A mast cell degranulation screening assay for the identification of novel mast cell activating agents

Staats, Herman F.; Kirwan, Shaun M.; Choi, Hae Woong; Shelburne, Christopher P.; Abraham, Soman N.; Leung, Gulice Y. C.; Chen, David Y.‐K.

doi:10.1039/c2md20073b

Cited by 19 publications

(14 citation statements)

References 41 publications

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“…For the assay, adapted from 53 , cells were incubated in fresh medium for 18 hours prior to the experiment. If testing IgE activation, cells were also incubated with biotin-labeled IgE during this time.…”

Section: Methodsmentioning

confidence: 99%

“…LAD2 cells were maintained in Stem-Pro34 medium (GIBCO) supplemented with StemPro34 Supplement (GIBCO), 2 mM L-Glutamine, 100 U/mL penicillin/streptomycin, and recombinant human stem cell factor (100 ng/ml) (Peprotech) as described in 52 . For the assay, adapted from 53 , cells were incubated in fresh medium for 18 hours prior to the experiment. If testing IgE activation, cells were also incubated with biotin-labeled IgE during this time.…”

Section: Methodsmentioning

confidence: 99%

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In silico design of novel probes for the atypical opioid receptor MRGPRX2

et al. 2017

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The primate-exclusive MRGPRX2 G protein-coupled receptor (GPCR) has been suggested to modulate pain and itch. Despite putative peptide and small molecule MRGPRX2 agonists, selective nanomolar potency probes have not yet been reported. To identify a MRGPRX2 probe, we first screened 5,695 small molecules and found many opioid compounds activated MRGPRX2, including (−)- and (+)-morphine, hydrocodone, sinomenine, dextromethorphan and the prodynorphin-derived peptides, dynorphin A, dynorphin B, and α- and β-neoendorphin. We used these to select for mutagenesis-validated homology models and docked almost 4 million small molecules. From this docking, we predicted ZINC-3573, which represents a potent MRGPRX2-selective agonist, showing little activity against 315 other GPCRs and 97 representative kinases, and an essentially inactive enantiomer. ZINC-3573 activates endogenous MRGPRX2 in a human mast cell line inducing degranulation and calcium release. MRGPRX2 is a unique atypical opioid-like receptor important for modulating mast cell degranulation, which can now be specifically modulated with ZINC-3573.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

In silico design of novel probes for the atypical opioid receptor MRGPRX2

et al. 2017

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“…MC/9 mast cells from mouse fetal liver (American Type Culture Collection, Manassas, VA, USA, catalog number: CRL‐8306) , which are typical cells for mast cell‐activating agent screening , were used for in vitro experiments. The maintenance of MC/9 cells was described elsewhere .…”

Section: Methodsmentioning

confidence: 99%

Mast cells partially contribute to mucosal adjuvanticity of surfactin in mice

Yoshino

Takeshita

Kawamura

et al. 2017

Immunity Inflam & Disease

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IntroductionSurfactin (SF) is a cyclic lipopeptide that has potent mucosal adjuvant properties. However, immunological mechanisms of SF adjuvant action have not yet been elucidated. As some cyclic lipopeptides, such as polymyxin, can stimulate histamine release from mast cells, we hypothesized that mast cell activation is critical for SF adjuvanticity.Methods/ResultsWe observed that following intranasal immunization with ovalbumin (OVA) plus SF, the titers of the OVA‐specific antibody (Ab) in the mucosal secretions and plasma of mast cell‐deficient mice were significantly lower than those in congenic normal mice, although OVA‐specific Ab did not entirely disappear from mast cell‐deficient mice. SF induced degranulation of mast cells and release of histamine in vitro. To investigate whether SF stimulated mast cells in vivo, we measured body temperature of mice immunized intranasally with OVA plus SF because histamine level affects body temperature. Following immunizations, body temperature of immunized congenic normal mice transiently decreased, whereas body temperature of mast cell‐deficient mice did not change. Plasma levels of OVA‐specific IgE Ab were not significantly different in mast cell‐deficient and congenic normal mice. These findings suggest that SF directly affected mast cells in an IgE Ab‐independent fashion. Furthermore, we analyzed the effects of SF on MC/9 mast cells cultured in vitro. MC/9 cells stimulated by SF released not only histamine but also leukotriene B4 and prostaglandin D2. Moreover, SF up‐regulated mRNA expression levels of Tnf, Ccr5, and Il4 genes in mast cells. These cytokines may play a facilitating role in OVA‐specific immune responses in mice.ConclusionOverall, our results showed that mast cell activation partially mediated SF adjuvanticity.

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“…22 On MC stimulation, β-hexosaminidase can be released instantly, and its enzymatic activity is stably retained, making β-hexosaminidase release a useful indicator of MC degranulation. 20 We performed HTS of commercially available compound libraries using an in vitro β-hexosaminidase release assay using the MC/9 line of murine MCs. The assay procedure used 384-well plates and automated liquid-handling systems to minimize bias and enhance reproducibility.…”

Section: Assessment Of Variability In the Screening Assaymentioning

confidence: 99%

“…The readout in these assays is the release of β-hexosaminidase, which can be detected by its ability to produce a colorimetric change [in optical density (OD)] due to its activity on its substrate, 4-nitrophenyl N-acetyl-β-D-glucosaminide. 20 Triton X-100 was used as a positive control to lyse the MCs to measure the maximum release of β-hexosaminidase. Tyrode's buffer was used as the negative control.…”

Section: Assessment Of Variability In the Screening Assaymentioning

confidence: 99%

Identification of Novel Mast Cell Activators Using Cell-Based High-Throughput Screening

et al. 2019

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Mast cells (MCs) are known to regulate innate and adaptive immunity. MC activators have recently been described as safe and effective vaccine adjuvants. Many currently known MC activators are inadequate for in vivo applications, however, and research on identifying novel MC activators is limited. In this study, we identified novel MC activators by using high-throughput screening (HTS) assays using approximately 55,000 small molecules. Data sets obtained by the primary HTS assays were statistically evaluated using quality control rules and the B-score calculation, and compounds with B-scores of >3.0 were chosen as mast cell activators (hits). These hits were re-evaluated with secondary and tertiary HTS assays, followed by further statistical analysis. From these hits, we selected 15 compounds that caused degranulation in murine and human MCs, with potential for flexible chemical modification for further study. Among these 15 compounds, ST101036, ST029248, and ST026567 exhibited higher degranulation potency than other hit compounds in both human and mouse MCs. In addition, the 15 compounds identified promote de novo synthesis of cytokines and induce the release of eicosanoids from human and mouse MCs. HTS enabled us to identify small-molecule MC activators with unique properties that may be useful as vaccine adjuvants.

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A mast cell degranulation screening assay for the identification of novel mast cell activating agents

Cited by 19 publications

References 41 publications

In silico design of novel probes for the atypical opioid receptor MRGPRX2

In silico design of novel probes for the atypical opioid receptor MRGPRX2

Mast cells partially contribute to mucosal adjuvanticity of surfactin in mice

Identification of Novel Mast Cell Activators Using Cell-Based High-Throughput Screening

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