-APPLIED BIOMATERIALS}, author={Chee, Eunice and Nandi, Seema and Nellenbach, Kimberly and Mihalko, Emily and Snider, Douglas B. and Morrill, Landon and Bond, Andrew and Sproul, Erin and Sollinger, Jennifer and Cruse, Glenn and et al.}, year={2020}, month={Aug}, pages={2599–2609} }
 @article{mishra_wheeler_pitake_ding_jiang_fukuyama_paps_ralph_coyne_parkington_et al._2020, title={Periostin Activation of Integrin Receptors on Sensory Neurons Induces Allergic Itch}, volume={31}, ISSN={["2211-1247"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-85082772179&partnerID=MN8TOARS}, DOI={10.1016/j.celrep.2020.03.036}, abstractNote={Chronic allergic itch is a common symptom affecting millions of people and animals, but its pathogenesis is not fully explained. Herein, we show that periostin, abundantly expressed in the skin of patients with atopic dermatitis (AD), induces itch in mice, dogs, and monkeys. We identify the integrin αVβ3 expressed on a subset of sensory neurons as the periostin receptor. Using pharmacological and genetic approaches, we inhibited the function of neuronal integrin αVβ3, which significantly reduces periostin-induced itch in mice. Furthermore, we show that the cytokine TSLP, the application of AD-causing MC903 (calcipotriol), and house dust mites all induce periostin secretion. Finally, we establish that the JAK/STAT pathway is a key regulator of periostin secretion in keratinocytes. Altogether, our results identify a TSLP-periostin reciprocal activation loop that links the skin to the spinal cord via peripheral sensory neurons, and we characterize the non-canonical functional role of an integrin in itch.}, number={1}, journal={CELL REPORTS}, author={Mishra, Santosh K. and Wheeler, Joshua J. and Pitake, Saumitra and Ding, Huiping and Jiang, Changyu and Fukuyama, Tomoki and Paps, Judy S. and Ralph, Patrick and Coyne, Jacob and Parkington, Michelle and et al.}, year={2020}, month={Apr} }
 @article{arthur_ehrhardt-humbert_snider_jania_tilley_metcalfe_cruse_2020, title={The FcεRIβ homologue, MS4A4A, promotes FcεRI signal transduction and store-operated Ca2+ entry in human mast cells}, volume={71}, url={https://doi.org/10.1016/j.cellsig.2020.109617}, DOI={10.1016/j.cellsig.2020.109617}, abstractNote={Members of the membrane spanning 4A (MS4A) gene family are clustered around 11q12-13, a region linked to allergy and asthma susceptibility. Other than the known functions of FcεRIβ (MS4A2) and CD20 (MS4A1) in mast cell and B cell signaling, respectively, functional studies for the remaining MS4A proteins are lacking. We thus explored whether MS4A4A, a mast cell expressed homologue of FcεRIβ, has related functions to FcεRIβ in FcεRI signaling. We establish in this study that MS4A4A promotes phosphorylation of PLCγ1, calcium flux and degranulation in response to IgE-mediated crosslinking of FcεRI. We previously demonstrated that MS4A4A promotes recruitment of KIT into caveolin-1-enriched microdomains and signaling through PLCγ1. Caveolin-1 itself is an important regulator of IgE-dependent store-operated Ca2+ entry (SOCE) and promotes expression of the store-operated Ca2+ channel pore-forming unit, Orai1. We thus further report that MS4A4A functions through interaction with caveolin-1 and recruitment of FcεRI and KIT into lipid rafts. In addition to proximal FcεRI signaling, we similarly show that MS4A4A regulates Orai1-mediated calcium entry downstream of calcium release from stores. Both MS4A4A and Orai1 had limited effects with compound 48/80 stimulation, demonstrating some degree of selectivity of both proteins to FcεRI receptor signaling over Mas-related G Protein coupled receptor X2 signaling. Overall, our data are consistent with the conclusion that MS4A4A performs a related function to the homologous FcεRIβ to promote PLCγ1 signaling, SOCE, and degranulation through FcεRI in human mast cells and thus represents a new target in the regulation of IgE-mediated mast cell activation.}, journal={Cellular Signalling}, publisher={Elsevier BV}, author={Arthur, Greer K. and Ehrhardt-Humbert, Lauren C. and Snider, Douglas B. and Jania, Corey and Tilley, Stephen L. and Metcalfe, Dean D. and Cruse, Glenn}, year={2020}, month={Jul}, pages={109617} }
 @article{mishra_wheeler_pitake_ding_jiang_fukuyama_paps_ralph_coyne_parkington_et al._2019, title={The Periostin Activation of Integrin Receptors on Sensory Neurons Induces Allergic Itch}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-85110898348&partnerID=MN8TOARS}, DOI={10.2139/ssrn.3459205}, abstractNote={Chronic allergic itch is a major symptom that affects millions of people and animals, but its pathogenesis is not fully explained. Here, we show that one of the mediators abundantly present in the skin of patients with atopic dermatitis, periostin, induces itch via sensory neurons in mice, dogs, and monkeys. We identified the periostin receptor, the integrin αvβ3 expressed on a subset of sensory neurons. Using pharmacological and genetic approaches, we inhibited the function of this neuronal integrin αvβ3, which significantly reduced periostin-mediated itch in mice. Furthermore, we showed that the cytokine TSLP, and the application of the AD-causing MC903 (calcipotriol) and house dust mites induced periostin secretion. Finally, we established that the JAK/STAT pathway is a key regulator of periostin secretion in keratinocytes. Overall, our results identified a TSLP-periostin reciprocal activation that links the skin to the central nervous system via peripheral sensory neurons, and we characterized the non-canonical functional role of a neuronal integrin in itch behavior.}, journal={SSRN}, author={Mishra, S.K. and Wheeler, J. and Pitake, S. and Ding, H. and Jiang, C. and Fukuyama, T. and Paps, J.S. and Ralph, P. and Coyne, J. and Parkington, M.K. and et al.}, year={2019} }
 @article{arthur_cruse_2018, title={Exon Skipping of Fc epsilon RI beta for Allergic Diseases}, volume={1828}, ISBN={["978-1-4939-8650-7"]}, ISSN={["1940-6029"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-85052966676&partnerID=MN8TOARS}, DOI={10.1007/978-1-4939-8651-4_33}, abstractNote={Mast cells are key effector cells in allergic inflammation and consequently are ideal targets for new therapeutics. The high-affinity IgE receptor complex, FcεRI, plays a critical role in mast cell and basophil activation by allergens to drive the immediate allergic inflammatory response. The β subunit of FcεRI is critical for trafficking the FcεRI complex to the cell membrane and amplifies the FcεRI signaling cascade. We have utilized splice switching antisense oligonucleotides to force expression of a truncated isoform of FcεRIβ, which we have shown does not associate with the FcεRI complex. This approach eliminates surface FcεRI expression in mast cells by targeting protein-protein interactions. Exon skipping has several therapeutic applications, and our findings demonstrate a novel application to alter receptor trafficking and dampen allergic inflammation. Here, we describe the methods of exon skipping in mast cells and the assays used to examine the responses of mast cells in vitro and in vivo.}, journal={EXON SKIPPING AND INCLUSION THERAPIES: METHODS AND PROTOCOLS}, author={Arthur, Greer K. and Cruse, Glenn}, year={2018}, pages={503–518} }
 @article{kim_beaven_kulinski_desai_cruse_prussin_komarow_carter_metcalfe_olivera_2016, title={Constitutive KIT Activity and IL-6 Production in Mast Cells Alters Levels of Reactive Oxygen Species (ROS) and the Scavenger Protein DJ-1 in Mastocytosis}, volume={137}, ISSN={0091-6749}, url={http://dx.doi.org/10.1016/J.JACI.2015.12.1168}, DOI={10.1016/J.JACI.2015.12.1168}, abstractNote={Mastocytosis is characterized by hyperproliferation of mast cells (MCs) which is mostly associated with activating mutations in KIT, the stem cell factor (SCF) receptor. DJ-1, a scavenger protein of ROS in MCs and other tissue cells, has been linked to oxidative damage in atopic dermatitis, cancer and neurogenerative diseases. We examined whether DJ-1 is dysregulated in indolent systemic mastocytosis (ISM) and the impact of KIT mutations on DJ-1 and ROS levels. Sera was collected from patients with ISM with tryptase ranging from 1-1000 ng/ml. P815 cells were injected i.v. into DBA/2 mice to induce SM. DJ-1 levels were measured by ELISA and ROS by a fluorescent assay. Patients with ISM showed increased ROS and diminished DJ-1 levels in serum. DJ-1 but not ROS levels reverted towards normal values in patients with advanced ISM. Long-term exposure to SCF or expression of constitutively active mutant KIT in human MC cultures enhanced DJ-1 degradation. In contrast, IL-6, a cytokine which increases in serum with disease severity, induced DJ-1 transcription and promoted ROS release. Injection of mastocytoma cells harboring mutant KIT into mice reproduced the effects of human disease with biphasic changes in serum DJ-1 and increasing elevations in ROS and IL-6 as disease progressed. These effects and disease severity were reversed with anti-IL-6 receptor blocking antibody. The link between IL-6 production in the context of aberrant KIT signaling to dysregulation of DJ-1 and ROS homeostasis suggests that IL-6 contributes to redox imbalance and worsening of ISM and provides potential targets for therapeutic intervention.}, number={2}, journal={Journal of Allergy and Clinical Immunology}, publisher={Elsevier BV}, author={Kim, Dokyun and Beaven, Michael A. and Kulinski, Joseph and Desai, Avanti and Cruse, Glenn and Prussin, Calman and Komarow, Hirsh D. and Carter, Melody C. and Metcalfe, Dean D. and Olivera, Ana}, year={2016}, month={Feb}, pages={AB281} }
 @article{cruse_yin_fukuyama_desai_arthur_baumer_beaven_metcalfe_2016, title={Exon skipping of Fc epsilon RI beta eliminates expression of the high-affinity IgE receptor in mast cells with therapeutic potential for allergy}, volume={113}, ISSN={["0027-8424"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-85002784626&partnerID=MN8TOARS}, DOI={10.1073/pnas.1608520113}, abstractNote={Significance We identified an innovative use for the technique of antisense oligonucleotide-mediated exon skipping to specifically target and down-regulate IgE receptor expression in mast cells. Exon skipping is typically used as part of personalized medicine, where a mutant exon is skipped after sequencing the patients’ affected genes. Our approach, however, targets a nonmutated gene and an exon that is critical for surface IgE receptor expression. It does not require a personalized approach with genetic sequencing or multiple iterations of oligonucleotides that would require clinical trials. Furthermore, the diseases to be treated with this technology are ideal for local delivery of the oligonucleotides by aerosols or topical cream formulations. Allergic diseases are driven by activation of mast cells and release of mediators in response to IgE-directed antigens. However, there are no drugs currently available that can specifically down-regulate mast cell function in vivo when chronically administered. Here, we describe an innovative approach for targeting mast cells in vitro and in vivo using antisense oligonucleotide-mediated exon skipping of the β-subunit of the high-affinity IgE receptor (FcεRIβ) to eliminate surface high-affinity IgE receptor (FcεRI) expression and function, rendering mast cells unresponsive to IgE-mediated activation. As FcεRIβ expression is restricted to mast cells and basophils, this approach would selectively target these cell types. Given the success of exon skipping in clinical trials to treat genetic diseases such as Duchenne muscular dystrophy, we propose that exon skipping of FcεRIβ is a potential approach for mast cell-specific treatment of allergic diseases.}, number={49}, journal={PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA}, author={Cruse, Glenn and Yin, Yuzhi and Fukuyama, Tomoki and Desai, Avanti and Arthur, Greer K. and Baumer, Wolfgang and Beaven, Michael A. and Metcalfe, Dean D.}, year={2016}, month={Dec}, pages={14115–14120} }
 @article{immunophenotypic and ultrastructural analysis of mast cells in hermansky-pudlak syndrome type-1: a possible connection to pulmonary fibrosis_2016, volume={11}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-84980047618&partnerID=MN8TOARS}, DOI={10.1371/journal.pone.0159177}, abstractNote={Hermansky-Pudlak Syndrome type-1 (HPS-1) is an autosomal recessive disorder caused by mutations in HPS1 which result in reduced expression of the HPS-1 protein, defective lysosome-related organelle (LRO) transport and absence of platelet delta granules. Patients with HPS-1 exhibit oculocutaneous albinism, colitis, bleeding and pulmonary fibrosis postulated to result from a dysregulated immune response. The effect of the HPS1 mutation on human mast cells (HuMCs) is unknown. Since HuMC granules classify as LROs along with platelet granules and melanosomes, we set out to determine if HPS-1 cutaneous and CD34+ culture-derived HuMCs have distinct granular and cellular characteristics. Cutaneous and cultured CD34+-derived HuMCs from HPS-1 patients were compared with normal cutaneous and control HuMCs, respectively, for any morphological and functional differences. One cytokine-independent HPS-1 culture was expanded, cloned, designated the HP proMastocyte (HPM) cell line and characterized. HPS-1 and idiopathic pulmonary fibrosis (IPF) alveolar interstitium showed numerous HuMCs; HPS-1 dermal mast cells exhibited abnormal granules when compared to healthy controls. HPS-1 HuMCs showed increased CD63, CD203c and reduced mediator release following FcɛRI aggregation when compared with normal HuMCs. HPM cells also had the duplication defect, expressed FcɛRI and intracytoplasmic proteases and exhibited less mediator release following FcɛRI aggregation. HPM cells constitutively released IL-6, which was elevated in patients’ serum, in addition to IL-8, fibronectin-1 (FN-1) and galectin-3 (LGALS3). Transduction with HPS1 rescued the abnormal HPM morphology, cytokine and matrix secretion. Microarray analysis of HPS-1 HuMCs and non-transduced HPM cells confirmed upregulation of differentially expressed genes involved in fibrogenesis and degranulation. Cultured HPS-1 HuMCs appear activated as evidenced by surface activation marker expression, a decrease in mediator content and impaired releasibility. The near-normalization of constitutive cytokine and matrix release following rescue by HPS1 transduction of HPM cells suggests that HPS-1 HuMCs may contribute to pulmonary fibrosis and constitute a target for therapeutic intervention.}, number={7}, journal={PLoS ONE}, year={2016} }
 @article{cruse_bradding_2016, title={Mast cells in airway diseases and interstitial lung disease}, volume={778}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-84929630909&partnerID=MN8TOARS}, DOI={10.1016/j.ejphar.2015.04.046}, abstractNote={Mast cells are major effector cells of inflammation and there is strong evidence that mast cells play a significant role in asthma pathophysiology. There is also a growing body of evidence that mast cells contribute to other inflammatory and fibrotic lung diseases such as chronic obstructive pulmonary disease and idiopathic pulmonary fibrosis. This review discusses the role that mast cells play in airway diseases and highlights how mast cell microlocalisation within specific lung compartments and their cellular interactions are likely to be critical for their effector function in disease.}, journal={European Journal of Pharmacology}, author={Cruse, G. and Bradding, P.}, year={2016}, pages={125–138} }
 @article{boyden_desai_cruse_young_bolan_scott_robin eisch_daniel long_lee_satorius_et al._2016, title={Vibratory urticaria associated with a missense variant in ADGRE2}, volume={374}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-84959017087&partnerID=MN8TOARS}, DOI={10.1056/NEJMoa1500611}, abstractNote={Patients with autosomal dominant vibratory urticaria have localized hives and systemic manifestations in response to dermal vibration, with coincident degranulation of mast cells and increased histamine levels in serum. We identified a previously unknown missense substitution in ADGRE2 (also known as EMR2), which was predicted to result in the replacement of cysteine with tyrosine at amino acid position 492 (p.C492Y), as the only nonsynonymous variant cosegregating with vibratory urticaria in two large kindreds. The ADGRE2 receptor undergoes autocatalytic cleavage, producing an extracellular subunit that noncovalently binds a transmembrane subunit. We showed that the variant probably destabilizes an autoinhibitory subunit interaction, sensitizing mast cells to IgE-independent vibration-induced degranulation. (Funded by the National Institutes of Health.).}, number={7}, journal={New England Journal of Medicine}, author={Boyden, S.E. and Desai, A. and Cruse, G. and Young, M.L. and Bolan, H.C. and Scott, L.M. and Robin Eisch, A. and Daniel Long, R. and Lee, C.-C.R. and Satorius, C.L. and et al.}, year={2016}, pages={656–663} }
 @article{kim_beaven_cruse_prussin_komarow_carter_metcalfe_olivera_2015, title={Constitutively Activated KIT in Mastocytosis Patients Is Associated with Decreased Levels of the Scavenger Protein DJ1 and Reciprocal Increases in Reactive Oxygen Species}, volume={135}, ISSN={0091-6749}, url={http://dx.doi.org/10.1016/J.JACI.2014.12.1900}, DOI={10.1016/J.JACI.2014.12.1900}, abstractNote={DJ1, a scavenger protein of reactive oxygen species (ROS) that facilitates mast cell (MC) function, is reduced in atopic dermatitis. Mastocytosis patients accumulate MC in organs due to clonal gain-of-function mutations in the stem cell factor (SCF) receptor KIT. Because DJ-1 is linked to MC function, MC-related diseases and other malignancies, we explored whether KIT activation regulates DJ1 and ROS in MC and the relationship of DJ1 levels with the progression of systemic mastocytosis (SM). Sera was collected from SM patients with tryptase values ranging from 1 to 1000 ng/ml. Measurement of DJ-1 levels was performed by ELISA. ROS levels were measured with the OxiSelect™ In Vitro ROS/RNS Assay Kit. LAD2 MC and MC carrying KIT mutations (HMC1) were used to study DJ1 and ROS regulation in vitro. DJ1 levels were decreased and ROS levels increased in SM patients with low to medium tryptase, but DJ1 levels were normal in patients with high tryptase. LAD2 MCs exposed long-term to SCF, or chronic activation of KIT in HMC1 showed reduced DJ1 levels due to increased proteosomal degradation. This reduction in DJ1 levels was reversed by IL-6, which induced DJ1 transcription, but not by IL-31 or histamine, all of which are mediators increased concomitantly with tryptase in SM patients. KIT hyperactivity causes DJ-1 dysregulation and ROS increases in SM, while high IL-6 in severe SM normalizes DJ1 levels. The findings raise the possibility that DJ1 and ROS contribute to SM symptoms and/or progression and may serve as markers of disease progression.}, number={2}, journal={Journal of Allergy and Clinical Immunology}, publisher={Elsevier BV}, author={Kim, Dokyun and Beaven, Michael A. and Cruse, Glenn and Prussin, Calman and Komarow, Hirsh and Carter, Melody C. and Metcalfe, Dean D. and Olivera, Ana}, year={2015}, month={Feb}, pages={AB389} }
 @book{cruse_gilfillan_smrz_2015, title={Flow cytometry-based monitoring of mast cell activation}, volume={1220}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-84921774989&partnerID=MN8TOARS}, DOI={10.1007/978-1-4939-1568-2_23}, abstractNote={Mast cell activation is a central process in the initiation of allergic disorders. As described elsewhere in this volume, this process can be readily monitored by biochemical, antibody-based, and enzyme-based formats when the cell population examined is homogenous. When dealing with mixed and transfected cell populations however, such approaches may not be appropriate. Hence alternative methods are required. Here we describe flow-cytometry-based assays that can be utilized to examine signaling processes and degranulation in both pure mast cell populations and, following appropriate selection, in populations where the mast cells of interest may only represent a fraction of the total cell population.}, journal={Methods in Molecular Biology}, author={Cruse, G. and Gilfillan, A.M. and Smrz, D.}, year={2015}, pages={365–379} }
 @article{kirshenbaum_bandara_desai_fischer_leerkes_metcalfe_cruse_2015, title={Mast Cells Expressing the Germline HPS1 16-Bp Duplication (c.1470_1486dup16, Hermansky-Pudlak Syndrome-1) Defect Produce Extracellular Matrix Components}, volume={135}, ISSN={0091-6749}, url={http://dx.doi.org/10.1016/J.JACI.2014.12.1497}, DOI={10.1016/J.JACI.2014.12.1497}, abstractNote={In the process of developing the Hermansky-Pudlak (HP) mastocyte (M) cell line from a patient with HPS-1, we noticed matrix production on culture flask surfaces which was reduced in clones transfected with wild type HPS1. Since human mast cells are associated with pulmonary fibrosis in patients with HPS-1, HPM cells were examined for production of matrix components. Stable transfection and HPS1 overexpression in HPM clones was performed using GeneCopoeia third generation HIV-based lentiviral vector system and human HPS-1 ORF cDNA lentiviral particles. Matrix ultrastructure was examined sequentially using scanning electron (SE) and transmission electron (TE) microscopy (M) of fixed control (cHPM) and transfected (tHPM) cells. Analysis of extracellular matrix-associated genes including collagen I, IV, V, laminin, fibronectin and galectin-3 from cHPM and tHPM was performed using cDNA and Affymetrix GeneChip RNA Array. Western blot (WB) was also performed for quantitation. SEM and TEM showed increasing formation of globular and fibrillar matrix forms over 8 weeks. Microarrays showed a shift in expression of genes coding for collagen, laminin, fibronectin and galectin-3 from cHPM when compared with tHPM. WB confirmed elevated fibronectin and galectin-3 from HPM cell lysates. HPM cells in culture are thus capable of producing fibronectin and galectin-3 extracellular matrix components which are down regulated in HPM cells transfected with a normal HPS-1 gene. These observations support the possibility in vivo that human HPS-1 mast cells may be contributing directly to pulmonary fibrosis in certain patients, and that treatment targeting the mast cell compartment should be considered.}, number={2}, journal={Journal of Allergy and Clinical Immunology}, publisher={Elsevier BV}, author={Kirshenbaum, Arnold S. and Bandara, Geethani and Desai, Avanti and Fischer, Elizabeth and Leerkes, Maarten and Metcalfe, Dean D. and Cruse, Glenn}, year={2015}, month={Feb}, pages={AB172} }
 @article{cruse_beaven_music_bradding_gilfillan_metcalfe_2015, title={The CD20 homologue MS4A4 directs trafficking of KIT toward clathrin-independent endocytosis pathways and thus regulates receptor signaling and recycling}, volume={26}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-84929484535&partnerID=MN8TOARS}, DOI={10.1091/mbc.E14-07-1221}, abstractNote={MS4A4 traffics through endocytic recycling pathways and stabilizes surface KIT expression by regulating endocytosis and recycling. Silencing MS4A4 reduces KIT recruitment to lipid raft microdomains and PLCg1 signaling while promoting AKT signaling, cell migration, and proliferation. This study is the first to describe functions for human MS4A4.}, number={9}, journal={Molecular Biology of the Cell}, author={Cruse, G. and Beaven, M.A. and Music, S.C. and Bradding, P. and Gilfillan, A.M. and Metcalfe, D.D.}, year={2015}, pages={1711–1727} }
 @article{cruse_beaven_music_bradding_metcalfe_2015, title={The Fcεriβ Homologue, MS4A4, Promotes Fcεri-Dependent Human Mast Cell Degranulation By Facilitating PLCγ1 Signaling}, volume={135}, ISSN={0091-6749}, url={http://dx.doi.org/10.1016/J.JACI.2014.12.1718}, DOI={10.1016/J.JACI.2014.12.1718}, abstractNote={RationaleMS4A4 is a member of the MS4A gene family that includes FcεRIβ and CD20. The MS4A gene family are clustered around 11q12, a region previously linked to atopy. FcεRIβ is critical for trafficking FcεRI complexes to the plasma membrane and promotes FcεRI-dependent signaling within lipid rafts via recruitment of Lyn kinase. We predicted analogous roles for the highly homologous MS4A4 protein.MethodsWe used lentiviral-delivered shRNA to silence MS4A4 expression in human LAD2 mast cells (MCs). Cells were monitored by measurement of receptor expression and apoptosis by flow cytometry, degranulation by β-hexoseaminidase release, protein trafficking by confocal microscopy and cell signaling using immunoblotting.ResultsMS4A4 expression increased as human MCs mature in culture. Silencing MS4A4 did not affect surface FcεRIα expression, but resulted in significant reduction in FcεRI-dependent degranulation without reducing receptor-independent degranulation induced by thapsigargin. We found that FcεRI-induced PLCγ1 and ERK signaling were also significantly reduced, but not signaling through Akt (an activation marker of the PI-3-Kinase pathway). MS4A4 also regulates KIT signaling and trafficking via recruitment to caveolin-1-enriched membrane microdomains and enables SCF-induced PLCγ1 signaling. In addition, MS4A4 silencing promoted proliferation and migration in response to SCF.ConclusionsMS4A4 promotes MC degranulation by facilitating PLCγ1 signaling pathways most likely within lipid rafts. MS4A4 expression may be involved in MC differentiation as indicated by increased expression during maturation and enhanced proliferation with knockdown. Indeed, silencing MS4A4 may convert MCs from a secretory to a less differentiated and migratory phenotype indicating a significant role for MS4A4 in MC function. RationaleMS4A4 is a member of the MS4A gene family that includes FcεRIβ and CD20. The MS4A gene family are clustered around 11q12, a region previously linked to atopy. FcεRIβ is critical for trafficking FcεRI complexes to the plasma membrane and promotes FcεRI-dependent signaling within lipid rafts via recruitment of Lyn kinase. We predicted analogous roles for the highly homologous MS4A4 protein. MS4A4 is a member of the MS4A gene family that includes FcεRIβ and CD20. The MS4A gene family are clustered around 11q12, a region previously linked to atopy. FcεRIβ is critical for trafficking FcεRI complexes to the plasma membrane and promotes FcεRI-dependent signaling within lipid rafts via recruitment of Lyn kinase. We predicted analogous roles for the highly homologous MS4A4 protein. MethodsWe used lentiviral-delivered shRNA to silence MS4A4 expression in human LAD2 mast cells (MCs). Cells were monitored by measurement of receptor expression and apoptosis by flow cytometry, degranulation by β-hexoseaminidase release, protein trafficking by confocal microscopy and cell signaling using immunoblotting. We used lentiviral-delivered shRNA to silence MS4A4 expression in human LAD2 mast cells (MCs). Cells were monitored by measurement of receptor expression and apoptosis by flow cytometry, degranulation by β-hexoseaminidase release, protein trafficking by confocal microscopy and cell signaling using immunoblotting. ResultsMS4A4 expression increased as human MCs mature in culture. Silencing MS4A4 did not affect surface FcεRIα expression, but resulted in significant reduction in FcεRI-dependent degranulation without reducing receptor-independent degranulation induced by thapsigargin. We found that FcεRI-induced PLCγ1 and ERK signaling were also significantly reduced, but not signaling through Akt (an activation marker of the PI-3-Kinase pathway). MS4A4 also regulates KIT signaling and trafficking via recruitment to caveolin-1-enriched membrane microdomains and enables SCF-induced PLCγ1 signaling. In addition, MS4A4 silencing promoted proliferation and migration in response to SCF. MS4A4 expression increased as human MCs mature in culture. Silencing MS4A4 did not affect surface FcεRIα expression, but resulted in significant reduction in FcεRI-dependent degranulation without reducing receptor-independent degranulation induced by thapsigargin. We found that FcεRI-induced PLCγ1 and ERK signaling were also significantly reduced, but not signaling through Akt (an activation marker of the PI-3-Kinase pathway). MS4A4 also regulates KIT signaling and trafficking via recruitment to caveolin-1-enriched membrane microdomains and enables SCF-induced PLCγ1 signaling. In addition, MS4A4 silencing promoted proliferation and migration in response to SCF. ConclusionsMS4A4 promotes MC degranulation by facilitating PLCγ1 signaling pathways most likely within lipid rafts. MS4A4 expression may be involved in MC differentiation as indicated by increased expression during maturation and enhanced proliferation with knockdown. Indeed, silencing MS4A4 may convert MCs from a secretory to a less differentiated and migratory phenotype indicating a significant role for MS4A4 in MC function. MS4A4 promotes MC degranulation by facilitating PLCγ1 signaling pathways most likely within lipid rafts. MS4A4 expression may be involved in MC differentiation as indicated by increased expression during maturation and enhanced proliferation with knockdown. Indeed, silencing MS4A4 may convert MCs from a secretory to a less differentiated and migratory phenotype indicating a significant role for MS4A4 in MC function.}, number={2}, journal={Journal of Allergy and Clinical Immunology}, publisher={Elsevier BV}, author={Cruse, Glenn and Beaven, Michael A. and Music, Stephen C. and Bradding, Peter and Metcalfe, Dean D.}, year={2015}, month={Feb}, pages={AB240} }
 @article{cruse_metcalfe_olivera_2014, title={Functional deregulation of KIT: Link to mast cell proliferative diseases and other neoplasms}, volume={34}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-84898818894&partnerID=MN8TOARS}, DOI={10.1016/j.iac.2014.01.002}, abstractNote={In this review, the authors discuss common gain-of-function mutations in the stem cell factor receptor KIT found in mast cell proliferation disorders and summarize the current understanding of the molecular mechanisms by which these transforming mutations may affect KIT structure and function leading to altered downstream signaling and cellular transformation. Drugs targeting KIT have shown mixed success in the treatment of mastocytosis and other hyperproliferative diseases. A brief overview of the most common KIT inhibitors currently used, the reasons for the varied clinical results of such inhibitors and a discussion of potential new strategies are provided.}, number={2}, journal={Immunology and Allergy Clinics of North America}, author={Cruse, G. and Metcalfe, D.D. and Olivera, A.}, year={2014}, pages={219–237} }
 @article{smrz_cruse_beaven_kirshenbaum_metcalfe_gilfillan_2014, title={Rictor negatively regulates high-affinity receptors for IgE-induced mast cell degranulation}, volume={193}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-84916920272&partnerID=MN8TOARS}, DOI={10.4049/jimmunol.1303495}, abstractNote={Rictor is a regulatory component of the mammalian target of rapamycin (mTOR) complex 2 (mTORC2). We have previously demonstrated that rictor expression is substantially downregulated in terminally differentiated mast cells as compared with their immature or transformed counterparts. However, it is not known whether rictor and mTORC2 regulate mast cell activation. In this article, we show that mast cell degranulation induced by aggregation of high-affinity receptors for IgE (FcεRI) is negatively regulated by rictor independently of mTOR. We found that inhibition of mTORC2 by the dual mTORC1/mTORC2 inhibitor Torin1 or by downregulation of mTOR by short hairpin RNA had no impact on FcεRI-induced degranulation, whereas downregulation of rictor itself resulted in an increased sensitivity (∼50-fold) of cells to FcεRI aggregation with enhancement of degranulation. This was linked to a similar enhancement in calcium mobilization and cytoskeletal rearrangement attributable to increased phosphorylation of LAT and PLCγ1. In contrast, degranulation and calcium responses elicited by the G protein–coupled receptor ligand, C3a, or by thapsigargin, which induces a receptor-independent calcium signal, was unaffected by rictor knockdown. Overexpression of rictor, in contrast with knockdown, suppressed FcεRI-mediated degranulation. Taken together, these data provide evidence that rictor is a multifunctional signaling regulator that can regulate FcεRI-mediated degranulation independently of mTORC2.}, number={12}, journal={Journal of Immunology}, author={Smrz, D. and Cruse, G. and Beaven, M.A. and Kirshenbaum, A. and Metcalfe, D.D. and Gilfillan, A.M.}, year={2014}, pages={5924–5932} }
 @article{cruse_beaven_ashmole_bradding_gilfillan_metcalfe_2013, title={A Truncated Splice-Variant of the FcεRIβ Receptor Subunit Is Critical for Microtubule Formation and Degranulation in Mast Cells}, volume={38}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-84878200358&partnerID=MN8TOARS}, DOI={10.1016/j.immuni.2013.04.007}, abstractNote={Human linkage analyses have implicated the MS4A2-containing gene locus (encoding FcεRIβ) as a candidate for allergy susceptibility. We have identified a truncation of FcεRIβ (t-FcεRIβ) in humans that contains a putative calmodulin-binding domain and thus, we sought to identify the role of this variant in mast cell function. We determined that t-FcεRIβ is critical for microtubule formation and degranulation and that it may perform this function by trafficking adaptor molecules and kinases to the pericentrosomal and Golgi region in response to Ca2+ signals. Mutagenesis studies suggest that calmodulin binding to t-FcεRIβ in the presence of Ca2+ could be critical for t-FcεRIβ function. In addition, gene targeting of t-FcεRIβ attenuated microtubule formation, degranulation, and IL-8 production downstream of Ca2+ signals. Therefore, t-FcεRIβ mediates Ca2+ -dependent microtubule formation, which promotes degranulation and cytokine release. Because t-FcεRIβ has this critical function, it represents a therapeutic target for the downregulation of allergic inflammation.}, number={5}, journal={Immunity}, author={Cruse, G. and Beaven, M. and Ashmole, I. and Bradding, P. and Gilfillan, A. and Metcalfe, D.}, year={2013}, pages={906–917} }
 @article{diminished allergic disease in patients with stat3 mutations reveals a role for stat3 signaling in mast cell degranulation_2013, volume={132}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-85027940079&partnerID=MN8TOARS}, DOI={10.1016/j.jaci.2013.08.045)}, abstractNote={[[{:Label=>"BACKGROUND", :NlmCategory=>"BACKGROUND"}, "Severe atopic conditions associated with elevated serum IgE are heterogeneous with few known causes. Nearly every patient with autosomal-dominant hyper-IgE syndrome (AD-HIES) due to signal transducer and activator of transcription 3 (STAT3) mutations has a history of eczematous dermatitis and elevated IgE; however, clinical atopy has never been systematically studied."], [{:Label=>"OBJECTIVE", :NlmCategory=>"OBJECTIVE"}, "Understanding of genetic determinants of allergic disease may lead to novel therapies in controlling allergic disease."], [{:Label=>"METHODS", :NlmCategory=>"METHODS"}, "We conducted clinical evaluation of the rates of food allergies and anaphylaxis in patients with AD-HIES, a cohort of patients with no STAT3 mutation but with similar histories of elevated IgE and atopic dermatitis, and healthy volunteers with no history of atopy. Morphine skin prick testing, ImmunoCAP assays for allergen-specific IgE, and basophil activation were measured. A model of systemic anaphylaxis was studied in transgenic mice carrying an AD-HIES mutation. STAT3 was silenced in LAD2 and primary human mast cells to study the role of STAT3 in signaling and degranulation after IgE cross-linking."], [{:Label=>"RESULTS", :NlmCategory=>"RESULTS"}, "Food allergies and anaphylaxis were markedly diminished in patients with AD-HIES compared with a cohort of patients with no STAT3 mutation but with similar histories of elevated IgE and atopic dermatitis. Morphine skin prick testing and basophil activation were diminished in patients with AD-HIES, whereas mice carrying an AD-HIES mutation were hyporesponsive to systemic anaphylaxis models. Rapid mast cell STAT3 serine727 phosphorylation was noted after IgE cross-linking, and inhibition of STAT3 signaling in mast cells lead to impaired FcεRI-mediated proximal and distal signaling, as well as reduced degranulation."], [{:Label=>"CONCLUSION", :NlmCategory=>"CONCLUSIONS"}, "This study serves as an example for how mutations in specific atopic pathways can lead to discrete allergic phenotypes, encompassing increased risk of some phenotypes but a relative protection from others."]]}, number={6}, journal={Journal of Allergy and Clinical Immunology}, year={2013} }
 @article{siegel_stone_cruse_lawrence_olivera_jung_barber_freeman_holland_o'brien_et al._2013, title={Diminished allergic disease in patients with STAT3 mutations reveals a role for STAT3 signaling in mast cell degranulation}, volume={132}, ISSN={0091-6749}, url={http://dx.doi.org/10.1016/J.JACI.2013.08.045}, DOI={10.1016/J.JACI.2013.08.045}, abstractNote={BackgroundSevere atopic conditions associated with elevated serum IgE are heterogeneous with few known causes. Nearly every patient with autosomal-dominant hyper-IgE syndrome (AD-HIES) due to signal transducer and activator of transcription 3 (STAT3) mutations has a history of eczematous dermatitis and elevated IgE; however, clinical atopy has never been systematically studied.ObjectiveUnderstanding of genetic determinants of allergic disease may lead to novel therapies in controlling allergic disease.MethodsWe conducted clinical evaluation of the rates of food allergies and anaphylaxis in patients with AD-HIES, a cohort of patients with no STAT3 mutation but with similar histories of elevated IgE and atopic dermatitis, and healthy volunteers with no history of atopy. Morphine skin prick testing, ImmunoCAP assays for allergen-specific IgE, and basophil activation were measured. A model of systemic anaphylaxis was studied in transgenic mice carrying an AD-HIES mutation. STAT3 was silenced in LAD2 and primary human mast cells to study the role of STAT3 in signaling and degranulation after IgE cross-linking.ResultsFood allergies and anaphylaxis were markedly diminished in patients with AD-HIES compared with a cohort of patients with no STAT3 mutation but with similar histories of elevated IgE and atopic dermatitis. Morphine skin prick testing and basophil activation were diminished in patients with AD-HIES, whereas mice carrying an AD-HIES mutation were hyporesponsive to systemic anaphylaxis models. Rapid mast cell STAT3 serine727 phosphorylation was noted after IgE cross-linking, and inhibition of STAT3 signaling in mast cells lead to impaired FcεRI-mediated proximal and distal signaling, as well as reduced degranulation.ConclusionThis study serves as an example for how mutations in specific atopic pathways can lead to discrete allergic phenotypes, encompassing increased risk of some phenotypes but a relative protection from others. Severe atopic conditions associated with elevated serum IgE are heterogeneous with few known causes. Nearly every patient with autosomal-dominant hyper-IgE syndrome (AD-HIES) due to signal transducer and activator of transcription 3 (STAT3) mutations has a history of eczematous dermatitis and elevated IgE; however, clinical atopy has never been systematically studied. Understanding of genetic determinants of allergic disease may lead to novel therapies in controlling allergic disease. We conducted clinical evaluation of the rates of food allergies and anaphylaxis in patients with AD-HIES, a cohort of patients with no STAT3 mutation but with similar histories of elevated IgE and atopic dermatitis, and healthy volunteers with no history of atopy. Morphine skin prick testing, ImmunoCAP assays for allergen-specific IgE, and basophil activation were measured. A model of systemic anaphylaxis was studied in transgenic mice carrying an AD-HIES mutation. STAT3 was silenced in LAD2 and primary human mast cells to study the role of STAT3 in signaling and degranulation after IgE cross-linking. Food allergies and anaphylaxis were markedly diminished in patients with AD-HIES compared with a cohort of patients with no STAT3 mutation but with similar histories of elevated IgE and atopic dermatitis. Morphine skin prick testing and basophil activation were diminished in patients with AD-HIES, whereas mice carrying an AD-HIES mutation were hyporesponsive to systemic anaphylaxis models. Rapid mast cell STAT3 serine727 phosphorylation was noted after IgE cross-linking, and inhibition of STAT3 signaling in mast cells lead to impaired FcεRI-mediated proximal and distal signaling, as well as reduced degranulation. This study serves as an example for how mutations in specific atopic pathways can lead to discrete allergic phenotypes, encompassing increased risk of some phenotypes but a relative protection from others.}, number={6}, journal={Journal of Allergy and Clinical Immunology}, publisher={Elsevier BV}, author={Siegel, Andrea M. and Stone, Kelly D. and Cruse, Glenn and Lawrence, Monica G. and Olivera, Ana and Jung, Mi-yeon and Barber, John S. and Freeman, Alexandra F. and Holland, Steven M. and O'Brien, Michelle and et al.}, year={2013}, month={Dec}, pages={1388–1396.e3} }
 @article{kirshenbaum_desai_bandara_cruse_fischer_gilfillan_gahl_metcalfe_2013, title={Establishment of the Hermansky Pudlak Mastocyte (HPM) Cell Line Which Has the HPS1 16-Bp Duplication (c.1470_1486dup16)}, volume={131}, ISSN={0091-6749}, url={http://dx.doi.org/10.1016/j.jaci.2012.12.1081}, DOI={10.1016/j.jaci.2012.12.1081}, abstractNote={Hermansky Pudlak Syndrome-1(HPS-1) is characterized by bleeding, tyrosinase positive oculocutaneous albinism, granulomatosis colitis, pulmonary fibrosis and mast cell (MC) activation due to a 16-bp duplication (c.1470_1486dup16) in the HPS1 gene affecting lysosome-related organelle trafficking. To further study the impact of disordered granule formation on MC function, we set about to establish a permanent cell line to facilitate such studies. MCs were cultured from CD34+ hematopoietic progenitor cells obtained by leukapheresis from patients with the HPS1 defect. We then selected cultures where MCs persisted in the absence of SCF/IL6, which we cloned using single-cell sorting. Clones were expanded and shown to express the HPS1 duplication. Cells were characterized by light and electron microscopy, flow cytometry, chemotaxis, degranulation and transfection. HPM clones have been maintained without cytokines for over 9 months, and re-established from frozen cultured cells, with a doubling time of 3-4 days. The cells have a normal karyotype and express FceRI, HLA-DR, CD13, CD36, CD49d, CD63, and to a lesser degree CD3, CD25, CD34, CD69, CD117 and CXCR4, but not basophil or dendritic cell specific markers. The cytoplasm/nuclear ratio increased in the presence of SCF/IL-6, and although hypogranulated compared to mature mast cells, granularity increased in the presence of aphidicolin. Cells are chemotactic to SCF and b-hex release was < 5% with/without SCF/IL-6 pretreatment. Cells were transfectable with a plasmid containing the normal HPS1 gene. We thus established the HPM cell line from single cell cloning of HPS-1 mast cells. HPM cells should facilitate the study of defects in granulation that alter MC function.}, number={2}, journal={Journal of Allergy and Clinical Immunology}, publisher={Elsevier BV}, author={Kirshenbaum, Arnold and Desai, Avanti and Bandara, Geethani and Cruse, Glenn and Fischer, Elizabeth and Gilfillan, Alasdair M. and Gahl, William and Metcalfe, Dean D.}, year={2013}, month={Feb}, pages={AB115} }
 @article{ombrello_remmers_sun_freeman_datta_torabi-parizi_subramanian_bunney_baxendale_martins_et al._2012, title={Cold urticaria, immunodeficiency, and autoimmunity related to PLCG2 deletions}, volume={366}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-84863030888&partnerID=MN8TOARS}, DOI={10.1056/NEJMoa1102140}, abstractNote={BACKGROUND
Mendelian analysis of disorders of immune regulation can provide insight into molecular pathways associated with host defense and immune tolerance.


METHODS
We identified three families with a dominantly inherited complex of cold-induced urticaria, antibody deficiency, and susceptibility to infection and autoimmunity. Immunophenotyping methods included flow cytometry, analysis of serum immunoglobulins and autoantibodies, lymphocyte stimulation, and enzymatic assays. Genetic studies included linkage analysis, targeted Sanger sequencing, and next-generation whole-genome sequencing.


RESULTS
Cold urticaria occurred in all affected subjects. Other, variable manifestations included atopy, granulomatous rash, autoimmune thyroiditis, the presence of antinuclear antibodies, sinopulmonary infections, and common variable immunodeficiency. Levels of serum IgM and IgA and circulating natural killer cells and class-switched memory B cells were reduced. Linkage analysis showed a 7-Mb candidate interval on chromosome 16q in one family, overlapping by 3.5 Mb a disease-associated haplotype in a smaller family. This interval includes PLCG2, encoding phospholipase Cγ(2) (PLCγ(2)), a signaling molecule expressed in B cells, natural killer cells, and mast cells. Sequencing of complementary DNA revealed heterozygous transcripts lacking exon 19 in two families and lacking exons 20 through 22 in a third family. Genomic sequencing identified three distinct in-frame deletions that cosegregated with disease. These deletions, located within a region encoding an autoinhibitory domain, result in protein products with constitutive phospholipase activity. PLCG2-expressing cells had diminished cellular signaling at 37°C but enhanced signaling at subphysiologic temperatures.


CONCLUSIONS
Genomic deletions in PLCG2 cause gain of PLCγ(2) function, leading to signaling abnormalities in multiple leukocyte subsets and a phenotype encompassing both excessive and deficient immune function. (Funded by the National Institutes of Health Intramural Research Programs and others.).}, number={4}, journal={New England Journal of Medicine}, author={Ombrello, M.J. and Remmers, E.F. and Sun, G. and Freeman, A.F. and Datta, S. and Torabi-Parizi, P. and Subramanian, N. and Bunney, T.D. and Baxendale, R.W. and Martins, M.S. and et al.}, year={2012}, pages={330–338} }
 @article{cruse_singh_duffy_doe_saunders_brightling_bradding_2011, title={Functional K<inf>Ca</inf>3.1 K<sup>+</sup> channels are required for human fibrocyte migration}, volume={128}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-82555164971&partnerID=MN8TOARS}, DOI={10.1016/j.jaci.2011.07.047}, abstractNote={BackgroundFibrocytes are bone marrow–derived CD34+ collagen I–positive cells present in peripheral blood that develop α-smooth muscle actin expression and contractile activity in tissue culture. They are implicated in the pathogenesis of tissue remodeling and fibrosis in both patients with asthma and those with idiopathic pulmonary fibrosis. Targeting fibrocyte migration might therefore offer a new approach for the treatment of these diseases. Ion channels play key roles in cell function, but the ion-channel repertoire of human fibrocytes is unknown.ObjectiveWe sought to examine whether human fibrocytes express the KCa3.1 K+ channel and to determine its role in cell differentiation, survival, and migration.MethodsFibrocytes were cultured from the peripheral blood of healthy subjects and patients with asthma. Whole-cell patch-clamp electrophysiology was used for the measurement of ion currents, whereas mRNA and protein were examined to confirm channel expression. Fibrocyte migration and proliferation assays were performed in the presence of KCa3.1 ion-channel blockers.ResultsHuman fibrocytes cultured from the peripheral blood of both healthy control subjects and asthmatic patients expressed robust KCa3.1 ion currents together with KCa3.1 mRNA and protein. Two specific and distinct KCa3.1 blockers (TRAM-34 and ICA-17043) markedly inhibited fibrocyte migration in transwell migration assays. Channel blockers had no effect on fibrocyte growth, apoptosis, or differentiation in cell culture.ConclusionsThe K+ channel KCa3.1 plays a key role in human fibrocyte migration. Currently available KCa3.1-channel blockers might therefore attenuate tissue fibrosis and remodeling in patients with diseases such as idiopathic pulmonary fibrosis and asthma through the inhibition of fibrocyte recruitment. Fibrocytes are bone marrow–derived CD34+ collagen I–positive cells present in peripheral blood that develop α-smooth muscle actin expression and contractile activity in tissue culture. They are implicated in the pathogenesis of tissue remodeling and fibrosis in both patients with asthma and those with idiopathic pulmonary fibrosis. Targeting fibrocyte migration might therefore offer a new approach for the treatment of these diseases. Ion channels play key roles in cell function, but the ion-channel repertoire of human fibrocytes is unknown. We sought to examine whether human fibrocytes express the KCa3.1 K+ channel and to determine its role in cell differentiation, survival, and migration. Fibrocytes were cultured from the peripheral blood of healthy subjects and patients with asthma. Whole-cell patch-clamp electrophysiology was used for the measurement of ion currents, whereas mRNA and protein were examined to confirm channel expression. Fibrocyte migration and proliferation assays were performed in the presence of KCa3.1 ion-channel blockers. Human fibrocytes cultured from the peripheral blood of both healthy control subjects and asthmatic patients expressed robust KCa3.1 ion currents together with KCa3.1 mRNA and protein. Two specific and distinct KCa3.1 blockers (TRAM-34 and ICA-17043) markedly inhibited fibrocyte migration in transwell migration assays. Channel blockers had no effect on fibrocyte growth, apoptosis, or differentiation in cell culture. The K+ channel KCa3.1 plays a key role in human fibrocyte migration. Currently available KCa3.1-channel blockers might therefore attenuate tissue fibrosis and remodeling in patients with diseases such as idiopathic pulmonary fibrosis and asthma through the inhibition of fibrocyte recruitment.}, number={6}, journal={Journal of Allergy and Clinical Immunology}, author={Cruse, G. and Singh, S.R. and Duffy, S.M. and Doe, C. and Saunders, R. and Brightling, C.E. and Bradding, P.}, year={2011} }
 @article{cruse_kaur_leyland_bradding_2010, title={A novel FcεRIβ-chain truncation regulates human mast cell proliferation and survival}, volume={24}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-77957829860&partnerID=MN8TOARS}, DOI={10.1096/fj.10-158378}, abstractNote={Mast cells contribute to allergy through IgE‐dependent activation via the high‐affinity IgE receptor FcεRI. The role of the FcεRIβ chain (MS4A2) in mast cell function is not understood fully, although it serves to amplify FcεRI‐dependent signaling. We demonstrate the expression of a novel MS4A2 truncation lacking exon 3 in human mast cells termed MS4A2trunc. MS4A2trunc gene expression was regulated negatively by the mast cell growth factor stem cell factor (SCF), and its expression was not detected in the SCF receptor gain‐of‐function human mast cell line HMC‐1. Unlike MS4A2, MS4A2trunc did not traffic to the cytoplasmic membrane but instead was associated with the nuclear membrane. Overexpression of MS4A2trunc induced human lung mast cell death and profoundly inhibited HMC‐1 cell proliferation by inducing G2‐phase cell cycle arrest and apoptosis. Thus, we have identified a novel splice variant of MS4A2 that might be important in the regulation of human mast cell proliferation and survival. This finding demonstrates that the MS4A2 gene has multiple roles, extending beyond the regulation of acute allergic responses. By understanding the mechanisms regulating its function, it might be possible to induce its expression in mast cells in vivo, which could lead to better treatments for diseases such as mastocytosis and asthma.—Cruse, G., Kaur, D., Leyland, M., Bradding, P. A novel FcεRIβ‐chain truncation regulates human mast cell proliferation and survival. FASEB J. 24, 4047–4057 (2010). www.fasebj.org}, number={10}, journal={FASEB Journal}, author={Cruse, G. and Kaur, D. and Leyland, M. and Bradding, P.}, year={2010}, pages={4047–4057} }
 @article{kajiwara_sasaki_bradding_cruse_sagara_ohmori_saito_ra_okayama_2010, title={Activation of human mast cells through the platelet-activating factor receptor}, volume={125}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-77951666148&partnerID=MN8TOARS}, DOI={10.1016/j.jaci.2010.01.056}, abstractNote={In human subjects platelet-activating factor (PAF) concentrations are markedly increased in the plasma after anaphylactic reactions, and these correlate strongly with the severity of the response. The mechanism for the systemic spread of mast cell (MC) activation in anaphylaxis is often assumed to relate to the hematogenous spread of allergen, but this is implausible, and amplification mechanisms need to be considered.We have investigated the ability of PAF to induce human MC degranulation using skin, lung, and peripheral blood (PB)-derived cultured MCs and the signaling pathways activated in PB-derived MCs in response to PAF.The expression of PAF receptor was investigated by means of RT-PCR and Western blot analysis. Cell-signaling pathways in PB-derived MCs in response to PAF were investigated by analyzing the effect of various inhibitors and the silencing of phospholipase C (PLC) mRNA on PAF-mediated histamine release.We show for the first time that PAF induces histamine release from human lung MCs and PB-derived MCs but not skin MCs. Activation of PAF receptor-coupled G(alphai) leads to degranulation through PLCgamma1 and PLCbeta2 activation in human MCs. PAF-induced degranulation was rapid, being maximal at 5 seconds, and was partially dependent on extracellular Ca(2+).Our findings provide a mechanism whereby PAF mediates an amplification loop for MC activation in the generation of anaphylaxis.}, number={5}, journal={Journal of Allergy and Clinical Immunology}, author={Kajiwara, N. and Sasaki, T. and Bradding, P. and Cruse, G. and Sagara, H. and Ohmori, K. and Saito, H. and Ra, C. and Okayama, Y.}, year={2010} }
 @article{kaur_hollins_saunders_woodman_sutcliffe_cruse_bradding_brightling_2010, title={Airway smooth muscle proliferation and survival is not modulated by mast cells}, volume={40}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-74049156319&partnerID=MN8TOARS}, DOI={10.1111/j.1365-2222.2009.03423.x}, abstractNote={Background Airway smooth muscle (ASM) hyperplasia and mast cell localization within the ASM bundle are important features of asthma. The cause of this increased ASM mass is uncertain and whether it is a consequence of ASM–mast cell interactions is unknown.}, number={2}, journal={Clinical and Experimental Allergy}, author={Kaur, D. and Hollins, F. and Saunders, R. and Woodman, L. and Sutcliffe, A. and Cruse, G. and Bradding, P. and Brightling, C.}, year={2010}, pages={279–288} }
 @article{cruse_yang_duffy_chachi_leyland_amrani_bradding_2010, title={Counterregulation of β<inf>2</inf>-adrenoceptor function in human mast cells by stem cell factor}, volume={125}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-73149091630&partnerID=MN8TOARS}, DOI={10.1016/j.jaci.2009.08.020}, abstractNote={Mast cells contribute to the pathophysiology of asthma with the sustained release of both preformed and newly generated mediators in response to allergens and other diverse stimuli. Stem cell factor (SCF) is the key human mast cell growth factor, but also primes mast cells for mediator release. SCF expression is markedly increased in asthmatic airways. Short-acting beta(2)-adrenoceptor drugs such as albuterol inhibit human lung mast cell (HLMC) degranulation in vitro in the absence of SCF, but their effect in the presence of SCF is not known.The aim of this study was to elucidate the effects of albuterol on HLMC function in the presence of SCF.Mediator release and K(Ca)3.1 ion channel activity were analyzed in purified HLMC. Intracellular signalling and beta(2)-adrenoceptor phosphorylation and internalization were analyzed in the HMC-1 human mast cell line.beta(2)-Adrenoceptor agonist-dependent inhibition of K(Ca)3.1 ion channels and HLMC mediator release was markedly attenuated in the presence of SCF. Remarkably, albuterol actually potentiated IgE-induced histamine release in a dose-dependent manner when both SCF and IgE were present. These effects were related to the SCF-dependent phosphorylation of Tyr350 on the beta(2)-adrenoceptor with immediate uncoupling of the receptor followed by receptor internalization.The potentially beneficial effects of beta(2)-adrenoceptor agonists in asthmatic airways may be blunted as a result of the high concentrations of SCF present.}, number={1-3}, journal={Journal of Allergy and Clinical Immunology}, author={Cruse, G. and Yang, W. and Duffy, S.M. and Chachi, L. and Leyland, M. and Amrani, Y. and Bradding, P.}, year={2010} }
 @article{cruse_fernandes_de salort_pankhania_marinas_brewin_andrew_bradding_kadioglu_2010, title={Human lung mast cells mediate pneumococcal cell death in response to activation by pneumolysin}, volume={184}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-77953629095&partnerID=MN8TOARS}, DOI={10.4049/jimmunol.0900802}, abstractNote={Mast cells are emerging as contributors to innate immunity. Mouse mast cells have a pivotal role in protection against bacterial infection, and human cord blood-derived mast cells reduce bacterial viability in culture. The objectives of this study were to determine whether human lung mast cells (HLMCs) might be protective against pneumococcal lung infection through direct antimicrobial activity. Tissue-derived HLMCs and the human mast cell lines HMC-1 and LAD2 were cocultured with wild-type and mutant pneumococci, and viability and functional assays were performed. Mast cells were also stimulated with purified pneumolysin. HLMCs killed wild-type serotype-2 (D39) pneumococci in coculture but had no effect on an isogenic pneumolysin-deficient (PLN-A) pneumococcus. D39 wild-type, but not PLN-A pneumococci, induced the release of leukotriene C4 from human mast cells in a dose-dependent manner, which was not accompanied by histamine release. Stimulation of mast cells with sublytic concentrations of purified pneumolysin replicated this effect. Furthermore, pneumolysin induced the release of the cathelicidin LL-37 from HLMCs, purified LL-37 reduced pneumococcal viability, and neutralizing Ab to LL-37 attenuated mast cell-dependent pneumococcal killing. In addition, at high concentrations, all pneumococcal strains tested reduced HLMC viability through a combination of pneumolysin and H2O2-dependent mechanisms. HLMCs exhibit direct antimicrobial activity to pneumococci through their activation by pneumolysin. This antimicrobial activity is mediated, in part, by the release of LL-37 from HLMCs. This suggests that mast cells provide an early warning system and potentially limit pneumococcal dissemination early in the course of invasive pulmonary pneumococcal disease.}, number={12}, journal={Journal of Immunology}, author={Cruse, G. and Fernandes, V.E. and De Salort, J. and Pankhania, D. and Marinas, M.S. and Brewin, H. and Andrew, P.W. and Bradding, P. and Kadioglu, A.}, year={2010}, pages={7108–7115} }
 @article{siddiqui_gupta_cruse_haldar_entwisle_mcdonald_whithers_hainsworth_coxson_brightling_2009, title={Airway wall geometry in asthma and nonasthmatic eosinophilic bronchitis}, volume={64}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-65549162207&partnerID=MN8TOARS}, DOI={10.1111/j.1398-9995.2009.01951.x}, abstractNote={Background:  Variable airflow obstruction and airway hyperresponsiveness (AHR) are features of asthma, which are absent in nonasthmatic eosinophilic bronchitis (EB). Airway remodelling is characteristic of both conditions suggesting that remodelling and airway dysfunction are disassociated, but whether the airway geometry differs between asthma and nonasthmatic EB is uncertain.}, number={6}, journal={Allergy: European Journal of Allergy and Clinical Immunology}, author={Siddiqui, S. and Gupta, S. and Cruse, G. and Haldar, P. and Entwisle, J. and McDonald, S. and Whithers, P.J. and Hainsworth, S.V. and Coxson, H.O. and Brightling, C.E.}, year={2009}, pages={951–958} }
 @article{siddiqui_cruse_mckenna_monteiro_mistry_wardlaw_brightling_2009, title={IL-13 expression by blood T cells and not eosinophils is increased in asthma compared to non-asthmatic eosinophilic bronchitis}, volume={9}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-68349109476&partnerID=MN8TOARS}, DOI={10.1186/1471-2466-9-34}, abstractNote={In asthma interleukin (IL)-13 is increased in the airway compared with non-asthmatic eosinophilic bronchitis. Whether this differential expression is specific to the airway or is more generalised is uncertain.We sought to examine IL-13 expression in peripheral blood T-cells and eosinophils in asthma and non-asthmatic eosinophilic bronchitis. Peripheral blood CD3+ cell and eosinophil intracellular IL-13 expression from subjects with asthma, non-asthmatic eosinophilic bronchitis and healthy controls was assessed. The effect of priming by asthmatic serum on the release of IL-13 by peripheral blood mononuclear cells from healthy subjects was examined and the serum from these subjects was analysed for a range of chemokines and cytokines.The median (IQR)% intracellular IL-13 expression by CD3+ cells was increased in asthma [5.3 (2.7-9.8)%; n = 12] compared to non-asthmatic eosinophilic bronchitis [1.1 (0.5-3)%; n = 7] and healthy controls [1.7 (0.2-3%); n = 9] (p = 0.02), but was not significantly different in eosinophils across the groups. IL-13 released from healthy peripheral blood mononuclear cells (n = 10) was increased by asthmatic serum [117 (47.8-198)pg/ml] compared to control [78.5 (42.6-128)pg/ml; p = 0.02), but was not affected by non-asthmatic serum.Our findings support the view that IL-13 expression is increased in peripheral blood-derived T cells in asthma and that asthmatic serum up-regulates IL-13 release from healthy peripheral blood mononuclear cells.}, journal={BMC Pulmonary Medicine}, author={Siddiqui, S. and Cruse, G. and Mckenna, S. and Monteiro, W. and Mistry, V. and Wardlaw, A. and Brightling, C.}, year={2009} }
 @inbook{bradding_cruse_2009, title={Mast Cells: Biological Properties and Role in Health and Allergic Diseases}, volume={1}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-77955890651&partnerID=MN8TOARS}, DOI={10.1002/9781444300918.ch11}, abstractNote={This chapter contains sections titled: Introduction Mast cell development Mast cell heterogeneity General morphology and biology Mechanisms of mast cell activation Role of mast cells in health Role of mast cells in allergic diseases Mast cells in asthma Mast cells in allergic rhinitis Mast cells in allergic conjunctivitis Mast cells in anaphylaxis Mast cells in atopic dermatitis (eczema) and urticaria Concluding remarks References}, booktitle={Allergy and Allergic Diseases, Second Edition}, author={Bradding, P. and Cruse, G.}, year={2009}, pages={217–257} }
 @article{duffy_cruse_cockerill_brightling_bradding_2008, title={Engagement of the EP<inf>2</inf> prostanoid receptor closes the K<sup>+</sup> channel K<inf>Ca</inf>3.1 in human lung mast cells and attenuates their migration}, volume={38}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-55249097262&partnerID=MN8TOARS}, DOI={10.1002/eji.200738106}, abstractNote={Human lung mast cells (HLMC) express the Ca2+‐activated K+ channel KCa3.1, which plays a crucial role in their migration to a variety of diverse chemotactic stimuli. KCa3.1 activation is attenuated by the β2‐adrenoceptor and the adenosine A2A receptor through a Gs‐coupled mechanism independent of cyclic AMP. Prostaglandin E2 promotes degranulation and migration of mouse bone marrow‐derived mast cells through the Gi‐coupled EP3 prostanoid receptor, and induces LTC4 and cytokine secretion from human cord blood‐derived mast cells. However, PGE2 binding to the Gs‐coupled EP2 receptor on HLMC inhibits their degranulation. We show that EP2 receptor engagement closes KCa3.1 in HLMC. The EP2 receptor‐specific agonist butaprost was more potent than PGE2 in this respect, and the effects of both agonists were reversed by the EP2 receptor antagonist AH6809. Butaprost markedly inhibited HLMC migration induced by chemokine‐rich airway smooth muscle‐conditioned media. Interestingly, PGE2 alone was chemotactic for HLMC at high concentrations (1 µM), but was a more potent chemoattractant for HLMC following EP2 receptor blockade. Therefore, the Gs‐coupled EP2 receptor closes KCa3.1 in HLMC and attenuates both chemokine‐ and PGE2‐dependent HLMC migration. EP2 receptor agonists with KCa3.1 modulating function may be useful for the treatment of mast cell‐mediated disease.}, number={9}, journal={European Journal of Immunology}, author={Duffy, S.M. and Cruse, G. and Cockerill, S.L. and Brightling, C.E. and Bradding, P.}, year={2008}, pages={2548–2556} }
 @article{hollins_kaur_yang_cruse_saunders_sutcliffe_berger_ito_brightling_bradding_2008, title={Human airway smooth muscle promotes human lung mast cell survival, proliferation, and constitutive activation: Cooperative roles for CADM1, stem cell factor, and IL-6}, volume={181}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-53149123285&partnerID=MN8TOARS}, DOI={10.4049/jimmunol.181.4.2772}, abstractNote={The microlocalization of mast cells within specific tissue compartments is thought to be critical for the pathophysiology of many diverse diseases. This is particularly evident in asthma where they localize to the airway smooth muscle (ASM) bundles. Mast cells are recruited to the ASM by numerous chemoattractants and adhere through CADM1, but the functional consequences of this are unknown. In this study, we show that human ASM maintains human lung mast cell (HLMC) survival in vitro and induces rapid HLMC proliferation. This required cell-cell contact and occurred through a cooperative interaction between membrane-bound stem cell factor (SCF) expressed on ASM, soluble IL-6, and CADM1 expressed on HLMC. There was a physical interaction in HLMC between CADM1 and the SCF receptor (CD117), suggesting that CADM1-dependent adhesion facilitates the interaction of membrane-bound SCF with its receptor. HLMC-ASM coculture also enhanced constitutive HLMC degranulation, revealing a novel smooth muscle-driven allergen-independent mechanism of chronic mast cell activation. Targeting these interactions in asthma might offer a new strategy for the treatment of this common disease.}, number={4}, journal={Journal of Immunology}, author={Hollins, F. and Kaur, D. and Yang, W. and Cruse, G. and Saunders, R. and Sutcliffe, A. and Berger, P. and Ito, A. and Brightling, C.E. and Bradding, P.}, year={2008}, pages={2772–2780} }
 @article{cruse_cockerill_bradding_2008, title={IgE alone promotes human lung mast cell survival through the autocrine production of IL-6}, volume={9}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-39849110433&partnerID=MN8TOARS}, DOI={10.1186/1471-2172-9-2}, abstractNote={Mast cells play a key role in asthma and recent evidence indicates that their ongoing activation in this disease is mediated, in part, via IgE in the absence of antigen. In this study we have examined whether IgE alone enhances human lung mast cell (HLMC) survival.Purified HLMC were cultured for 4 weeks and survival assays then performed over 10 days following cytokine withdrawal in the presence or absence of human myeloma IgE. Quantitative real time RT-PCR was carried out to examine IL-6 mRNA expression and IL-6 protein was measured in HLMC supernatants by ELISA.IgE alone promoted the survival of HLMC in a dose-dependent manner following cytokine withdrawal. IgE-induced survival was eliminated with the addition of neutralising anti-IL-6 antibody but not by the addition of neutralising anti-stem cell factor. IgE sensitisation initiated profound upregulation of IL-6 mRNA in HLMC, and IL-6 concentrations were also raised in the culture supernatants of IgE-exposed cells.These data taken together suggest that IgE in the absence of antigen promotes HLMC survival through the autocrine production of IL-6. This provides a further mechanism through which IL-6 and IgE contribute to the pathogenesis of asthma, and through which anti-IgE therapy might achieve its therapeutic effect.}, journal={BMC Immunology}, author={Cruse, G. and Cockerill, S. and Bradding, P.}, year={2008} }
 @article{woodman_siddiqui_cruse_sutcliffe_saunders_kaur_bradding_brightling_2008, title={Mast cells promote airway smooth muscle cell differentiation via autocrine up-regulation of TGF-β1}, volume={181}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-58149280835&partnerID=MN8TOARS}, number={7}, journal={Journal of Immunology}, author={Woodman, L. and Siddiqui, S. and Cruse, G. and Sutcliffe, A. and Saunders, R. and Kaur, D. and Bradding, P. and Brightling, C.}, year={2008}, pages={5001–5007} }
 @article{duffy_cruse_brightling_bradding_2007, title={Adenosine closes the K<sup>+</sup> channel K<inf>Ca</inf>3.1 in human lung mast cells and inhibits their migration via the adenosine A<inf>2A</inf> receptor}, volume={37}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-34250701701&partnerID=MN8TOARS}, DOI={10.1002/eji.200637024}, abstractNote={Human lung mast cells (HLMC) express the Ca2+‐activated K+ channel KCa3.1, which opens following IgE‐dependent activation. This hyperpolarises the cell membrane and potentiates both Ca2+ influx and degranulation. In addition, blockade of KCa3.1 profoundly inhibits HLMC migration to a variety of diverse chemotactic stimuli. KCa3.1 activation is attenuated by the β2adrenoceptor through a Gαs‐coupled mechanism independent of cyclic AMP. Adenosine is an important mediator that both attenuates and enhances HLMC mediator release through the Gαs‐coupled A2A and A2B adenosine receptors, respectively. We show that at concentrations that inhibit HLMC degranulation (10–5–10–3 M), adenosine closes KCa3.1 both dose‐dependently and reversibly. KCa3.1 suppression by adenosine was reversed partially by the selective adenosine A2A receptor antagonist ZM241385 but not by the A2B receptor antagonist MRS1754, and the effects of adenosine were mimicked by the selective A2A receptor agonist CGS21680. Adenosine also opened a depolarising current carried by non‐selective cations. As predicted from the role of KCa3.1 in HLMC migration, adenosine abolished HLMC chemotaxis to asthmatic airway smooth muscle‐conditioned medium. In summary, the Gαs‐coupled adenosine A2A receptor closes KCa3.1, providing a clearly defined mechanism by which adenosine inhibits HLMC migration and degranulation. A2A receptor agonists with channel‐modulating function may be useful for the treatment of mast cell‐mediated disease.}, number={6}, journal={European Journal of Immunology}, author={Duffy, S.M. and Cruse, G. and Brightling, C.E. and Bradding, P.}, year={2007}, pages={1653–1662} }
 @article{shepherd_duffy_harris_cruse_schuliga_brightling_neylon_bradding_stewart_2007, title={K<inf>Ca</inf>3.1 Ca<sup>2+</sup>-activated K<sup>+</sup> channels regulate human airway smooth muscle proliferation}, volume={37}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-35848939416&partnerID=MN8TOARS}, DOI={10.1165/rcmb.2006-0358OC}, abstractNote={Airway smooth muscle cell hyperplasia contributes to airway remodeling and hyperreactivity characteristic of asthma. Changes to potassium channel activity in proliferating human airway smooth muscle (HASM) cells have been described, but no regulatory role in proliferation has been attributed to them. We sought to investigate the expression of the intermediate conductance calcium-activated potassium channel K(Ca)3.1 in HASM cells and investigate its role in proliferation. Smooth muscle cells derived from human airways were grown in vitro and K(Ca)3.1 channel expression was measured using Western blot, RT-PCR, and patch clamp electrophysiology. Pharmacologic inhibitors of the channel were used in assays of cellular proliferation, and flow cytometry was used to identify cell cycle regulation. HASM cells expressed K(Ca)3.1 channel mRNA, protein, and activity with up-regulation evident after transforming growth factor-beta stimulation. Pharmacologic inhibition of K(Ca)3.1 led to growth arrest in cells stimulated to proliferate with mitogens. These inhibitors did not cause cellular toxicity or induce apoptosis. We have demonstrated, for the first time, the expression of K(Ca)3.1 channels in HASM cells. In addition, we have shown that K(Ca)3.1 channels are important in HASM cell proliferation, making these channels a potential therapeutic target in airway remodeling.}, number={5}, journal={American Journal of Respiratory Cell and Molecular Biology}, author={Shepherd, M.C. and Duffy, S.M. and Harris, T. and Cruse, G. and Schuliga, M. and Brightling, C.E. and Neylon, C.B. and Bradding, P. and Stewart, A.G.}, year={2007}, pages={525–531} }
 @article{cruse_duffy_brightling_bradding_2006, title={Functional K<inf>Ca</inf>3.1 K<sup>+</sup> channels are required for human lung mast cell migration}, volume={61}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-33750019000&partnerID=MN8TOARS}, DOI={10.1136/thx.2006.060319}, abstractNote={Background: Mast cell recruitment and activation are critical for the initiation and progression of inflammation and fibrosis. Mast cells infiltrate specific structures in many diseased tissues such as the airway smooth muscle (ASM) in asthma. This microlocalisation of mast cells is likely to be key to disease pathogenesis. Human lung mast cells (HLMC) express the Ca2+ activated K+ channel KCa3.1 which modulates mediator release, and is proposed to facilitate the retraction of the cell body during migration of several cell types. A study was undertaken to test the hypothesis that blockade of KCa3.1 would attenuate HLMC proliferation and migration. Methods: HLMC were isolated and purified from lung material resected for bronchial carcinoma. HLMC proliferation was assessed by cell counts at various time points following drug exposure. HLMC chemotaxis was assayed using standard Transwell chambers (8 μm pore size). Ion currents were measured using the single cell patch clamp technique. Results: KCa3.1 blockade with triarylmethane-34 (TRAM-34) did not inhibit HLMC proliferation and clotrimazole had cytotoxic effects. In contrast, HLMC migration towards the chemokine CXCL10, the chemoattractant stem cell factor, and the supernatants from tumour necrosis factor α stimulated asthmatic ASM was markedly inhibited with both the non-selective KCa3.1 blocker charybdotoxin and the highly specific KCa3.1 blocker TRAM-34 in a dose dependent manner. Although KCa3.1 blockade inhibits HLMC migration, KCa3.1 is not opened by the chemotactic stimulus, suggesting that it must be involved downstream of the initial receptor-ligand interactions. Conclusions: Since modulation of KCa3.1 can inhibit HLMC chemotaxis to diverse chemoattractants, the use of KCa3.1 blockers such as TRAM-34 could provide new therapeutic strategies for mast cell mediated diseases such as asthma.}, number={10}, journal={Thorax}, author={Cruse, G. and Duffy, S.M. and Brightling, C.E. and Bradding, P.}, year={2006}, pages={880–885} }
 @article{cruse_kaur_yang_duffy_brightling_bradding_2005, title={Activation of human lung mast cells by monomeric immunoglobulin E}, volume={25}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-18744401130&partnerID=MN8TOARS}, DOI={10.1183/09031936.05.00091704}, abstractNote={The mechanism of chronic mast cell activation in asthma is unclear. Monomeric immunoglobulin (Ig)E in the absence of allergen induces mediator release from rodent mast cells, indicating a possible role for IgE in the continued activation of mast cells within the asthmatic bronchial mucosa. In this study it was investigated whether monomeric IgE induces Ca2+ influx and mediator release from human lung mast cells (HLMC). Purified HLMC were cultured for 4 weeks and then exposed to monomeric human myeloma IgE. Ratiometric Ca2+ imaging was performed on single fura-2-loaded cells. Histamine release was measured by radioenzymatic assay; leukotriene C4 (LTC4) and interleukin (IL)-8 were measured by ELISA. At concentrations experienced in vivo, monomeric IgE induced dose-dependent histamine release, LTC4 production and IL-8 synthesis. This was associated with a rise in cytosolic free Ca2+. Enhanced histamine release was still evident 1 week after initial exposure to IgE suggesting that continued exposure maintains enhanced secretion. Monomeric immunoglobulin E alone activates cultured human lung mast cells initiating Ca2+ influx, degranulation, arachidonic acid metabolism and cytokine synthesis. These findings support the hypothesis that immunoglobulin E loading of mast cells within the asthmatic airway contributes to the disordered airway physiology of this disease.}, number={5}, journal={European Respiratory Journal}, author={Cruse, G. and Kaur, D. and Yang, W. and Duffy, S.M. and Brightling, C.E. and Bradding, P.}, year={2005}, pages={858–863} }
 @article{hart_dransfield_bradding_cruse_2005, title={Activation of human lung mast cells by monomeric immunoglobulin E (multiple letters) [2]}, volume={26}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-26844502512&partnerID=MN8TOARS}, DOI={10.1183/09031936.05.00061105}, abstractNote={To the Editors: 

Immunoglobulin (Ig) preparations contain variable proportions of Ig aggregates 1, which behave like true immune complexes and are able to activate inflammatory cells by crosslinking Fc receptors 2. The validity of the study by Cruse et al. 3, which appeared in a previous issue of the European Respiratory …}, number={4}, journal={European Respiratory Journal}, author={Hart, S.P. and Dransfield, I. and Bradding, P. and Cruse, G.}, year={2005}, pages={744–746} }
 @article{duffy_cruse_lawley_bradding_2005, title={β<inf>2</inf>-adrenoceptor regulation of the K<sup>+</sup> channel iK<inf>Ca</inf>1 in human mast cells}, volume={19}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-20444471589&partnerID=MN8TOARS}, DOI={10.1096/fj.04-3439fje}, abstractNote={Human mast cells express the intermediate conductance Ca2+‐activated K+ channel iKCa1, which opens following IgE‐dependent activation. This results in cell membrane hyperpolarization and potentiation of both Ca2+ influx and degranulation. Mast cell activation is attenuated following exposure to β2‐adrenoceptor agonists such as salbutamol, an effect postulated to operate via intracellular cyclic AMP. In this study, we show that salbutamol closes iKCa1 in mast cells derived from human lung and peripheral blood. Salbutamol (1–10 µM) inhibited iKCa1 currents following activation with both anti‐IgE and the iKCa1 opener 1‐EBIO, and was reversed by removing salbutamol or by the addition of the selective β2‐adrenoceptor antagonist and inverse agonist ICI 118551. Interestingly, ICI 118551 consistently opened iKCa1 in quiescent cells, suggesting that constitutive β2‐receptor signaling suppresses channel activity. Manipulation of intracellular cAMP, Gαi, and Gαs demonstrates that the β2‐adrenergic effects are consistent with a membrane‐delimited mechanism involving Gαs. This is the first demonstration that gating of the iKCa1 channel is regulated by a G protein‐coupled receptor and provides a clearly defined mechanism for the mast cell “stabilizing” effect of β2‐agonists. Furthermore, the degree of constitutive β2‐receptor “tone” may control the threshold for human mast cell activation through the regulation of iKCa1.}, number={8}, journal={FASEB Journal}, author={Duffy, S.M. and Cruse, G. and Lawley, W.J. and Bradding, P.}, year={2005}, pages={1006–1008} }
 @article{mark duffy_berger_cruse_yang_bolton_bradding_2004, title={The K <sup>+</sup> channel iK <inf>CA</inf>1 potentiates Ca <sup>2+</sup> influx and degranulation in human lung mast cells}, volume={114}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-3442890222&partnerID=MN8TOARS}, DOI={10.1016/j.jaci.2004.04.005}, abstractNote={Human lung and blood-derived mast cells express a Ca2+-activated K+ channel (KCA) that has electrophysiological properties resembling the intermediate conductance KCA (iKCA1). This channel is predicted to enhance IgE-dependent mast cell responses.To confirm the identity of this channel as iKCA1 in human lung mast cells and to examine the effect of an iKCA1 opener, 1-ethyl-2-benzimidazolinone (1-EBIO), on Ca2+ influx and degranulation after IgE-dependent activation.iKCA1 expression was examined by using RT-PCR. Ion currents were measured by using the patch clamp technique in human peripheral blood-derived mast cells, freshly isolated human lung mast cells (HLMCs), and long-term cultured HLMCs (LTHLMCs). Currents were manipulated with the specific iKCA1 opener 1-EBIO and the iKCA1 blockers clotrimazole and TRAM-34. Ratiometric Ca2+ imaging was performed on single fura-2-loaded cells, and histamine release was measured by radioenzymatic assay.Both fresh HLMCs and LTHLMCs expressed iKCA1 mRNA. The iKCA1 opener 1-EBIO induced iKCA1 currents in 89% of human peripheral blood-derived mast cells, 12% of fresh HLMCs, and 67% of LTHLMCs, which were blocked by the iKCA1 blockers clotrimazole and TRAM-34. After cell activation with a suboptimal concentration of anti-IgE, 1-EBIO enhanced the IgE-dependent rise in cytosolic-free Ca2+ and potentiated IgE-dependent histamine release.Opening of iKCA1 enhances IgE-dependent Ca2+ influx and histamine release in HLMCs. Inhibition of iKCA1 may provide a novel approach to the treatment of mast cell-mediated disease.}, number={1}, journal={Journal of Allergy and Clinical Immunology}, author={Mark Duffy, S. and Berger, P. and Cruse, G. and Yang, W. and Bolton, S.J. and Bradding, P.}, year={2004}, pages={66–72} }