Rat Basophilic Leukemia Cells Research Articles

The Effect of In Vitro Digestion on the Anti-allergic, Anti-Inflammatory and Antioxidant Properties of Purple Rice and Purple Barley Phenolic Extracts in Caco-2 and RBL-2H3 Cells

Gastrointestinal diseases are associated with increased oxidative stress and inflammation. Pigmented cereal polyphenols are believed to have therapeutic potential. However, the impact of digestion on their bioactivity has not been fully elucidated. This study investigated the impact of digestion on the ability of purple rice extract (PRx) and purple barley (PBx) to alleviate oxidative stress-induced intestinal epithelial cell death and allergic inflammation. The study used a human colon adenocarcinoma (Caco-2) cell line to simulate intestinal oxidative stress for 4 h using 1 mM hydrogen peroxide. To evaluate the protective effects of digested and undigested cereal extracts, cells were first pre-treated with varying concentrations (50, 100, 200, and 500 μg/ml) of both purple rice (PRx) and purple barley (PBx) extracts. A rat basophilic leukemia (RBL-2H3) cell line was used to investigate the ability of the extracts to prevent calcium ionophore-induced histamine and cytokine release. The results demonstrated that both digested and undigested phenolic extracts prevented oxidative stress-induced cell death in Caco-2 cells and reduced Interleukin (IL)-8 secretion. PRx showed greater cytoprotective effects than PBx. Both digested and undigested extracts showed a time-dependent antihistamine effect, with the greatest inhibitory effects observed after 2 h and 4 h of treatment with PRx. Additionally, the extracts significantly attenuated the release of several cytokines including IL-2, IL-4, IL-13, monocyte chemoattractant protein-1, interferon-gamma, and tumour necrosis factor-alpha. Overall, the study demonstrates that both digested and undigested pigmented rice and barley extracts are potential sources of polyphenols which may promote intestinal health and alleviate allergic inflammation.

Identification of Potential Tryptase Inhibitors from FDA-Approved Drugs Using Machine Learning, Molecular Docking, and Experimental Validation.

This study explores the innovative use of machine learning (ML) to identify novel tryptase inhibitors from a library of FDA-approved drugs, with subsequent confirmation via molecular docking and experimental validation. Tryptase, a significant mediator in inflammatory and allergic responses, presents a therapeutic target for various inflammatory diseases. However, the development of effective tryptase inhibitors has been challenging due to the enzyme's complex activation and regulation mechanisms. Utilizing a machine learning model, we screened an extensive FDA-approved drug library to identify potential tryptase inhibitors. The predicted compounds were then subjected to molecular docking to assess their binding affinity and conformation within the tryptase active site. Experimental validation was performed using RBL-2H3 cells, a rat basophilic leukemia cell line, where the efficacy of these compounds was evaluated based on their ability to inhibit tryptase activity and suppress β-hexosaminidase activity and histamine release. Our results demonstrated that several FDA-approved drugs, including landiolol, laninamivir, and cidofovir, significantly inhibited tryptase activity. Their efficacy was comparable to that of the FDA-approved mast cell stabilizer nedocromil and the investigational agent APC-366. These findings not only underscore the potential of ML in accelerating drug repurposing but also highlight the feasibility of this approach in identifying effective tryptase inhibitors. This research contributes to the field of drug discovery, offering a novel pathway to expedite the development of therapeutics for tryptase-related pathologies.

Synthesis and Antiallergic Activity of Dicoumarin Derivatives.

Allergies are one of the diseases whose incidence rates have increased in recent years due to the greenhouse effect and extreme climate change. Therefore, the development of new antiallergic drugs has attracted the interest of researchers in chemistry and pharmacy fields. Dicoumarin is a coumarin derivative with various biological activities, but its antiallergic activity has not been evaluated. In this study, 14 different dicoumarin derivatives were synthesized by diethylamine-catalyzed condensation reactions of 4-hydroxycoumarin with 14 different aldehydes, and they were identified on the basis of their spectral data. The dicoumarin derivatives were subjected to studies on the degranulation of rat basophilic leukemia cells (RBL-2H3 cells) and mouse bone-marrow-derived mast cells (mBMMCs), and some of them showed good inhibitory effects on the degranulation of the two types of mast cells, demonstrating their good antiallergic activity. This study presents a new method of developing new antiallergic drugs.

AYA22A Aptamers Mitigate Peanut Allergenicity: Insights from Degranulation Assays and Modulating Immune Responses

Food allergy, particularly peanut allergy (PA), is a growing health concern affecting millions globally. PA can lead to severe reactions, including fatal anaphylaxis. Despite the availability of FDA-approved therapies like Palforzia, a cure remains elusive. Current immunotherapies show promise but lack a definitive cure. This study applies an established computational biology tool to design aptamers targeting Ara h1 and Ara h2. The in silico design aims to streamline the selection process, enabling cost-effective and rapid identification of aptamer candidates. The developed aptamers (AYA22A, including AYA22AR321, AYA22AR211, and AYA22AR524), demonstrated efficacy in inhibiting degranulation of RBL-2H3 cells (rat basophilic leukemia cell line) in vitro. They showed promise in neutralizing peanut allergen-induced immune responses. The selected aptamers inhibited degranulation in RBL-2H3 cells, addressing concerns in raw peanuts. Moreover, these aptamers demonstrated stability and effectiveness in peanut plant seeds and commercial products. Our aptamers exhibited potential in modulating immune responses associated with peanut allergy. They influenced Th1/Th2 balance, indicating a role in cytokine regulation. In vitro studies also showed the aptamers’ impact on immune cell expression and cytokine production, resembling responses observed with established immunotherapies. The findings suggest AYA22A aptamers as a potential therapeutic option for peanut allergy, providing a basis for further in vivo investigations.

The peels of sacred Lotus (Nelumbo nucifera) rhizomes suppress allergic reactions by inhibiting the A23187-induced degranulation in rat basophilic leukemia (RBL[sbnd]2H3) cells

We isolated ten compounds from methanolic extract of the peels of sacred lotus (Nelumbo nucifera) rhizomes which were identified as β-sitosterol linoleate 1, β-sitosterol 2, lupeol 3, stigmasterol 3-O-β-D-glucoside 4, oleanolic acid 5, betulinic acid 6, pinoresinol 7, 4-hydroxybenzoic acid 8, catechin 9 and gallocatechin 10. All of the isolated compounds from the peels of sacred lotus rhizomes are reported for the first time, and were investigated for their anti-allergic activity. We found that three of them, stigmasterol 3-O-β-D-glucoside 4, oleanolic acid 5 and pinoresinol 7, were capable of inhibiting A23187-induced degranulation in RBL-2H3 cells with IC50 values 0.18 ± 0.01 mM, 0.28 ± 0.06 mM, and 0.27 ± 0.01 mM, respectively. With an exception to 4, compounds 5 and 7 achieved the anti-allergic effect without affecting the cells viability even at higher concentrations with their selectivity indices (SI) being >5. By reducing A23187-induced degranulation, it is suggestive of a mechanism attenuation of Ca2+ elevation. Our findings suggest that, the peels of sacred lotus rhizomes would be beneficial for providing an inexpensive source for the production of bioactive compounds with anti-allergic effect.

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.

Surface charge, but not size, of liposomes is involved in the suppression of rat basophilic leukemia (RBL-2H3) cell degranulation mediated by Akt phosphorylation.

Cationic liposomes composed of cholesteryl-3β-carboxyamidoethylene-N-hydroxyethylamine (OH-chol) and 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE) inhibit mast cell degranulation mediated via crosslinking of high-affinity IgE receptors (FcεRI). Although the inhibitory efficiency of mast cell degranulation is altered by modifying the ratio of OH-chol and DOPE in cationic liposomes, the manner in which physicochemical properties, such as surface charge and size, influence suppression is not clear. We observed that positive surface charge, but not the size, of liposomes plays a role in suppressing rat basophilic leukemia (RBL-2H3) cell activation. Pretreatment with middle-ratio OH-chol liposomes (zeta potential, 62.2 ± 0.5 mV; diameter, 325.4 ± 7.3 nm) exhibited a larger suppression of RBL-2H3 cell degranulation evoked by FcεRI crosslinking compared with that by low-ratio OH-chol liposomes (zeta potential, 48.6 ± 1.9 mV; diameter, 344.4 ± 25.0 nm), although both liposomes were similarly attached to RBL-2H3 cells. Preparation of middle-ratio OH-chol liposomes, classified roughly by size using an extrusion method, revealed that the liposomal size did not affect the inhibitory efficiency of RBL-2H3 cell activation. Mechanistically, we found that middle-ratio OH-chol liposomes increased the inhibition of antigen-induced Akt phosphorylation compared to low-ratio OH-chol liposomes. We measured the phosphorylation of linker for activation of T cells (LAT) and paxillin, which are important proteins in FcεRI- and focal adhesions (FAs)-mediated signaling, respectively. Middle ratio OH-chol liposomes significantly suppressed antigen-induced paxillin phosphorylation, but did not affect LAT phosphorylation, suggesting that middle-ratio OH-chol liposomes attached to RBL-2H3 cells suppress the degranulation by impairing FA-mediated Akt phosphorylation evoked by FcεRI crosslinking.

Sulfur dioxide inhibits mast cell degranulation by sulphenylation of galectin-9 at cysteine 74.

Mast cell (MC) degranulation is a key process in allergic reactions and inflammatory responses. Aspartate aminotransferase 1 (AAT1)-derived endogenous sulfur dioxide (SO2) is an important regulator of MC function. However, the mechanism underlying its role in MC degranulation remains unclear. This study aimed to investigate the mechanism by which endogenous SO2 controlled MC degranulation. HMC-1 and Rat basophilic leukemia cell MC line (RBL-2H3) were used in the cell experiments. SO2 content was detected by in situ fluorescent probe. MC degranulation represented by the release rate of MC β-hexosaminidase was determined using a colorimetric assay. Sulfenylation of galectin-9 (Gal-9) in MCs and purified protein was detected using a biotin switch assay. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to determine the exact sulfenylation sites of Gal-9 by SO2. Animal models of passive cutaneous anaphylaxis (PCA) and hypoxia-driven pulmonary vascular remodeling were used to investigate the effect of SO2 on mast cell activation in vivo. Site-directed mutation of Gal-9 was conducted to confirm the exact site of SO2 and support the significance of SO2/Gal-9 signal axis in the regulation of MC degranulation. Degranulation was increased in AAT1-knockdowned MCs, and SO2 supplementation reversed the increase in MC degranulation. Furthermore, deficiency of endogenous SO2 contributed to IgE-mediated degranulation in vitro. Besides, SO2 inhibited IgE-mediated and hypoxia-driven MC degranulation in vivo. Mechanistically, LC-MS/MS analysis and site-directed mutation results showed that SO2 sulfenylated Gal-9 at cysteine 74. Sulfenylation of the 74th cysteine of Gal-9 protein was required in the SO2-inhibited MC degranulation under both physiological and pathophysiological conditions. These findings elucidated that SO2 inhibited MC degranulation via sulfenylating Gal-9 under both physiological and pathophysiological conditions, which might provide a novel treatment approach for MC activation-related diseases.

ERβ activation improves nonylphenol-induced depression and neurotransmitter secretion disruption via the TPH2/5-HT pathway

The aim of this study is to investigate the role of estrogen receptor β (ERβ) in nonylphenol (NP) - induced depression - like behavior in rats and its impact on the regulation of the TPH2/5-HT pathway. In the in vitro experiment, rat basophilic leukaemia cells (RBL-2H3) cells were divided into the four groups: blank group, NP group (20 μM), ERβ agonist group (0.01 μM), and NP＋ERβ agonist group (20 μM＋0.01 μM). For the in vivo experiment, 72 adult male Sprague-Dawley rats were randomly divided into following six groups: the Control, NP (40 mg/kg) group, ERβ agonist (2 mg/kg, Diarylpropionitrile (DPN)) group, ERβ inhibitor (0.1 mg/kg, 4-(2-phenyl-5,7-bis(trifluoromethyl)pyrazolo[1,5-a]pyrimidin-3-yl) phenol (PHTPP)) group, NP+ERβ agonist (40 mg/kg NP ＋ 2 mg/kg DPN) group, and NP+ERβ inhibitor (40 mg/kg NP + 0.1 mg/kg PHTPP) group, with 12 rats in each group. Each rat in drug group were given NP by gavage and/or received a single intraperitoneal injection of DPN 2 mg/kg or PHTPP 0.1 mg/kg. Both in vivo and in vitro, NP group showed a decrease in the expression levels of ERβ, tryptophan hydroxylase (TPH1), and tryptophan hydroxylase-2 (TPH2) genes and proteins, and reduced levels of DA, NE, and 5-hydroxytryptophan (5-HT) neurotransmitters. RBL-2H3 cells showed signs of cell shrinkage, with rounded cells, increased suspension and more loosely arranged cells. The effectiveness of the ERβ agonist stimulation exhibited an increase exceeding 60% in RBL-2H3 cells. The application of ERβ agonist resulted in an alleviation the aforementioned alterations. ERβ agonist activated the TPH2/5-HT signaling pathways. Compared to the control group, the NP content in the brain tissue of the NP group was significantly increased. The latency to eat for the rats was longer and the amount of food consumed was lower, and the rats had prolonged immobility time in the behavioral experiment of rats. The expression levels of ERβ, TPH1, TPH2, 5-HT and 5-HITT proteins were decreased in the NP group, suggesting NP-induced depression-like behaviours as well as disturbances in the secretion of serum hormones and monoamine neurotransmitters. In the NP group, the midline raphe nucleus showed an elongated nucleus with a dark purplish-blue colour, nuclear atrophy, displacement and pale cytoplasm. ERβ might ameliorate NP-induced depression-like behaviors, and secretion disorders of serum hormones and monoamine neurotransmitters via activating TPH2/5-HT signaling pathways.

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.

Nur77 Mediates Anaphylaxis by Regulating miR-21a.

Nur77 belongs to the NR4A subfamily of orphan nuclear hormone receptors. It has been shown to play important roles in metabolism, cancer progression, cellular differentiation, and the regulation of immune process. However, there has yet to be research reporting on the role of Nur77 in allergic inflammations such as anaphylaxis. This study aimed to identify molecules that could mediate allergic inflammations. To this end, we performed RNA sequencing analysis employing bone marrow-derived mast cells (BMMCs). Antigen (DNP-HSA) stimulation increased the expression levels of transcription factors such as Nr4a3 (NOR1), Nr4a1 (Nur77), and Nr4a2 (Nurr1). We focused our study on Nur77. Antigen stimulation increased the expression of Nur77 in a time- and dose-dependent manner in rat basophilic leukemia cells (RBL2H3). The downregulation of Nur77 prevented both antigen-induced increase in β-hexosaminidase activity as well as hallmarks of allergic reactions such as HDAC3, COX2, and MCP1 in RBL2H3 cells. Nur77 was necessary for both passive cutaneous anaphylaxis (PCA) and passive systemic anaphylaxis (PSA). TargetScan analysis predicted that miR-21a would be a negative regulator of Nur77. miR-21a mimic negatively regulated PCA and PSA by inhibiting the hallmarks of allergic reactions. ChIP assays showed that c-JUN could bind to the promoter sequences of Nur77. Antigen stimulation increased the expression of c-JUN in RBL2H3 cells. Altogether, our findings demonstrate the regulatory role played by Nur77-miR-21a loop in allergic inflammations such as anaphylaxis, making this the first report to present the role played by Nur77 in an allergic inflammation. Our results suggest that Nur77 and miR-21 might serve as targets for developing anti-allergy drugs.

Degranulation of RBL-2H3 rat basophilic leukemia cells is synergistically inhibited by combined treatment with nobiletin and lactoferrin.

The aim of this study was to elucidate the anti-allergic effects of polymethoxyflavonoids in combination with milk proteins and the mechanism of inhibition. Three polymethoxyflavonoids and two milk proteins were exposed to the rat basophilic leukemia cell line RBL-2H3. β-hexosaminidase was used as an indicator of degranulation inhibition. The mechanism of inhibition was examined by measuring intracellular Ca2+ levels and western blot method. In the degranulation inhibition test with polymethoxyflavonoids and milk proteins alone, nobiletin was the strongest inhibitor in the polymethoxyflavonoid group and lactoferrin in the milk protein group. Next, co-stimulation with nobiletin and lactoferrin showed stronger synergistic degranulation inhibition than treatment with nobiletin or lactoferrin alone. Western blot analysis showed that co-stimulation with nobiletin and lactoferrin significantly downregulated the induction of phospholipase Cγ 1 phosphorylation. The degranulation response in RBL-2H3 cells was synergistically suppressed by co-stimulation of nobiletin and lactoferrin acting on both Ca2+-dependent and Ca2+-independent pathways.

Mechanism of Action of Yikun Yitong Ping-Containing Serum on Mast Cells and its Possible Involvement in Endometriosis-Related Dysmenorrhea

Background Previously, we showed that Yikun Yitong Ping (YKYTP) can effectively and safely relieve endometriosis-related dysmenorrhea; however, the underlying mechanism remained unclear. This study aimed to assess the effects of YKYTP-containing serum on rat basophilic leukemia cell line, as mast cells (MCs) analog, and investigate the mechanisms by which YKYTP alleviates endometriosis-related dysmenorrhea. Method In this study, YKYTP drug-containing serum was used to treat rat basophilic leukemia cell line (RBL2H3). The effect of YKYTP-containing serum on the expression of ER-α, ER-β, NGF, and NGFRp75 in RBL2H3 cells was evaluated using enzyme-linked immunosorbent assay, quantitative real-time polymerase reaction, and Western blotting. Results Different concentrations (5%-40%) of YKYTP-containing serum reduced ER expression in RBL2H3 cells, and the optimum concentration was 40%. Compared to the blank group, the expression of ER and NGF significantly increased in the E2 group ( P < .01). After co-administration with YKYTP-containing serum, the expression of ER-α, ER-β, NGF, and P75 significantly decreased ( P < .01). Conclusion YKYTP-containing serum can efficiently inhibit the expression of ER-α, ER-β, NGF, and P75 in RBL2H3 cells. YKYTP may alleviate endometriosis-related dysmenorrhea by downregulating ER expression in MCs.

Antiallergic Effect of the Alpha-Cyclodextrin Moringin Complex in Rat Basophilic Leukaemia (RBL-2H3) Cell Line.

Allergic diseases (ADs) are a major concern when it comes to public well-being. Moringa oleifera Lam is a tropical plant that is used in traditional medicine due to the presence of isothiocyanate. The present study investigated the antiallergic properties of 4-(α-L-rhamnopyranosyloxy)-benzyl isothiocyanate or moringin isolated from Moringa oleifera seeds in the form of alpha-cyclodextrin-moringin (α-CD/MG) complex on rat basophilic leukaemia (RBL-2H3) cell line at both the early and late stages of an allergic reaction. The α-CD/MG complex was initially elucidated using nuclear magnetic resonance (NMR) followed by the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium inner salt proliferation assay to evaluate the cytotoxicity and cell viability with respect to ketotifen fumarate (KF) and α-CD/MG. The release of beta-hexosaminidase (β-hexosaminidase) and histamine was used to determine the level of inhibition in the early stage while the suppression of the release of prostaglandin (PGD2), tumour necrosis factor-alpha (TNF-α), and interleukin (IL-4) was considered in the late stage. Higher concentrations of α-CD/MG (5 μM, p < 0.001) in mast cell degranulation significantly inhibited the expression of β-hexosaminidase, histamine, TNF-α, PGD2, and IL-4 in both the early and late stages. Thus, α-CD/MG can potentially be developed as an antiallergic drug as it has the ability to inhibit allergic responses in the late and early stages.

Onopordopicrin from the Japanese Leaf Burdock Exerts Antiallergic Effects through the Inhibition of I Kappa B Kinase β

Onopordopicrin (OPP), found in burdock leaves and stems, exerts antiallergic properties whose mechanism remains elusive. We aimed to elucidate its mechanism using rat basophilic leukemia cells in vitro. Purified OPP demonstrated concentration‐dependent inhibition of degranulation after allergen or PMA/A23187 stimulation. OPP effectively suppressed TNF‐α and PGD2 releases. However, OPP did not suppress the increase in intracellular Ca2+ concentration after allergen stimulation. The α, β‐unsaturated carbonyl structure of OPP suggests potential electrophilic reactivity with polyfunctional thiol‐trapping agents, such as cysteine residues. Indeed, the degranulation‐inhibiting effect of OPP disappeared with the addition of cysteamine, which possesses a thiol group. I kappa B kinase β (IKKβ), which regulates degranulation in an NF‐κB‐independent manner, possesses a cysteine residue between the activation loop. Moreover, IKKβ plays an important role in TNF‐α and PGD2 production. OPP was found to reduce IKKβ activity in a concentration‐dependent manner. Together, our findings suggest that OPP exerts its antiallergic action via binding to cysteine residues in signal proteins such as IKKβ, thereby inhibiting their activation.

Crude Polysaccharides from Mushrooms Elicit an Anti-Allergic Effect Against Type 1 Allergy In Vitro.

Type 1 allergic disease is a global challenge, hence the search for alternative therapies. Mushrooms have several medicinal and health benefits. However, scant data exist on the anti-allergic properties of polysaccharides from fruiting bodies (FB) and mycelia of mushrooms. We used an in vitro co-culture system comprising Caco-2 cells (intestinal epithelial colorectal carcinoma cell line) and RBL-2H3 cells (cell line from rat basophilic leukemia cells). Reduction in degranulation of mast cells indicated anti-allergy properties. The inhibitory effect of crude polysaccharides from different mushroom FB and mycelia on β-hexosaminidase release from RBL-2H3 cells was measured. Results showed that crude polysaccharides from the FB of Inonotus obliquus exhibited a significant inhibitory effect on β-hexosaminidase release and lowered it by 16%. Polysaccharides from the FB of Lentinus squarrosulus, and Pleurotus ostreatus did not exhibit a significant reduction in β-hexosaminidase. However, crude polysaccharides from their mycelia had a significant inhibitory effect, resulting in up to a 23% reduction in β-hexosaminidase activity. Among fungi showing degranulation properties, crude polysaccharides from their mycelia showed more potent action against degranulation than their corresponding FB. Polysaccharides extracted from FB and or mycelia, of selected mushrooms, possess anti-allergic properties that could be harnessed for use in alternative allergy therapies.

The target region focused imaging method for scanning ion conductance microscopy

Scanning ion conductance microscopy (SICM) has developed rapidly and has wide applications in biomedicine, single-cell science and other fields. SICM scanning speed is limited by the conventional raster-type scanning method, which spends most of time on imaging the substrate and does not focus enough on the target area. In order to solve this problem, a target region focused (TRF) method is proposed, which can effectively avoid the scanning of unnecessary substrate areas and enables SICM to image the target area only to achieve high-speed and effective local scanning. TRF method and conventional hopping mode scanning method are compared in the experiments using breast cancer cells and rat basophilic leukemia cells as experimental materials. It was demonstrated that our method can reduce the scanning time for a single sample image significantly without losing scanning information or compromising the quality of imaging. The TRF method developed in this paper can provide an efficient and fast scanning strategy for improving the imaging performance of SICM systems, which can be applied to the dynamic features of cell samples in the fields of biology and pharmacology analysis.

Methiothepin downregulates SNAP-23 and inhibits degranulation of rat basophilic leukemia cells and mouse bone marrow-derived mast cells.

In the present study, we found that methiothepin (a nonselective 5-hydroxytryptamine [5-HT] receptor antagonist) inhibited antigen-induced degranulation in rat basophilic leukemia cells and mouse bone marrow-derived mast cells. Although antigen stimulation induces release of histamine and serotonin (5-HT) by exocytosis and mast cells express several types of 5-HT receptor, the detailed role of these receptors remains unclear. Here, pretreatment of cells with methiothepin attenuated increased intracellular Ca2+ concentration, phosphorylated critical upstream signaling components (Src family tyrosine kinases, Syk, and PLCγ1), and suppressed TNF-α secretion via inhibition of Akt (a Ser/Thr kinase activated by PI3K)and ERK phosphorylation. Furthermore, it inhibited PMA/ionomycin-induced degranulation; this finding suggested that methiothepin affected downstream signaling. IκB kinase β phosphorylates synaptosomal associated protein 23, which regulates the fusion events of the secretory granule/plasma membrane after mast cell activation, resulting in degranulation. We showed that methiothepin blocked PMA/ionomycin-induced phosphorylation of synaptosomal associated protein 23 by inhibiting its interaction with IκB kinase β. Together with the results of selective 5-HT antagonists, it is suggested that methiothepin inhibits mast cell degranulation by downregulating upstream signaling pathways and exocytotic fusion machinery through mainly 5-HT1A receptor. Our findings provide that 5-HT antagonists may be used to relieve allergic reactions.

Natural human Bet v 1-specific IgG antibodies recognize non-conformational epitopes whereas IgE reacts with conformational epitopes.

The nature of epitopes on Bet v 1 recognized by natural IgG antibodies of birch pollen allergic patients and birch pollen-exposed but non-sensitized subjects has not been studied in detail. To investigate IgE and IgG recognition of Bet v 1 and to study the effects of natural Bet v 1-specific IgG antibodies on IgE recognition of Bet v 1 and Bet v 1-induced basophil activation. Sera from birch pollen allergic patients (BPA, n = 76), allergic patients without birch pollen allergy (NBPA, n = 40) and non-allergic individuals (NA, n = 48) were tested for IgE, IgG as well as IgG1 and IgG4 reactivity to folded recombinant Bet v 1, two unfolded recombinant Bet v 1 fragments comprising the N-terminal (F1) and C-terminal half of Bet v 1 (F2) and unfolded peptides spanning the corresponding sequences of Bet v 1 and the apple allergen Mal d 1 by ELISA or micro-array analysis. The ability of Bet v 1-specific serum antibodies from non-allergic subjects to inhibit allergic patients IgE or IgG binding to rBet v 1 or to unfolded Bet v 1-derivatives was assessed by competition ELISAs. Furthermore, the ability of serum antibodies from allergic and non-allergic subjects to modulate Bet v 1-induced basophil activation was investigated using rat basophilic leukaemia cells expressing the human FcεRI which had been loaded with IgE from BPA patients. IgE antibodies from BPA patients react almost exclusively with conformational epitopes whereas IgG, IgG1 and IgG4 antibodies from BPA, NBPA and NA subjects recognize mainly unfolded and sequential epitopes. IgG competition studies show that IgG specific for unfolded/sequential Bet v 1 epitopes is not inhibited by folded Bet v 1 and hence the latter seem to represent cryptic epitopes. IgG reactivity to Bet v 1 peptides did not correlate with IgG reactivity to the corresponding Mal d 1 peptides and therefore does not seem to be a result of primary sensitization to PR10 allergen-containing food. Natural Bet v 1-specific IgG antibodies inhibited IgE binding to Bet v 1 only poorly and could even enhance Bet v 1-specific basophil activation. IgE and IgG antibodies from BPA patients and birch pollen-exposed non-sensitized subjects recognize different epitopes. These findings explain why natural allergen-specific IgG do not protect against allergic symptoms and suggest that allergen-specific IgE and IgG have different clonal origin.

Isolation and Quantification of 2-O-Caffeoyl-hydroxycitric Acid, an Active Component in Hot Water Extracts of Coriander (Coriandrum sativum L.), Which Inhibits Degranulation of RBL-2H3 Cells

Objectives: Natural compounds that can effectively prevent and alleviate allergic symptoms, such as hay fever, are widely sought after from sources such as natural ingredients that can be ingested daily. Coriander extracts have been reported to show anti-allergic activity; however, hot water extracts have not been tested previously. Here, we found that a hot water extract of the aerial parts of coriander inhibited the degranulation of rat basophilic leukemia cell line RBL-2H3 cells and identified the active compounds. Methods: Chromatographic methods were used to isolate compounds from the hot water extract of coriander. The chemical structures were elucidated by analyses of high-resolution electrospray ionization mass spectrum and nuclear magnetic resonance spectral data. The degranulation activity of compounds was evaluated using the β-hexosaminidase release assay. Results: We analyzed the components to identify active compounds. 2 S,3 S-2- O-caffeoyl-hydroxycitric acid (1), and 2- O-caffeoyl-hydroxycitric acid 6′- O-methyl ester (2) were isolated from the hot water extract of coriander, and both compounds showed degranulation inhibitory activity in RBL-2H3 cells at a concentration below 2.5 mM. The content of compound 1 in the freeze-dried coriander powder was calculated to be 2.0% via HPLC quantification. Conclusions: Compound 1 was considered to be the primary active contributor in the hot water extract of coriander.