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      Mast cells derived from systemic mastocytosis exhibit an increased responsiveness to hyperosmolarity


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          CONFLICT OF INTEREST The authors report no conflict of interest. To the Editor, Systemic mastocytosis (SM) is a disease characterized by increased number of aberrant mast cells in one or several organs and increased systemic levels of mast cell (MC) mediators. 1 Indolent SM (ISM) is the most common form of SM, constituting approximately 80% of the patients diagnosed with SM. Individuals with ISM often have mediator mediated symptoms, most commonly from the skin, the gastrointestinal tract, cardiovascular, and respiratory system, but also in the form of anaphylaxis. Although basal mediator levels, including serum tryptase and metabolites of histamine and prostaglandin D2 in the urine, are increased at steady state, 1 , 2 , 3 the symptoms often come as spells without any obvious trigger suggesting an intrinsic defect causing a hyper‐reactive state of the mast cells, or an endogenous trigger. We have previously addressed the hypothesis of a hyper‐reactive MC phenotype in ISM by in vivo provocation. 2 None of the used triggers mounted a response that was different between ISM patients and healthy volunteers (HV). To further investigate the hypothesis of a hyper‐reactive MC phenotype, we also developed MCs in vitro from 14 ISM patients and 13 HV (same subjects as included in 2 ) (Tables S1 and S2). Peripheral blood was obtained, and CD34‐selected progenitor cells were cultured under MC promoting conditions 4 (see supplement). When the cells were mature, they were plated and exposed to IgE‐receptor activation, morphine, or mannitol‐induced hyperosmolarity, representing three distinct activation pathways (see supplement). Histamine (as a measurement of degranulation) and PGD2 (newly synthesized lipid mediator); that is, two prominent MC mediators, released through two different routes that are increased in ISM, were measured. We did not observe any difference in in vitro growth and development of MCs over a 6‐week period between cells from ISM patients and HV (Figure 1). This result stands in contrast to a study where a significant increase in MC growth from CD34‐selected progenitor cells from ISM patients was described. 5 An explanation could be the different culture protocols used in the two studies. 5 The in vitro developed MCs (>90% tryptase positive) were plated and exposed to different MC secretagogues: the calcium ionophore A23187, morphine, anti‐IgE, and mannitol. The release of histamine was comparable between MCs derived from ISM and HV in response to all tested secretagogues (Figure 2A). In contrast, MCs derived from ISM showed a significantly increased release of PGD2 in response to mannitol, but not to the other tested triggers (Figure 2B). It has been reported previously that the release of β‐hexosaminidase (released through degranulation) after IgE‐receptor activation is the same from MCs derived from ISM as from HV. 5 However, in that study, they neither investigated the secretion of PGD2, nor other type of secretagogues. FIGURE 1 In vitro growth and maturation of cells over a 42‐day period. CD34‐selected peripheral blood cells from healthy volunteers (n = 13) or individuals with indolent systemic mastocytosis (n = 14) were cultured under conditions that promote mast cell development. Mean ± SEM FIGURE 2 Release of histamine and PGD2 from activated in vitro developed mast cells. Mast cells were treated for 30 minutes with calcium ionophore A23187, morphine, anti‐IgE, or mannitol, and the release of histamine (A) and PGD2 (B) was measured in the cell free supernatant. Healthy volunteers (red boxes) (n = 6–10) and systemic mastocytosis (purple boxes) (n = 7–13). Results are shown as box and whiskers; the box extends from the 25th to 75th percentiles and the whiskers min to max. * p < 0.05 Our study provides the first evidence that MCs derived from ISM exhibit an aberrant response profile to mannitol‐induced hyperosmolarity, with no change in degranulation but an increased synthesis and secretion of PGD2, the main eicosanoid produced by MCs. Mannitol is clinically used to measure bronchoconstriction in individuals with asthma. Individuals with mastocytosis have not been reported to have increased risk for asthma and airway hyperresponsiveness, and in our previously published study, we did not observe any increased bronchoconstriction after mannitol challenge in those with mastocytosis. 2 Here, we used mannitol as a stimulus to mimic hyperosmolarity. Cells sense physical changes through the receptor family transient receptor potential vanilloid type 1–4 (TRPV). 6 Thus, one could speculate that MCs sense osmolarity changes through TRPV and that this pathway is altered in mastocytosis patients, resulting in an increased synthesis and release of PGD2. A hyper‐reactive MC phenotype in ISM is still elusive, but our data indicate that an intrinsic defect in these cells could affect other signaling pathways than the commonly studied downstream of the IgE‐receptor and that other mediator releasing systems than degranulation, that is, newly synthesized mediators, should be studied. Supporting information Supplementary Material Click here for additional data file.

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          Proposed diagnostic algorithm for patients with suspected mastocytosis: a proposal of the European Competence Network on Mastocytosis.

          Mastocytosis is an emerging differential diagnosis in patients with more or less specific mediator-related symptoms. In some of these patients, typical skin lesions are found and the diagnosis of mastocytosis can be established. In other cases, however, skin lesions are absent, which represents a diagnostic challenge. In the light of this unmet need, we developed a diagnostic algorithm for patients with suspected mastocytosis. In adult patients with typical lesions of mastocytosis in the skin, a bone marrow (BM) biopsy should be considered, regardless of the basal serum tryptase concentration. In adults without skin lesions who suffer from mediator-related or other typical symptoms, the basal tryptase level is an important parameter. In those with a slightly increased tryptase level, additional investigations, including a sensitive KIT mutation analysis of blood leucocytes or measurement of urinary histamine metabolites, may be helpful. In adult patients in whom (i) KIT D816V is detected and/or (ii) the basal serum tryptase level is clearly increased (>25-30 ng/ml) and/or (iii) other clinical or laboratory features suggest the presence of 'occult' mastocytosis or another haematologic neoplasm, a BM investigation is recommended. In the absence of KIT D816V and other signs or symptoms of mastocytosis or another haematopoietic disease, no BM investigation is required, but the clinical course and tryptase levels are monitored in the follow-up. In paediatric patients, a BM investigation is usually not required, even if the tryptase level is increased. Although validation is required, it can be expected that the algorithm proposed herein will facilitate the management of patients with suspected mastocytosis and help avoid unnecessary referrals and investigations.
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            A protocol for generating high numbers of mature and functional human mast cells from peripheral blood.

            Mast cells (MCs) are multi-functional effector cells with an essential role in innate immunity and host defence, and under several pathological conditions, such as allergy. Here, we aimed at defining the culture conditions that would allow efficient generation of mature and functional human MCs from their progenitor cells. Human peripheral blood-derived CD34(+) progenitor cells were cultured in vitro under serum-free conditions with human stem cell factor for 9 weeks. Growth and differentiation of the cells into MCs were optimized by selected cytokines and a combination of hypoxic and normoxic conditions. MCs were phenotypically characterized by immunocytochemistry, their preformed mediators were quantified, and their functional ability to degranulate and release histamine was tested. On average, 20 x 10(6) mature MCs were generated from 0.5 x 10(6) progenitor cells during 9 weeks of culture, i.e. at least a 40-fold increase in cell number was achieved. The mature MCs had oval-shaped non-lobular nuclei, contained histamine, heparin, tryptase, chymase, and cathepsin G in their secretory granules, and strongly expressed c-kit (CD117) and Fc epsilon receptor I on their surface. Histamine release from the cells could be brought about by IgE-anti-IgE cross-linkage, compound 48/80, substance P, and anaphylatoxin C3a. The MCs remained functional for several weeks after their maturation. This study describes an efficient protocol for generating mature MCs from human peripheral blood with a functional phenotype of connective tissue-type MCs. Use of these cultured human MCs will increase our knowledge and understanding about human MC development and biology in human disease.
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              A distinct biomolecular profile identifies monoclonal mast cell disorders in patients with idiopathic anaphylaxis

              Background Clonal mast cell disorders are known to occur in a subset of patients with systemic reactions to Hymenoptera stings. This observation has prompted the question as to whether clonal mast cell disorders also occur in patients with idiopathic anaphylaxis (IA). Objective We sought to determine the prevalence of clonal mast cell disorders among patients with IA, criteria to identify those patients who require a bone marrow biopsy and whether the pathogenesis of IA involves a hyper-responsive mast cell compartment. Methods We prospectively enrolled patients with IA (≥3 episodes/yr) and who then underwent a medical evaluation that included a serum tryptase determination, allele-specific quantitative polymerase chain reaction (ASqPCR) for KIT D816V and a bone marrow examination. Mast cells were cultured from peripheral blood CD34+ cells and examined for releasibility following FcεRI aggregation. Results Clonal mast cell disease was diagnosed in 14% of patients referred with IA. ASqPCR for the KIT D816V mutation was a useful adjunct in helping identify those with systemic mastocytosis (SM) but not monoclonal mast cell activation syndrome (MMAS). A modified overall clonal prediction model was developed using clinical findings, a serum tryptase determination and ASqPCR. There was no evidence of a hyper-responsive mast cell phenotype in patients with IA. Conclusion Patients with clonal mast cell disease may present as idiopathic anaphylaxis. Distinct clinical and laboratory features may be used to select those patients more likely to have an underlying clonal mast cell disorder (MMAS or SM) and thus candidates for a bone marrow biopsy.

                Author and article information

                John Wiley and Sons Inc. (Hoboken )
                15 March 2022
                June 2022
                : 77
                : 6 ( doiID: 10.1111/all.v77.6 )
                : 1909-1911
                [ 1 ] Division of Immunology and Allergy Department of Medicine Solna Karolinska Institutet Stockholm Sweden
                [ 2 ] Clinical Immunology and Transfusion Medicine Karolinska University Hospital Stockholm Sweden
                [ 3 ] Department of Respiratory Medicine and Allergy Karolinska University Hospital Stockholm Sweden
                [ 4 ] Department of Medicine Huddinge Karolinska Institutet Stockholm Sweden
                [ 5 ] Department of Medical Sciences Uppsala University Uppsala Sweden
                Author notes
                [*] [* ] Correspondence

                Gunnar Nilsson, Division of Immunology and Allergy, Department of Medicine, J7:30 Bioclinicum Karolinska University Hospital, Karolinska Institutet, 171 64 Solna, Sweden.

                Email: gunnar.p.nilsson@ 123456ki.se

                © 2022 The Authors. Allergy published by European Academy of Allergy and Clinical Immunology and John Wiley & Sons Ltd.

                This is an open access article under the terms of the http://creativecommons.org/licenses/by-nc-nd/4.0/ License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non‐commercial and no modifications or adaptations are made.

                Page count
                Figures: 2, Tables: 0, Pages: 3, Words: 1214
                Funded by: the Konsul TH C Bergh foundation
                Funded by: Ellen, Walter and Lennart Hesselman Foundation for Scientific Research , doi 10.13039/501100013493;
                Funded by: the regional agreement on medical training and clinical research (ALF) between Stockholm Country Council and the Karolinska Institutet
                Funded by: Vetenskapsrådet , doi 10.13039/501100004359;
                Funded by: The Swedish Heart and Lung foundation
                Funded by: The Swedish Cancer Society
                Custom metadata
                June 2022
                Converter:WILEY_ML3GV2_TO_JATSPMC version:6.1.7 mode:remove_FC converted:26.07.2022

                histamine,ige,mast cells
                histamine, ige, mast cells


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