Β-hexosaminidase Release Research Articles

P16Ink4a-induced senescence in cultured mast cells as a model for ageing reveals significant morphological and functional changes

BackgroundMast cells (MCs) are tissue resident cells of the immune system, mainly known for their role in allergy. However, mounting evidence indicates their involvement in the pathology of age-related diseases, such as Alzheimer’s disease, Parkinson’s disease, and cancer. MC numbers increase in aged tissues, but how ageing affects MCs is poorly understood.ResultsWe show that MC ageing is associated with the increased expression of the cell cycle inhibitor p16 Ink4a, a marker and inducer of cellular senescence. Relying on this observation and the tight association of ageing with senescence, we developed a model of inducible senescence based on doxycycline-induced expression of p16Ink4a in cultured bone marrow derived MCs (BMMCs). Using this model, we show that senescent MCs upregulate IL-1β, TNF-α and VEGF-A. We also demonstrate that senescence causes marked morphological changes that impact MC function. Senescent MCs are larger, contain a larger number of secretory granules (SGs) and have less membrane protrusions. Particularly striking are the changes in their SGs, reflected in a significant reduction in the number of electron dense SGs with a concomitant increase in lucent SGs containing intraluminal vesicles. The changes in SG morphology are accompanied by changes in MC degranulation, including a significant increase in receptor-triggered release of CD63-positive extracellular vesicles (EVs) and the exteriorisation of proteoglycans, as opposed to a gradual inhibition of the release of β-hexosaminidase.ConclusionsThe inducible expression of p16Ink4a imposes MC senescence, providing a model for tracking the autonomous changes that occur in MCs during ageing. These changes include both morphological and functional alterations. In particular, the increased release of small EVs by senescent MCs suggests an enhanced ability to modulate neighbouring cells.

Quercetin Attenuates MRGPRX2-Mediated Mast Cell Degranulation via the MyD88/IKK/NF-κB and PI3K/AKT/ Rac1/Cdc42 Pathway.

CMRF35-like molecule-1 (CLM-1) is a receptor of the CD300 family that inhibits MRGPRX2-mediated mast cell degranulation. Understanding the role and mechanism of CLM-1 agonist has significant implications for the treatment of allergic disease. Quercetin is a natural small molecule compound derived from plants and vegetables that has been shown to prevent histamine release by immune cells. This study aims to examine the inhibitory effects of quercetin on MRGPRX2-mediated mast cell degranulation via CLM-1. We found that C48/80 stimulation resulted in significantly increased release of β-hexosaminidase, histamine and Ca2+ in CLM-1-knockdown LAD2 cells than in NC-LAD2 cells. Surface plasmon resonance (SPR) and molecular docking analyses revealed high-affinity binding between quercetin and CLM-1 (K D = 2.962×10-5 mol/L) mediated by the formation of hydrogen bonds. In addition, quercetin can selectively bind to CLM-1 on mast cells, leading to SHP-1 phosphorylation and subsequent inhibition of downstream MyD88/IKK/NF-κB signaling. Furthermore, activation of CLM-1 modulated the surface expression of MRGPRX2 by inhibiting F-actin, leading to internalization of the MRGPRX2 receptor via the PI3K/AKT/ Rac1/Cdc42 pathway. Quercetin is a promising treatment for allergic diseases by acting as a CLM-1 agonist that inhibits MRGPRX2-mediated mast cell degranulation.

Scutellaria baicalensis ameliorates allergic airway inflammation through agonism and transcriptional regulation of TAS2Rs

Ethnopharmacological relevanceScutellaria baicalensis Georgi (SCB, Huangqin) is a traditional medicinal plant used to treat fever and respiratory diseases. SCB has a good therapeutic effect on asthma and anti-inflammation in traditional clinic use. However, the molecular mechanism and targets of SCB in treating asthma are still unclear. Aim of the studyCombining transcriptomic analysis and in vitro experimental validation, this study aimed to reveal the molecular mechanism and targets of SCB in treating asthma. Materials and methodsThe anti-asthmatic effects of SCB and its active components, scutellarin and oroxylin A, were evaluated in ovalbumin (OVA)-induced rats by analysis of pulmonary function and pathology. The signaling pathways in rat pulmonary tissue were analyzed using transcriptomics and protein interaction network analysis. Calcium mobilization assay and molecular docking were utilized to discover the active compounds from SCB with agonism activity of type 2 taste receptors (TAS2Rs). The anti-asthmatic effect and transcriptional regulation of TAS2Rs regulated by SCB and its active components were analyzed in vitro. ResultsExtracts of SCB (ESB), scutellarin, and oroxylin A ameliorated airway function and inflammation in OVA-induced rats. The anti-asthma mechanism of ESB, scutellarin and oroxylin A was highly related to immune and taste transduction pathways based on transcriptomic analysis, especially the TAS2Rs signaling pathway. ESB was the direct agonist of TAS2R4 and TAS2R14 with EC50 of 209.1 and 217.2 μg/mL based on calcium mobilization assay, respectively. Baicalein was the main active component for TAS2R4 agonism activity, and scutellarin and oroxylin A had weak agonism activity of TAS2R4 and TAS2R14 through calcium mobilization assay and molecular docking. However, scutellarin and oroxylin A significantly upregulated the gene expression of Tas2r108 (the mouse ortholog of the TAS2R4) in lung tissue. ESB, scutellarin, and oroxylin A inhibited LPS-induced lactate dehydrogenase release and gene expression of TNF through transcriptional regulation of TAS2R4 and TAS2R14 on bronchial epithelial cells. ESB and oroxylin A ameliorated IgE-induced β-hexosaminidase release and gene expression of Il4 and Tnf and upregulated gene expression of Tas2r108. ConclusionThese results provided new insight into the anti-asthmatic mechanism of SCB and active components, scutellarin and oroxylin A, through agonism and transcriptional regulation of TAS2Rs to ameliorate allergic airway inflammation.

Guaijaverin and Epigallocatechin Gallate Complex Modulate Th1 and Th2 Cytokine-Mediated Allergic Responses Through STAT1/T-bet and STAT6/GATA3 Pathways.

We aimed to determine the in vitro and invivo synergistic antiallergic effect of guaijaverin and epigallocatechin gallate (EGCG) complex (GEC), and the antiallergic rhinitis (AR) properties of guaijaverin-rich Psidium guajava and EGCG-rich Camellia sinensis (ILS-F-2301). GEC showed synergistic inhibition of β-hexosaminidase by 4.20% and interleukin (IL)-4, -5, and -13 by 4.08%, 0.67%, and 4.71%, respectively, while increasing interferon (IFN)-γ by 12.43%, compared with EGCG only. In addition, 50 μg/mL of ILS-F-2301 inhibited β-hexosaminidase release, and inhibited IL-4, -5, and -13 by 61.54%, 58.79%, and 59.25%, respectively, while increasing IFN-γ (showing 133.14% activation). Moreover, 50 μg/mL of ILS-F-2301 suppressed p-STAT6 and GATA3, while p-STAT1 and T-bet increased, and 0.039 μg/mL of guaijaverin or 5.275 μg/mL of EGCG modulated T helper (Th)1- and Th2-related proteins. These data suggested that guaijaverin and EGCG in ILS-F-2301 was the main active compound involved in Th1/Th2 modulation. In the AR mouse model, the administration of ILS-F-2301 inhibited ovalbumin (OVA)-specific IgE, histamine in serum; it also inhibited IL-4 and -5 by 28.23% and 47.15%, respectively, while increasing IFN-γ (showing 37.11% activation), compared with OVA/Alu-treated mice. Taken together, our findings suggest that ILS-F-2301 is a functional food for alleviating anti-AR.

Nutraceutical formulation based on a synergic combination of melatonin and palmitoylethanolamide for the management of allergic events.

The management of allergic events is a growing global health issue, especially in industrialized countries. This disease is an immune-mediated process, regulated by the interaction of IgE with an allergen, resulting in mast cell activation, which concerns the release of several immune-inflammatory modulators, i.e., histamine, β-hexosaminidase, COX-2, IL-6, and TNF-α, responsible for the main allergic-reaction associated symptoms. The aim of the present study was the efficacy evaluation of an alternative remedy, an innovative nutraceutical formulation (NF) based on the synergic combination of melatonin (MEL) and palmitoylethanolamide (PEA) for the prevention and treatment of immune disease. At first, the intestinal bioaccessibility of PEA and MEL in NF was assessed at 1.6 and 36%, respectively. Then the MEL and PEA ability to modulate the release of immune-inflammatory modulators in the human mast cell line (HMC-1.2) at their bioaccessible concentration was investigated. Our results underline that NF treatment was able to reduce COX-2 mRNA transcription levels (-30% vs. STIM, p < 0.0001) in stimulated HMC-1.2 and to contract COX-2 enzymatic activity directly (IC50: 152 μg/mL). Additionally, NF showed valuable ability in reducing histamine and β-hexosaminidase release in stimulated HMC-1.2, as well as in decreasing TNF-α and IL-6 mRNA transcription levels and protein production.

Nematode Galectin Inhibits Basophilic Leukaemia RBL-2H3 Cells Apoptosis in IgE-Mediated Activation.

Mast cells are essential immune cells involved in the host's defence against gastrointestinal nematodes. To evade the immune response, parasitic nematodes produce a variety of molecules. Galectin 1, produced by Teladorsagia circumcincta (Tci-gal-1), reduces mast cell degranulation and selectively regulates mediator production and release in an IgE-dependent manner. To uncover the activity of Tci-gal-1, we have examined the effect of the protein on gene expression, protein production, and apoptosis in activated basophilic leukaemia RBL-2H3 cells. Rat RBL-2H3 cells were activated with anti-DNP IgE and DNP-HSA, and then treated with Tci-gal-1. Microarray analysis was used to examine gene expression. The levels of several apoptosis-related molecules and cytokines were determined using antibody arrays and ELISA. Early and late apoptosis was evaluated cytometrically. Degranulation of cells was determined by a β-hexosaminidase release assay. Treatment of activated RBL-2H3 cells with Tci-gal-1 resulted in inhibited apoptosis and decreased degranulation, although we did not detect significant changes in gene expression. The production of pro-apoptotic molecules, receptor for advanced glycation end products (RAGE) and Fas ligand (FasL), and the cytokines IL-9, IL-10, IL-13, TNF-α, and IL-2 was strongly inhibited. Tci-gal-1 modulates apoptosis, degranulation, and production of cytokines by activated RBL-2H3 cells without detectable influence on gene transcription. This parasite protein is crucial for modulation of the protective immune response and the inhibition of chronic inflammation driven by mast cell activity.

Shiikuwasha leaf and peel extracts inhibit allergic reactions by suppressing degranulation in RBL-2H3 rat basophilic leukemia cells and immunoglobulin production in mouse spleen lymphocytes.

This study aims to investigate the antiallergic effects of Shiikuwasha (Citrus depressa Hayata) leaf and peel extracts by examining the regulation of degranulation and inflammatory cytokine production from rat basophilic leukemia (RBL-2H3) cells and antigen-specific antibody production in sensitized mouse spleen lymphocytes. In vivo antiallergic activity was evaluated using the passive cutaneous anaphylaxis (PCA) reaction model. Extracts of Shiikuwasha leaves and peel were prepared using 80% methanol and dissolved in dimethylsulfoxide. The dinitrophenyl-human serum albumin-induced β-hexosaminidase levels in immunoglobulin (Ig) E-sensitized RBL-2H3 cells were assessed using enzymatic assays. Cytokine production was measured by enzyme-linked immunosorbent assay. Antibody production capacity was evaluated using lymphocytes isolated from spleens of type I allergy model mice. Lymphocytes were cultured for 72 h with Shiikuwasha extracts, and ovalbumin-specific IgE, IgG1, and IgG2a levels were measured. Shiikuwasha leaf and peel extract significantly reduced β-hexosaminidase release and suppressed interleukin-4 and tumor necrosis factor-α production from RBL-2H3 cells. Ovalbumin-specific IgE and IgG1 production decreased in Shiikuwasha extract-treated lymphocytes. These extracts also significantly suppressed the PCA reaction. Shiikuwasha leaf and peel extract reduce degranulation in RBL-2H3 cells and antibody production in spleen-derived lymphocytes and therefore exhibit antiallergic effects.

Exposure to Volatile Organic Compounds May Contribute to Atopic Dermatitis in Adults.

Volatile organic compounds (VOC) are major indoor air pollutants. Previous studies reported an association between VOC exposure and allergic diseases. Here, we aimed to explore the relationship between VOC exposure and atopic dermatitis (AD) in adults. We prospectively enrolled 31 adult AD patients and 11 healthy subjects as controls. Urine metabolite levels of VOCs, including 1.3-butadiene, acrylamide, benzene, toluene, and xylene, were all determined with liquid chromatography-mass spectrometry. The relationship between AD and log-transformed urine levels of VOC metabolites were examined using a multivariate linear regression model adjusted for age and sex. We also treated mouse bone marrow-derived cells (BMMCs) with 1,3-butadiene and toluene and measured the release of β-hexosaminidase. Our results demonstrated that creatinine-corrected urine levels of N-Acetyl-S- (3,4-dihydroxybutyl)-L-cysteine (DHBMA), N-Acetyl-S-(2-carbamoyl-2-hydroxyethyl)-L-cysteine (GAMA), and N-Acetyl-S-(benzyl)-L-cysteine (BMA) were all elevated in AD patients compared with controls. In a multivariate linear regression model, creatinine-corrected urine levels of BMA (a toluene metabolite) and DHBMA (a 1,3-butadiene metabolite) appeared elevated in AD patients, although statistical significance was not reached after correction for multiple comparisons. In addition, 1,3-butadiene and toluene could stimulate BMMCs to degranulate as much as compound 48/80. Some VOCs, such as 1,3-butadiene and toluene, might be associated with AD pathogenesis in adults.

Inhibitory effects of compounds isolated from Geranium wilfordii on IL-4 production and β-hexosaminidase release in RBL-2H3 cells

This study aimed to investigate the anti-allergic activity of compounds isolated from Geranium wilfordii Maxim. and to suggest potential therapeutic agents for allergies. Nine compounds were isolated from an ethanolic G. wilfordii extract using chromatographic methods and identified chemically and by spectroscopic analysis. These compounds were identified using reported literature data as brevifolin carboxylic acid (1), chlorogenic acid (2), corilagin (3), ellagic acid (4), geraniol (5), kaempferol 3-O-dirhamnoside (6), kaempferol 3-O-neohesperidoside (7), protocatechuic acid (8), and gallic acid (9). All nine identified compounds were assessed for including IL-4 mRNA expression and β-hexosaminidase release in RBL-2H3 cells stimulated with PMA/ionomycin or IgE + DNP-BSA. IL-4 gene expression assay showed that corilagin (3) potently inhibited IL-4 production, and β-hexosaminidase release assay showed that protocatechuic acid (8) markedly reduced histamine release. The study shows that of the nine compounds isolated from G. wilfordii, corilagin (3), and protocatechuic acid (8) are potential treatments for allergy-related diseases.

Inhibition of pseudo-allergic reactions by vitamin K3 directly targeting GAB1 in mast cells

BackgroundVitamin K3 (VK3), a fat-soluble synthetic analog of the vitamin K family, has coagulant, anti-inflammatory, antibacterial, and anticancer properties. Pseudo allergy is a IgE-independent immune response associated with mast cells. This study investigated the role of VK3 in IgE-independent mast cell activation. MethodsSubstance P (SP) was used to induce LAD2-cell activation in order to analyze the effects of VK3 in vitro. Cutaneous allergy and systemic allergy mouse models were used to analyze the anti-pseudo-allergic effects of VK3. Proteome microarray assays were used to analyze VK3-binding protein. Biolayer interferometry and immunoprecipitation were used to verify interaction between VK3 and its key targets. RNA interference was used to determine the role of GAB1 in LAD2cell activation. ResultsVK3 inhibited SP-induced LAD2-cell activation, and resulted in the release of β-hexosaminidase, histamine and cytokines; VK3 inhibited SP-induced pseudo allergic reactions in mice, and serum histamine and TNF-α levels decreased. Degranulation of skin mast cells was reduced; GAB1 in mast cells was stably bound to VK3. GAB1 participated in SP-induced LAD2-cell activation. GAB1 knockdown in LAD2 cells prevented SP-induced β-hexosaminidase release, calcium mobilization and cell skeletal remodeling. VK3 directly binds to GAB1 and reduces its expression to inhibited SP-induced LAD2 cell activation. ConclusionThe anti-pseudo-allergic activity of VK3 was confirmed in vitro and in vivo. VK3 can inhibit SP-induced mast cell activation by directly targeting GAB1. This study provides new insights on the activity of VK3 and the mechanism of pseudoallergic reaction.

Ameliorative effects of Wikstroemia trichotoma 95% EtOH extract on a mouse model of DNCB-induced atopic dermatitis

Ethnopharmacological relevanceThe genus Wikstroemia has been extensively utilized in traditional Chinese medicine (TCM) for the management of conditions such as coughs, edema, arthritis, and bronchitis. Studies have indicated that the crude extracts of Wikstroemia exhibit anti-inflammatory, anti-allergy, anti-aging, skin psoriasis, anti-cancer, and antiviral properties. In addition, these extracts are known to contain bioactive substances, including flavonoids, coumarins, and lignans. However, few studies have investigated the anti-inflammatory or anti-allergic activities of Wikstroemia trichotoma (Thunb.) Makino against atopic dermatitis (AD). Aim of the studyThe study aimed to explore the potential of a 95% ethanol extract of W. trichotoma (WTE) on the dysfunction of skin barrier and immune system, which are primary symptoms of AD, in 2,4-dinitrochlorobenzene (DNCB)-induced SKH-1 hairless mice and phorbol 12-myristate 13-acetate (PMA)/ionomycin or immunoglobulin E (IgE) + 2,4-dinitrophenylated bovine serum albumin (DNP-BSA) stimulated rat basophilic leukemia cell line (RBL-2H3). Furthermore, we sought to identify the chemical contents of WTE using high-performance liquid chromatography equipped with a photodiode array detector (HPLC-PDA). Materials and methodsAn in vitro study was conducted using RBL-2H3 cells stimulated with PMA/ionomycin or IgE + DNP-BSA to assess the inhibitory effects of WTE on mast cell degranulation and interleukin-4 (IL-4) mRNA expression levels. For the in vivo study, AD was induced in SKH-1 hairless mice by applying 1% DNCB to the dorsal skin daily for 7 days. Subsequently, 0.1% DNCB solution was applied on alternate days, and mice were orally administered WTE (at 30 or 100 mg/kg/day) dissolved in 0.5% carboxymethyl cellulose (CMC) daily for 2 weeks. Transepidermal water loss (TEWL), skin hydration, skin pH, and total serum IgE levels were measured. ResultsIn DNCB-stimulated SKH-1 hairless mice, WTE administration significantly improved AD symptoms and ameliorated dorsal skin inflammation. Oral administration of WTE led to a significant decrease in skin thickness, infiltration of mast cells, and level of total serum IgE, thus restoring skin barrier function in the DNCB-induced skin lesions. In addition, WTE inhibited β-hexosaminidase release and reduced IL-4 mRNA levels in RBL-2H3 cells. Chemical profile analysis of WTE confirmed the presence of three phenolic compounds, viz. chlorogenic acid, miconioside B, and matteucinol-7-O-β-apiofuranosyl (1 → 6)-β-glucopyranoside. ConclusionsWTE ameliorates AD symptoms by modulating in the skin barrier and immune system dysfunction. This suggests that W. trichotoma extract may offer therapeutic benefits for managing AD.

Role of ESCCAL-1 in regulating exocytosis of AuNPs in human esophageal squamous carcinoma cells

Exocytosis is a critical factor for designing efficient nanocarriers and determining cytotoxicity. However, the research on the exocytosis mechanism of nanoparticles, especially the role of long non-coding RNAs (lncRNAs), has not been reported. In this study, the exocytosis of AuNPs in the KYSE70 cells and the involved molecular pathways of exocytosis are analyzed. It demonstrates that nanoparticles underwent time-dependent release from the cells by exocytosis, and the release of β-hexosaminidase confirms that AuNPs are excreted through lysosomes. Mechanistic studies reveal that lncRNA ESCCAL-1 plays a vital role in controlling the exocytosis of AuNPs through activation of the MAPK pathway, including the phosphorylation of ERK and JNK. The study implies that the ESCCAL-1-mediated pathway plays an important role in the exocytosis of AuNPs in KYSE70 cells. This finding has implications for the role of ESCCAL-1 on the drug resistance of esophagus cancer by controlling lysosome-mediated exocytosis.

Traditional cooking methods decreased the allergenicity of egg proteins.

Egg allergy is one of the most common food allergies globally. This study aimed to assess the impact of four traditional cooking methods on the allergenicity of egg proteins using a comprehensive strategy, including simulated gastrointestinal digestion in vitro, serology experiments, a rat basophilic leukemia (RBL)-2H3 cell degranulation model, and a passive cutaneous anaphylaxis (PCA) mice model, and the structure changes were detected by circular dichroism (CD)spectra and ultraviolet (UV) spectra. The results showed that the processed egg proteins were more readily digested compared to raw egg proteins. The serological experiments revealed a significant reduction in immunoglobulin E binding of egg proteins after thermal treatments (p<0.05), particularly after frying. Subsequently, the RBL-2H3 cell degranulation experiment demonstrated a marked decrease in the level of egg allergens-induced β-hexosaminidase release after cooking (p<0.05). Moreover, the results from the PCA mice model indicated that the increase in vascular permeability was effectively relieved in the treated groups, especially in frying group (p<0.05). Additionally, the α-helix and β-turn contents of processed egg proteins were significantly decreased (p<0.05) compared with native egg proteins. The UV spectra findings showed that all cooking treatments caused significant alterations in the tertiary structure, and fluorescence analysis indicated that cooking decreased the surface hydrophobicity of egg proteins. In conclusion, four traditional cooking methods reduced the allergenicity of egg proteins, particularly frying, and this reduction was associated with structural changes that could contribute to the destruction or masking of epitopes of egg allergens. PRACTICAL APPLICATION: Egg allergy has a serious impact on public health, and there is no ideal treatment method at present. This study demonstrated that four traditional cooking methods (boiling, steaming, baking, and frying) reduced the allergenicity of egg proteins, especially frying, and the results will provide a basis for the development of hypoallergenic egg products.

Se-rich tea polysaccharide extracted by high hydrostatic pressure attenuated anaphylaxis by improving gut microbiota and metabolic regulation

Selenium-rich tea polysaccharides (Se-TPS) were extracted via high hydrostatic pressure technology with a pressure of 400 MPa (200–500 MPa) for 10 min (3–20 min) at a material-to-solvent ratio of 1:40 (1:20–1:50). Subsequently, Se-TPS1–4 were isolated and purified, with Se-TPS3–4 as the main components. A spectral analysis proved that Se, which has antioxidant activity, existed. An in vitro study found that among Se-TPS, Se-TPS3–4 attenuated the release of β-hexosaminidase, histamine, and interleukin (IL)-4. Furthermore, in vivo experiments revealed that treatment with Se-TPS downregulated IL-4 levels and upregulated TGF-β and interferon-γ levels to improve imbalanced Th1/Th2 immunity in tropomyosin-sensitized mice. Moreover, Se-TPS promoted Lactobacillus and norank_f_Muribaculaceaek growth and upregulated metabolites such as genipin and coniferyl alcohol. Overall, these results showed the strong anti-allergy potential of Se-TPS by regulating mast cell-mediated allergic inflammatory responses and microbiota regulation, highlighting the potential of Se-TPS as a novel therapeutic agent to regulate allergy-associated metabolic disorders.

Mast cell degranulation is greater when exposed to dialysate collected from exercising skeletal muscle

The effect of histamine is multifaceted and potentially triggers a broad range of cellular adaptations contributing to vascular function, cellular maintenance, metabolism, and inflammation. During exercise, histamine is released from skeletal muscle. This response and the subsequent actions of histamine appear important in obtaining many of the beneficial adaptations to exercise. Increases in exercising skeletal muscle histamine release, in part, occur due to mast cell degranulation. However, the direct effect of exercise on mast cell degranulation has yet to be elucidated. To that end, this preliminary study aimed to quantify in vitro mast cell degranulation when cells are exposed to dialysate taken from exercising and non-exercising skeletal muscle. We hypothesized that greater mast cell degranulation would occur when exposed to dialysate collected from exercising skeletal muscle compared to dialysate collected from non-exercising muscle. Ten subjects (5M/5F, age 26 ± 5 years, weight 76 ± 17 kg, height 168 ± 12 cm) completed 60 minutes of single-leg dynamic knee extension exercise. Prior to exercise, a microdialysis probe was inserted into the vastus lateralis of the exercising and non-exercising legs and perfused with saline at a rate of 5 uL/min. Dialysate was collected before and every 20 minutes during exercise and the subsequent one-hour recovery period. In the exercising skeletal muscle, mast cell degranulation (quantified via b-hexosaminidase release) was greater when exposed to dialysate during exercise versus dialysate collected pre-exercise (P &lt; 0.05). Compared to non-exercising leg dialysate, degranulation was greater for exercising leg dialysate taken between minutes 20 and 40 into exercise (20.6 ± 2.7% vs 15.8 ± 2.0%, P &lt; 0.05) and 40 and 60 into exercise (24.0 ± 2.6 % vs 15.0 ± 2.7 %, P &lt; 0.001) and remained greater for dialysate taken in the first 20 minutes of recovery (21.4 ± 2.8% vs 15.2 ± 3.0%, P &lt; 0.05). These data suggest that there is a soluble factor released from exercising skeletal muscle that causes mast cell degranulation in vitro. These preliminary findings establish a bioassay for further exploration of the identity of the soluble factor released from exercising skeletal muscle, which is responsible for mast cell degranulation in vivo. This research was supported in part by National Institutes of Health Grants AG072805 and HL115027. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Benastatin K, a chlorinated benastatin-related antibiotic from Streptomyces sp. HGTA384.

Benastatin K (1), a new chlorinated benastatin derivative, was isolated from the culture broth of the actinomycete Streptomyces sp. HGTA384. The structure of 1 was determined on the basis of spectroscopic analysis, including 1D and 2D NMR, as well as HRESI-MS, UV and IR, and comparison with data reported in the literature. Compound 1 and benastatins A and B exhibited inhibitory activity against Micrococcus luteus (MIC 7.8, 31.3, and 3.9 μM, respectively), and IgE-mediated β-hexosaminidase release in RBL-2H3 cells with IC50 values of 42, 79, and 19 μM, respectively.

Comparative analyses of various IgE-mediated and non-IgE-mediated inducers of mast cell degranulation for in vitro study.

In vitro investigations of mast cell (MC) degranulation are essential for studying many diseases, particularly allergy and urticaria. Many MC-degranulation inducers are currently available. However, there is no previous systematic comparative analysis of these available inducers in term of their efficacies to induce MC degranulation. Herein, we performed systematic comparisons of efficacies of five well-known and commonly used MC-degranulation inducers. RBL-2H3 cells were sensitized with 50ng/ml anti-DNP IgE or biotinylated IgE followed by stimulation with 100ng/ml DNP-BSA or streptavidin, respectively. For non-IgE-mediated inducers, the cells were treated with 5µg/ml substance P, compound 48/80, or A23187. At 15-, 30-, 45- and 60-min post-induction, several common MC-degranulation markers (including intracellular [Ca2+], β-hexosaminidase release, tryptase expression by immunofluorescence staining, cellular tryptase level by immunoblotting, secretory tryptase level by immunoblotting, CD63 expression by immunofluorescence staining, and CD63 expression by flow cytometry) were evaluated. The data showed that all these markers significantly increased after activation by all inducers. Among them, A23187 provided the greatest degrees of increases in intracellular [Ca2+] and β-hexosaminidase release at all time-points and upregulation of CD63 at one time-point. These data indicate that all these IgE-mediated (anti-DNP IgE/DNP-BSA and biotinylated IgE/streptavidin) and non-IgE-mediated (substance P, compound 48/80, and A23187) inducers effectively induce MC degranulation, while A23187 seems to be the most effective inducer for MC degranulation.

PTGS2: A potential immune regulator and therapeutic target for chronic spontaneous urticaria

AimsChronic spontaneous urticaria (CSU) is a common and debilitating skin disease that is difficult to control with existing treatments, and the pathogenesis of CSU has not been fully revealed. The aim of this study was to explore the underlying mechanisms of CSU and identify potential treatments. Materials and methodsMicroarray datasets of CSU were obtained from Gene Expression Omnibus database. Differentially expressed genes between skin lesions of CSU and normal controls (LNS-DEGs) were identified, and the enrichment analyses of LNS-DEGs were performed. Hub genes of LNS-DEGs were selected by protein–protein interaction analysis. The co-expression and transcriptional regulatory networks of hub genes were conducted using GeneMANIA and TRRUST database, respectively. CIBERSORT was utilized for immune cell infiltration analysis. Experimental validation was performed by β-hexosaminidase release examination and passive cutaneous anaphylaxis (PCA) mouse model. Key findingsA total of 247 LNS-DEGs were identified, which were enriched in cell migration, cell chemotaxis, and inflammatory pathways such as TNF and interleukin (IL) -17 signaling pathway. Among LNS-DEGs, seven upregulated (PTGS2, CCL2, IL1B, CXCL1, IL6, VCAM1, ICAM1) and one downregulated hub gene (PECAM1) were selected. Immune infiltration analysis identified eight different immune cells, such as activated/resting mast cells and neutrophils. Furthermore, PTGS2, encoding cyclooxygenase 2 (COX2), was selected for further validation. COX2 inhibitor, celecoxib, significantly inhibited mast cell degranulation, and reduced vascular permeability and inflammatory cytokine expression in PCA mouse model. SignificancePTGS2 may be a potential regulator of immunity and inflammation in CSU. Targeting PTGS2 is a new perspective for CSU treatment.

Fucosterol isolated from Sargassum horneri attenuates allergic responses in immunoglobulin E/bovine serum albumin-stimulated mast cells and passive cutaneous anaphylaxis in mice

Allergic diseases have become a serious problem worldwide and occur when the immune system overreacts to stimuli. Sargassum horneri is an edible marine brown alga with pharmacological relevance in treating various allergy-related conditions. Therefore, this study aimed to investigate the effect of fucosterol (FST) isolated from S. horneri on immunoglobulin E(IgE)/bovine serum albumin (BSA)-stimulated allergic reactions in mouse bone marrow-derived cultured mast cells (BMCMCs) and passive cutaneous anaphylaxis (PCA) in BALB/c mice. The in silico analysis results revealed the binding site modulatory potential of FST on the IgE and IgE-FcεRI complex. The findings of the study revealed that FST significantly suppressed the degranulation of IgE/BSA-stimulated BMCMCs by inhibiting the release of β-hexosaminidase and histamine in a dose-dependent manner. In addition, FST effectively decreased the expression of FcεRI on the surface of BMCMCs and its IgE binding. FST dose-dependently downregulated the expression of allergy-related cytokines (interleukin (IL)-4, -5, -6, -13, tumor necrosis factor (TNF)-α, and a chemokine (thymus and activation-regulated chemokine (TARC)) by suppressing the activation of nuclear factor-κB (NF-κB) and Syk-LAT-ERK-Gab2 signaling in IgE/BSA-stimulated BMCMCs. As per the histological analysis results of the in vivo studies with IgE-mediated PCA in BALB/c mice, FST treatment effectively attenuated the PCA reactions. These findings suggest that FST has an immunopharmacological potential as a naturally available bioactive compound for treating allergic reactions.

Anti-allergic effect of vitamin C through inhibiting degranulation and regulating TH1/TH2 cell polarization.

Food allergy has become a global public health problem. This study aimed to explore the possible anti-allergic effect of vitamin C (VC). A rat basophilic leukemia (RBL)-2H3 cell degranulation model was used to assess the effect of VC on degranulation in vitro, and an ovalbumin (OVA)-induced BALB/c mouse allergy model was used to assess the anti-allergy effect of VC in vivo. In vitro, VC significantly attenuated the release of β-hexosaminidase, tryptase and histamine, and also reduced cytokine production (interleukins 4 and 6, tumor necrosis factor α) significantly (P < 0.05), with the inhibitory effect demonstrating a positive correlation with VC dose. In vivo, compared with the OVA group, the levels of serum immunoglobulins E and G1 of the VC low-dose (VCL) group (50 mg kg-1) and high-dose (VCH) group (200 mg·kg-1) were significantly reduced (P < 0.05). Furthermore, the plasma histamine level was also significantly decreased (P < 0.05). Moreover, TH2 cell polarization in mice of the VCL and VCH groups was significantly inhibited (P < 0.05), promoting the TH1/TH2 cell polarization balance. Additionally, VC treatment enhanced the expression of CD80 (P < 0.05) in spleen and small intestine tissues, while significantly inhibiting the expression of CD86 (P < 0.05); notably, high-dose VC treatment was more effective. VC exerted an anti-allergic effect through inhibiting degranulation and regulating TH1/TH2 cell polarization balance. © 2024 Society of Chemical Industry.