Rat Basophilic Leukemia Research Articles

Cationic liposomes bearing Bet v 1 by coiled coil-formation are hypo-allergenic and induce strong immunogenicity in mice.

Although aluminum hydroxide (alum) is widely accepted and used as safe vaccine adjuvant, there is some concern about possible toxicity upon long-lasting repeated exposure during subcutaneous allergen immunotherapy (SCIT). Our objective was to evaluate allergen-bearing liposomes as possible alternative for alum-adsorption in SCIT. A self-assembling, coiled-coil forming peptide pair was used to anchor the major birch pollen allergen Bet v 1 to the surface of cationic liposomes. The resulting nanoparticulate liposomes were characterized with respect to their physicochemical, allergenic and immunological properties. Allergenicity was studied by ImmunoCAP inhibition and rat basophil leukemia (RBL) cell assays. Immunogenicity (immunoglobulin responses) and immune skewing (cytokine responses) were evaluated upon immunization of naïve mice, and compared to alum-adsorbed Bet v 1. Bet v 1-bearing cationic liposomes with a diameter of ∼200 nm showed a positive zeta potential. The coiled-coil conjugation of Bet v 1 to the surface of liposomes resulted in about a 15-fold lower allergenicity than soluble Bet v 1 as judged by RBL assays. Moreover, the nanoparticles induced Bet v 1-specific IgG1/IgG2a responses in mice that were several orders of magnitude higher than those induced by alum-adsorbed Bet v 1. This strong humoral response was accompanied by a relatively strong IL-10 induction upon PBMC stimulation with Bet v 1. In conclusion, their hypo-allergenic properties, combined with their capacity to induce a strong humoral immune response and a relatively strong IL-10 production, makes these allergen-covered cationic liposomes a promising alternative for aluminum salt-adsorption of allergen currently used in SCIT.

Generation and Characterization of Native and Sialic Acid-Deficient IgE.

Efficient characterization of IgE antibodies and their glycan structures is required for understanding their function in allergy and in the emerging AllergoOncology field for antibody immunotherapy. We report the generation, glyco-profiling and functional analysis of native and sialic acid-deficient glyco-engineered human IgE. The antibodies produced from human embryonic kidney cells were purified via a human IgE class-specific affinity matrix and structural integrity was confirmed by SDS-PAGE and size-exclusion chromatography (SEC). Purified IgEs specific for the tumor-associated antigens Chondroitin Sulfate Proteoglycan 4 (CSPG4-IgE) and Human Epidermal Growth Factor Receptor 2 (HER2-IgE) were devoid of by-products such as free light chains. Using neuraminidase-A, we generated sialic acid-deficient CSPG4-IgE as example glyco-engineered antibody. Comparative glycan analyses of native and glyco-engineered IgEs by Hydrophilic interaction liquid chromatography (HILIC)-high performance liquid chromatography (HPLC) indicated loss of sialic acid terminal residues and differential glycan profiles. Native and glyco-engineered CSPG4-IgEs recognized Fc receptors on the surface of human FcεRI-expressing rat basophilic leukemia RBL-SX38 cells, and of CD23/FcεRII-expressing human RPMI-8866 B-lymphocytes and bound to CSPG4-expressing A2058 human melanoma cells, confirming Fab-mediated recognition. When cross-linked on the cell surface, both IgEs triggered RBL-SX38 degranulation. We demonstrate efficient generation and functional competence of recombinant native and sialic acid-deficient IgEs.

Adenosine cyclic phosphate with ultrasonic-assisted pectinase extraction alleviated allergic reactions in RBL-2H3 through inhibiting the influx of intracellular Ca2+

Jujube contains abundant cyclic adenosine monophosphate (cAMP) and the ultrasonic-assisted pectinase extraction (UAPE) conditions for obtaining the maximum cAMP yield from jujube were optimized. Orthogonal array design was applied to evaluate the effects of 4 variables by UAPE on cAMP yield. The results showed that the optimal cAMP yield (783.0 μg/g) was derived at ratio of liquid to solid 5 mL/g, ratio of pectinase to raw material 1.5 %, time 60 min and temperature 40 °C. Moreover, the effect of cAMP on the anti-allergic function of action induced by immunoglobulin E (IgE) and its meschanism was investigated through establishing the sensitized cell model in rat basophilic leukemia (RBL-2H3) cells using dinitrophenylated (DNP)-bovine serum albumin (BSA)-IgE. The results showed that cAMP interfered with sensitized cells, effectively inhibited the occurrence of basophil degranulation in dose dependence, and significantly reduced the activity of β-hexosamindase (β-hex), at the optimal concentration of 50 μg/mL. The level of anti-inflammatory factor interleukin-10 (IL-10) was promoted and the content of pro-inflammatory factor tumor necrosis factor-α (TNF-α) was suppressed by cAMP. In addition, influx of intracellular Ca2+ was repressed effectively. Our results demonstrate that jujube cAMP regulated the cytokine balance in the allergy pathway through blocking the influx of extracellular Ca2+, with the prevention of allergy symptoms.

GRK2 inhibitors, paroxetine and CCG258747, attenuate IgE-mediated anaphylaxis but activate mast cells via MRGPRX2 and MRGPRB2.

G protein-coupled receptor (GPCR) kinase 2 (GRK2), which phosphorylates agonist-occupied GPCRs to promote their desensitization, has been investigated as an attractive therapeutic target for cardiovascular and metabolic diseases. Several GRK2-targeted inhibition strategies have been reported including the use of direct pharmacological inhibitors such as paroxetine (a widely prescribed antidepressant) and its analogs such as compound CCG258747. Cross-linking of high affinity IgE receptor (FcϵRI) on mast cells (MCs) and the resulting degranulation causes anaphylaxis and allergic asthma. Using gene silencing strategy, we recently showed that GRK2 contributes to FcεRI signaling and MC degranulation. The purpose of this study was to determine if the GRK2 inhibitors paroxetine and CCG258747 modulate FcεRI-mediated MC responses in vitro and in vivo. Utilizing rat basophilic leukemia (RBL-2H3) cells and primary mouse lung MCs (LMCs), we found that paroxetine and CCG258747 inhibit FcϵRI-mediated calcium mobilization and degranulation. Furthermore, intravenous administration of paroxetine and CCG258747 in mice resulted in substantial reduction of IgE-mediated passive cutaneous anaphylaxis. Unlike LMCs, human cutaneous MCs abundantly express a novel GPCR known as MRGPRX2 (mouse; MRGPRB2). We found that in contrast to their inhibitory effects on FcεRI-mediated MC responses, both paroxetine and CCG258747 induce calcium mobilization and degranulation in RBL-2H3 cells stably expressing MRGPRX2 but not in untransfected cells. Furthermore, paroxetine and CCG258747 induced degranulation in peritoneal MCs from Wild-type (WT) mice in vitro and caused increased cutaneous vascular permeability in vivo, but these responses were substantially reduced in Mrgprb2−/− mice. Additionally, upon intradermal injection, paroxetine also induced neutrophil recruitment in WT but not Mrgprb2−/− mice. These findings suggest that in addition to their potential therapeutic utility against cardiovascular and metabolic disorders, paroxetine-based GRK2-inhibitors may serve to modulate IgE-mediated anaphylaxis and to enhance cutaneous host defense by harnessing MC’s immunomodulatory property through the activation of MRGPRX2/MRGPRB2.

Betv1-displaying elastin-like polypeptide nanoparticles induce a strong humoral and weak CD4+ T-cell response against Betv1 in a murine immunogenicity model.

There is growing concern about the toxicity of colloidal aluminum salts used as adjuvants in subcutaneous allergen immunotherapy (SCIT). Therefore, alternative adjuvants and delivery systems are being explored to replace alum in SCIT. We applied micellar elastin-like polypeptides (ELPs), a type of self-assembling protein, to replace alum as vaccine adjuvant in birch pollen SCIT. ELP and an ELP-Bet v 1 fusion protein were expressed in E. coli and purified by immuno-affinity chromatography and inverse-transition cycling (ITC). Nanoparticles self-assembled from ELP and a 9:1 ELP/ELP-Bet v 1 mixture were characterized by using dynamic light scattering and atomic force microscopy. Allergenicity was assessed by measuring mediator release from rat basophilic leukemia cells transformed with the human FcϵR1 and sensitized with sera derived from human birch pollen allergic patients. Humoral and T-cell immunity were investigated by immunizing naïve mice with the ELP/ELP-Bet v 1 nanoparticles or alum-adsorbed Bet v 1, both containing 36 µg Bet v 1. ELP and ELP/ELP-Bet v 1 self-assembled at 37°C into spherically shaped micelles with a diameter of ~45 nm. ELP conjugation made Bet v 1 hypo-allergenic (10-fold). Compared to alum-adsorbed Bet v 1, ELP/ELP-Bet v 1 nanoparticles induced stronger IgG responses with an earlier onset. Additionally, ELP/ELP-Bet v 1 did not induce Th2 skewing cytokines and IgE. The hypoallergenic character and strong humoral immune response in the absence of a Th2-skewing T-cell response make ELP-based nanoparticles a promising candidate to replace alum in SCIT.

Novel peanut-specific human IgE monoclonal antibodies enable screens for inhibitors of the effector phase in food allergy.

Background10% of US residents have food allergies, including 2% with peanut allergy. Mast cell mediators released during the allergy effector phase drive allergic reactions. Therefore, targeting sensitized mast cells may prevent food allergy symptoms.ObjectiveWe used novel, human, allergen-specific, IgE monoclonal antibodies (mAbs) created using human hybridoma techniques to design an in vitro system to evaluate potential therapeutics targeting sensitized effector cells.MethodsTwo human IgE mAbs specific for peanut, generated through human hybridoma techniques, were used to sensitize rat basophilic leukemia (RBL) SX-38 cells expressing the human IgE receptor (FcϵRI). Beta-hexosaminidase release (a marker of degranulation), cytokine production, and phosphorylation of signal transduction proteins downstream of FcϵRI were measured after stimulation with peanut. Degranulation was also measured after engaging inhibitory receptors CD300a and Siglec-8.ResultsPeanut-specific human IgE mAbs bound FcϵRI, triggering degranulation after stimulation with peanut in RBL SX-38 cells. Sensitized RBL SX-38 cells stimulated with peanut increased levels of phosphorylated SYK and ERK, signal transduction proteins downstream of FcϵRI. Engaging inhibitory cell surface receptors CD300a or Siglec-8 blunted peanut-specific activation.ConclusionAllergen-specific human IgE mAbs, expressed from human hybridomas and specific for a clinically relevant food allergen, passively sensitize allergy effector cells central to the in vitro models of the effector phase of food allergy. Peanut reproducibly activates and induces degranulation of RBL SX-38 cells sensitized with peanut-specific human IgE mAbs. This system provides a unique screening tool to assess the efficacy of therapeutics that target allergy effector cells and inhibit food allergen-induced effector cell activation.

Identification of components in coriander (Coriandrum sativum L.) inhibiting degranulation of RBL-2H3 cells

We found that a water-soluble extract of coriander (Coriandrum sativum L.) (leaves, petioles and stems) inhibits antigen-induced degranulation of RBL-2H3 cells, a rat basophil leukemia cell line. The aim of this study was to elucidate the anti-degranulation active components in the extract. The methanol-eluate fraction obtained by fractionation of the water-soluble extract using MCI gel column chromatography had strong activity, and eight components were isolated and identified. Two of them were identified as new compounds, (3S)-3-methyl-6-hydroxyisocoumarin 8-O-β-D-glucopyranoside (compound 1) and (7S,8R)-7,8-dihydro-8-β-D-glucopyranosyloxy-4-methoxy-7-methyl-5H-fro[2,3-g][2]benzopyran-5-one (compound 2). As a result of evaluation of anti-degranulation activity of eight components, seven of them, such as tryptophan, phenylalanine, dihydroxycoumarin glucoside, quercetin glycoside, rutin, compound 1, and compound 2, had the activity. These results indicated that the water-soluble extract of coriander contains several anti-degranulation substances.

The Humanised NPY-mRFP RBL Reporter Cell Line Is a Fast and Inexpensive Tool for Detection of Allergen-Specific IgE in Human Sera.

Rat basophilic leukaemia (RBL) cells have been used for decades as a model of high-affinity Immunoglobulin E (IgE) receptor (FcεRI) signalling. Here, we describe the generation and use of huNPY-mRFP, a new humanised fluorescent IgE reporter cell line. Fusion of Neuropeptide Y (NPY) with monomeric red fluorescent protein (mRFP) results in targeting of fluorescence to the granules and its fast release into the supernatant upon IgE-dependent stimulation. Following overnight sensitisation with serum, optimal release of fluorescence upon dose-dependent stimulation with allergen-containing extracts could be measured after 45 min, without cell lysis or addition of any reagents. Five substitutions (D194A, K212A, K216A, K226A, and K230A) were introduced into the FcεRIα cDNA used for transfection, which resulted in the removal of known endoplasmic reticulum retention signals and high surface expression of human FcεRIα* in huNPY-mRFP cells (where * denotes the penta-substituted variant), comparable to the ~500,000 FcεRIα molecules per cell in the RS-ATL8 humanised luciferase reporter, which is a human FcεRIα/FcεRIγ double transfectant. The huNPY-mRFP reporter was used to demonstrate engagement of specific IgE in sera of Echinococcus granulosus-infected individuals by E. granulosus elongation factor EgEF-1β and, to a lesser extent, by EgEF-1δ, which had been previously described as IgE-immunoreactive EgEF-1β/δ.

Polystyrene micro(nano)plastics damage the organelles of RBL-2H3 cells and promote MOAP-1 to induce apoptosis

The ubiquity of microplastics increases the exposure risks and health threats to humans. In this study, rat basophilic leukemia (RBL-2H3) cells were exposed to polystyrene particles (PS-particles) of 50 nm, 500 nm and 5 µm to investigate organelle damage and the mechanism of cell death. PS-particles induced oxidative stress, which in turn led to mitochondrial and lysosomal damage, arrested the cell cycle in the G0/G1 phase, and finally caused apoptosis. Anti-apoptotic genes (Bcl-2) were down regulated, and pro-apoptotic genes (Bax) and a key gene (caspase-3) in apoptosis were upregulated. The molecular mechanism of apoptosis was further explored via the combination of transcriptome sequencing, RT-qPCR verification and small interfering RNA (siRNA) technology. The modulator of apoptosis-1 (MOAP-1) was significantly upregulated, and apoptosis was abolished by knocking down MOAP-1. This finding clarifies that PS-particles promote MOAP-1 to induce apoptosis. Hence, PS-particles may promote the binding of MOAP-1 and Bax, which ultimately activates caspase-3 and causes apoptosis through the mitochondrial pathway. The 50-nm PS-particles resulted in the most serious mitochondrial damage and apoptosis. Eventually, PS-particles cause oxidative stress, damage organelles and induce apoptosis by promoting MOAP-1. Altogether, our study emphasizes the need to assess the cytotoxicity of micro(nano)plastics and helps to predict the health risks.

Umbelliferose Isolated from Cuminum cyminum L. Seeds Inhibits Antigen-Induced Degranulation in Rat Basophilic Leukemia RBL-2H3 Cells

Cuminum cyminum L. (cumin) is an annual plant of the Umbelliferae family native to Egypt. We previously showed that the aqueous extract of cumin seeds suppresses degranulation by downregulating the activation of antigen-induced intracellular signaling molecules in rat basophilic leukemia RBL-2H3 cells. However, the active substances in the extract have not yet been identified. Accordingly, herein, we aimed to ascertain the water-soluble substances present in cumin seeds that inhibit degranulation, which led to the identification of umbelliferose, a characteristic trisaccharide present in plants of the Umbelliferae family. Our study is the first to reveal the degranulation-suppressing activity of umbelliferose, and quantification studies suggest that cumin seed powder contains 1.6% umbelliferose. Raffinose, an isomer of umbelliferose, was also found to significantly suppress antigen-induced degranulation, but less so than umbelliferose. Both umbelliferose and raffinose contain sucrose subunits in their structures, with galactose moieties bound at different sites. These differences in structure suggest that the binding of galactose to the sucrose subunit at the α1-2 bond contributes to its strong degranulation-inhibiting properties.

Anti-pseudo-allergic components in licorice extract inhibit mast cell degranulation and calcium influx.

Pseudo-allergic reactions (PARs) widely occur upon application of drugs or functional foods. Anti-pseudo-allergic ingredients from natural products have attracted much attention. This study aimed to investigate anti-pseudo-allergic compounds in licorice. The anti-pseudo-allergic effect of licorice extract was evaluated in rat basophilic leukemia 2H3 (RBL-2H3) cells. Anti-pseudo-allergic compounds were screened by using RBL-2H3 cell extraction and the effects of target components were verified further in RBL-2H3 cells, mouse peritoneal mast cells (MPMCs) and mice. Molecular docking and human MRGPRX2-expressing HEK293T cells (MRGPRX2-HEK293T cells) extraction were performed to determine the potential ligands of MAS-related G protein-coupled receptor-X2 (MRGPRX2), a pivotal target for PARs. Glycyrrhizic acid (GA) and licorice chalcone A (LA) were screened and shown to inhibit Compound48/80-induced degranulation and calcium influx in RBL-2H3 cells. GA and LA also inhibited degranulation in MPMCs and increase of histamine and TNF-α in mice. LA could bind to MRGPRX2, as determined by molecular docking and MRGPRX2-HEK293T cell extraction. Our study provides a strong rationale for using GA and LA as novel treatment options for PARs. LA is a potential ligand of MRGPRX2.

Antiallergic Properties of Biflavonoids Isolated from the Flowers of Mesua ferrea Linn.

The methanolic extract from the flowers of Mesua ferrea Linn. (Calophyllaceae) showed significant hyaluronidase inhibitory activity. Following a bioassay-guided separation of the extract, two biflavonoids, viz., mesuaferrone-A (1) and mesuaferrone-B (2), were isolated, along with ten flavonoids (3–12), two xanthones (13 and 14), three triterpenes (15–17), a phenylpropanoid (18), and five aromatics (19–24). Among the isolates, 1 and 2 (IC50 = 51.1 µM and 54.7 µM, respectively) exhibited hyaluronidase inhibitory activity equivalent to that of the commercially available antiallergic agents disodium cromoglycate (64.8 μM) and ketotifen fumarate (76.5 μM). These biflavonoids (1 and 2) are 8-8″ linked dimers that are composed of naringenin (1a) or apigenin (3), with their corresponding monomers lacking inhibitory activity (IC50 > 300 μM). In addition, 1 and 2 (IC50 = 49.4 µM and 49.2 µM, respectively) inhibited the release of β-hexosaminidase, which is a marker of antigen-IgE-mediated degranulation, in rat basophilic leukemia (RBL-2H3) cells. These inhibitory activities were more potent than those of the antiallergic agents tranilast and ketotifen fumarate (IC50 = 282 μM and 158 μM, respectively), as well as one of the corresponding monomers (1a; IC50 > 100 μM). Nonetheless, these effects were weaker than those of the other monomer (3; IC50 = 6.1 μM).

Effects of different cooking methods on peanut allergenicity

Peanut allergy is a type of serious food allergy worldwide. In this study, peanuts were treated with two different cooking methods. The effect of cooking on digestion stability was evaluated by simulated gastrointestinal digestions. The effects of cooking on the allergenicity were assessed by immunoblotting, passive cutaneous anaphylaxis (PCA) mice model and rat basophilic leukemia (RBL)-2H3 cell model. Results showed that raw peanuts were more stable to digestion than boiled and fried peanuts. SDS-PAGE showed that the quantities of allergens Ara h 1 (64 kDa), Ara h 2 (20 kDa), and Ara h 3 (38 kDa) in the boiled and fried peanuts were all reduced. Meanwhile, immunoblotting revealed the IgE binding capacities of boiled and fried peanuts were decreased significantly, especially the fried peanuts. In the PCA mice model, the vascular permeability of allergic mice, as well as the release of histamine, were both alleviated in mice gavaged with boiled and fried peanuts compared with raw peanuts. Lastly, the release of β-hexosaminidase of RBL-2H3 cells treated with boiled and fried peanuts was significantly decreased compared with the raw peanuts. In addition, the analyses of circular dichroism (CD) spectra showed that the α-helical content of peanut proteins was decreased after boiling and frying, indicating the structure stability of peanut allergens decreased. The analyses of ultraviolet (UV) spectra showed that aromatic amino acid residues were exposed, causing the change of the tertiary structure of peanut allergens after boiling and frying. In conclusion, the allergenicity of peanuts was significantly reduced after boiling and frying, especially frying.

Mangifera Indica leaf extracts promote hair growth via activation of Wnt signaling pathway in human dermal papilla cells

ABSTRACT The crosstalk between androgens and Wnt signaling pathways is critical in the hair growth cycle. Therefore, natural products that target these two pathways for the inhibition of hair loss are sought after. In this study, we investigated the effect of water extracts of Mangifera indica leaves (WEML) on hair growth. WEML treatment significantly reduced the expression levels of both dickkopf-1 (DKK1) and type 2 5α-reductase (SRD5A2) involved in Wnt signal suppression activity and dihydrotestosterone (DHT) synthesis, respectively, in human follicle dermal papilla cells (HFDP). In addition, WEML treatment effectively upregulated Wnt target genes and downregulated DKK1 expression that was increased by DHT treatment. Degranulation analysis in rat basophilic leukemia mast cell line (RBL-2H3) using β-hexosaminidase release assay confirmed that WEML did not exhibit allergenic activity. Furthermore, hair growth was significantly enhanced in in vivo mice model treated with WEML. These results suggest that M. indica leave extract contains bioactive materials that can be used to treat hair loss.

Implementation of flexible virtual microchannels based on optically induced dielectrophoresis

Micro-nano particle manipulation methods in liquid environments have been widely used in the fields such as medicine, biology and material science. Nevertheless, the methods usually rely on pre-prepared physical microfluidic channels. In this work, virtual electrodes based on the optically induced dielectrophoresis (ODEP) method were used as virtual microchannels instead of traditional physical microfluidic channels. Virtual microchannels with different shapes were implemented by the designs of projected light patterns, which made the virtual microchannels have great flexibility and controllability. The theory of ODEP was verified by simulation and analysis of electric field distributions. The relationship between the manipulation force and the alternating current (AC) voltage or the AC frequency exerted on the cells was assessed. The experimental results indicated that the manipulation force was increased with the increase of the AC voltage, and it was reduced with the increase of the AC frequency. Moreover, different virtual microchannels were designed to carry out the transportation, aggregation and sorting of yeast cells and rat basophilic leukemia cells (RBL-2H3 cells) and the survival rate of the cells was evaluated. This work shows that the virtual microchannels can be flexibly realized by ODEP in liquid environments.

A novel core skeleton design and synthesis of N‐alkyl‐1′‐(substituted sulfonyl)spiro[chromene‐2,4′‐piperidin]‐6‐amine derivatives as 5‐lipoxygenase inhibitors

Abstract5‐LO inhibitors can potentially be employed for the treatment of various inflammatory disorders. In this study, we have designed and synthesized new N‐alkyl‐1′‐(substituted sulfonyl)spiro[chromene‐2,4′‐piperidin]‐6‐amine‐based library as potential and novel 5‐LO inhibitors. In vitro results showed that several synthesized compounds exhibited high 5‐LO inhibitory activity, in parallel with the inhibition of leukotriene B4 (LTB4) production in the rat basophilic leukemia (RBL‐1) cells. Among the synthesized compounds, 8l was selected for in vivo study using a mouse ear edema model: oral administration of 8l (100 mg/kg) inhibited arachidonic acid‐induced ear edema, myeloperoxidase (MPO) activity, and LTB4 synthesis. SAR analysis and molecular docking studies demonstrated the allosteric binding mode between 5‐LO and the synthesized compounds including 8l.

Plant-Derived Molecule 4-Methylumbelliferone Suppresses FcεRI-Mediated Mast Cell Activation and Allergic Inflammation.

Mast cells (MCs) are an important treatment target for high-affinity IgE Fc receptor (FcεRI)-mediated allergic diseases. The plant-derived molecule 4-methylumbelliferone (4-MU) has beneficial effects in animal models of inflammation and autoimmunity diseases. The aim of this study was to examine 4-MU effects on MC activation and probe the underlying molecular mechanism(s). We sensitized rat basophilic leukemia cells (RBLs) and mouse bone marrow-derived mast cells (BMMCs) with anti-dinitrophenol (DNP) immunoglobulin (Ig)E antibodies, stimulated them with exposure to DNP-human serum albumin (HSA), and then treated stimulated cells with 4-MU. Signaling-protein expression was determined by immunoblotting. In vivo allergic responses were examined in IgE-mediated passive cutaneous anaphylaxis (PCA) and ovalbumin (OVA)-induced active systemic anaphylaxis (ASA) mouse models. 4-MU inhibited β-hexosaminidase activity and histamine release dose-dependently in FcεRI-activated RBLs and BMMCs. Additionally, 4-MU reduced cytomorphological elongation and F-actin reorganization while down-regulating IgE/Ag-induced phosphorylation of SYK, NF-κB p65, ERK1/2, p38, and JNK. Moreover, 4-MU attenuated the PCA allergic reaction (i.e., less ear thickening and dye extravasation). Similarly, we found that 4-MU decreased body temperature, serum histamine, and IL4 secretion in OVA-challenged ASA model mice. In conclusion, 4-MU had a suppressing effect on MC activation both in vitro and in vivo and thus may represent a new strategy for treating IgE-mediated allergic conditions.

The Alleviating Effect of Lagerstroemia indica Flower Extract on Stretch Marks through Regulation of Mast Cells.

Striae distensae (SD) or stretch marks are common linear scars of atrophic skin with disintegrating extracellular matrix (ECM) structures. Although fibroblasts contribute to the construction of ECM structure in SD, some studies have reported that mast cell degranulation causes the disruption of ECM in early SD lesions. Lagerstroemia indica flower (LIF) has traditionally been used in India as a diuretic. However, little is known about the effect and molecular action of Lagerstroemia indica flower extract (LIFE) on alleviating SD. This study evaluated the effects of LIFE on mast cell degranulation and the synthesis of ECM components in fibroblasts. LIFE inhibits the adhesion of rat basophilic leukemia (RBL) cells, RBL-2H3 on fibronectin (FN) and the expression of integrin, a receptor for FN, thereby reducing focal adhesion kinase (FAK) phosphorylation. In addition, LIFE attenuated the allergen-induced granules and cytokine interleukin 3 (IL-3) through the adhesion with FN. Moreover, the conditioned medium (CM) of activated mast cells decreases the synthesis of ECM components, and LIFE restores the abnormal expressions induced by activated mast cells. These results demonstrate that LIFE suppresses FN-induced mast cell activation and promotes the synthesis of ECM components in fibroblast, which indicates that LIFE may be a useful cosmetic agent for SD treatment.

The Critical Role Played by Mitochondrial MITF Serine 73 Phosphorylation in Immunologically Activated Mast Cells.

In recent years, growing evidence has indicated the pivotal role of mitochondria in mast cell immunological activation. We have previously reported a decrease in degranulation and cytokine secretion following the inhibition of pyruvate dehydrogenase (PDH) either by CPI-613 (PDH inhibitor/anti-cancer drug) or through its interaction with mitochondrial microphthalmia-associated transcription factor (MITF). In the present study, we further explored the role played by mitochondrial MITF in mast cell exocytosis using rat basophil leukemia cells [RBL], as well as mouse bone marrow-derived mast cells (BMMCs). Here, we report that mast cell degranulation, cytokine secretion and oxidative phosphorylation (OXPHOS) activities were associated with phosphorylation of Serine 73 of mitochondrial MITF, controlled by extracellular signals regulated by protein kinase (ERK1/2) activity. Also, we report here that decreased OXPHOS activity following ERK1/2 inhibition (U0126 treatment) during IgE-Ag activation was mediated by the dephosphorylation of Serine 73 mitochondrial MITF, which inhibited its association with PDH. This led to a reduction in mast cell reactivity. In addition, a phosphorylation-mimicking mitochondrial MITF-S73D positively regulated the mitochondrial activity, thereby supporting mast cell degranulation. Thus, the present research findings highlight the prominence of mitochondrial MITF Serine 73 phosphorylation in immunologically activated mast cells.

Sargahydroquinoic acid isolated from Sargassum serratifolium as inhibitor of cellular basophils activation and passive cutaneous anaphylaxis in mice

Basophils and mast cells are characteristic effector cells in allergic reactions. Sargahydorquinoic acid (SHQA), a compound isolated from Sargassum serratifolium (marine alga), possesses various biochemical properties, including potent antioxidant activities. The objective of the present study was to investigate inhibitory effects of SHQA on the activation of human basophilic KU812F cells induced by phorbol myristate acetate and A23187 (PMACI), a calcium ionophore. Furthermore, we confirmed the inhibitory effects of SHQA on the activation of rat basophilic leukemia (RBL)–2H3 cells induced by compound 48/80 (com 48/80), bone marrow-derived mast cells (BMCMCs) induced by anti-dinitrophenyl(DNP)-immunoglobulin E (IgE)/DNP-bovine serum albumin (BSA), DNP/IgE and on the reaction of passive cutaneous anaphylaxis (PCA) mediated by IgE. SHQA reduced PMACI-induced intracellular reactive oxygen species (ROS) and calcium levels. Western blot analysis revealed that SHQA downregulated the activation of ERK, p38, and NF-κB in a dose-dependent manner. Moreover, SHQA suppressed the production and gene expression of various cytokines, including interleukin (IL)-1 β, IL-4, IL-6, and IL-8 in PMACI-induced KU812F cells and IL-4 and tumor necrosis factor (TNF)- α in com 48/80-induced RBL-2H3 cells. It also determined the inhibition of PMACI, com 48/80- and IgE/DNP-induced degranulation by reducing the release of β -hexosaminidase. Furthermore, it attenuated the IgE/DNP-induced PCA reaction in the ears of BALB/c mice. These results suggest that SHQA isolated from S. serratifolium is a potential therapeutic functional food material for inhibiting effector cell activation in allergic reactions and anaphylaxis in animal model.