Beta-hexosaminidase Release Research Articles

The effect of Bruton’s tyrosine kinase (BTK) inhibitor in the eosinophilic asthma model of mouse

Bruton’s Tyrosine kinase (BTK) plays a pivotal role as the key mediator in B cell signaling. Recent research has revealed that it is also expressed in cells critical to asthma development, such as T cells, and eosinophils. This study aims to investigate the potential of BTK inhibitor in eosinophilic asthma mouse model. BALB/c mice were sensitized with ovalbumin (OVA) via intraperitoneal injections and followed by OVA nebulizations. The mice were treated with 250 µg/ml or 500 µg/ml of ibrutinib before the second intraperitoneal injection and the first nebulization. Two days after the last OVA challenge, airway hyperresponsiveness (AHR) was assessed with methacholine, and differential cell count in bronchoalveolar lavage fluid (BALF) was performed. The cytokines were measured in BALF, and serum OVA-specific IgE and IgG antibody levels were evaluated by ELISA. The inhibitory effect of ibrutinib was also evaluated in splenic mononuclear cells, mast cells, eosinophils, and T cells in vitro. Treatment with ibrutinib significantly attenuated AHR and airway inflammation, compared to the OVA-induced positive control. The treatment also reduced IL-4, IL-5, IL-13 and IFN-γ cytokine levels and suppressed OVA-specific IgE and IgG production compared to the OVA-induced positive control. Additionally, ibrutinib decreased beta-hexosaminidase release from mast cells, type 2 cytokine productions from mononuclear cells and T cells, and eosinophilic activation markers in vitro. The results of this study suggest that ibrutinib treatment could exert anti-allergic effects by inactivating B cells and other BTK-expressing cells. Further studies are needed to investigate the potential therapeutic effect of ibrutinib on allergic diseases.

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.

Formononetin inhibits IgE by huPlasma/PBMCs and mast cells/basophil activation via JAK/STAT/PI3-Akt pathways.

Food allergy is a prevalent disease in the U.S., affecting nearly 30 million people. The primary management strategy for this condition is food avoidance, as limited treatment options are available. The elevation of pathologic IgE and over-reactive mast cells/basophils is a central factor in food allergy anaphylaxis. This study aims to comprehensively evaluate the potential therapeutic mechanisms of a small molecule compound called formononetin in regulating IgE and mast cell activation. In this study, we determined the inhibitory effect of formononetin on the production of human IgE from peripheral blood mononuclear cells of food-allergic patients using ELISA. We also measured formononetin's effect on preventing mast cell degranulation in RBL-2H3 and KU812 cells using beta-hexosaminidase assay. To identify potential targets of formononetin in IgE-mediated diseases, mast cell disorders, and food allergies, we utilized computational modeling to analyze mechanistic targets of formononetin from various databases, including SEA, Swiss Target Prediction, PubChem, Gene Cards, and Mala Cards. We generated a KEGG pathway, Gene Ontology, and Compound Target Pathway Disease Network using these targets. Finally, we used qRT-PCR to measure the gene expression of selected targets in KU812 and U266 cell lines. Formononetin significantly decreased IgE production in IgE-producing human myeloma cells and PBMCs from food-allergic patients in a dose-dependent manner without cytotoxicity. Formononetin decreased beta-hexosaminidase release in RBL-2H3 cells and KU812 cells. Formononetin regulates 25 targets in food allergy, 51 in IgE diseases, and 19 in mast cell diseases. KEGG pathway and gene ontology analysis of targets showed that formononetin regulated disease pathways, primary immunodeficiency, Epstein-Barr Virus, and pathways in cancer. The biological processes regulated by formononetin include B cell proliferation, differentiation, immune response, and activation processes. Compound target pathway disease network identified NFKB1, NFKBIA, STAT1, STAT3, CCND1, TP53, TYK2, and CASP8 as the top targets regulated at a high degree by formononetin. TP53, STAT3, PTPRC, IL2, and CD19 were identified as the proteins mostly targeted by formononetin. qPCR validated genes of Formononetin molecular targets of IgE regulation in U266 cells and KU812 cells. In U266 cells, formononetin was found to significantly increase the gene expression of NFKBIA, TP53, and BCL-2 while decreasing the gene expression of BTK TYK, CASP8, STAT3, CCND1, STAT1, NFKB1, IL7R. In basophils KU812 cells, formononetin significantly increased the gene expression of NFKBIA, TP53, and BCL-2 while decreasing the gene expression of BTK, TYK, CASP8, STAT3, CCND1, STAT1, NFKB1, IL7R. These findings comprehensively present formononetin's mechanisms in regulating IgE production in plasma cells and degranulation in mast cells.

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.

Nematode galectin binds IgE and modulates mast cell activity

Mast cell degranulation is the major mechanism influencing establishment and survival of the abomasal nematode Teladorsagia circumcincta and probably many other gastrointestinal nematodes. Host galectins-3 and -9 have been shown to bind IgE and positively and negatively influence mast cell degranulation. As incoming nematodes produce large amounts of galectin, we hypothesised that nematode galectin competes with host galectin and inhibits mast cell degranulation. ELISA was used to show that nematode galectin reduced total IgE activity. Galectin also reduced the binding of sheep IgE to the surface of a mast cell line and decreased the release of LCT-4 and Beta hexosaminidase but not MMP-9. These results indicate that nematode galectin influences mast cell degranulation and identify a potential immunomodulatory mechanism used by nematodes to enhance their establishment and survival.

Atopic Dermatitis Inhibitory Effect of Chondria crassicaulis Ethanol Extract

This study investigated the effect of Chondria crassicaulis ethanol extract on degranulation of rat basophilic leukemia-2H3 cells and 2,4-dinitrochlorobenzene-induced atopic dermatitis-like skin lesions in Bagg Albino/c mice. General properties of Chondria crassicaulis (proximate analysis, total phenol and total flavonoid) were measured; moreover, beta-hexosaminidase release and cytotoxicity in rat basophilic leukemia-2H3 cells, severity of skin dermatitis, production of cytokines, and total immunoglobulin E content in an atopic dermatitis-like mouse model were estimated. Chondria crassicaulis ethanol extract decreased the secretion of beta-hexosaminidase without cytotoxicity in rat basophilic leukemia-2H3 cells and decreased the total immunoglobulin E content in serum. In addition, Chondria crassicaulis ethanol extract decreased the production of interleukin-4 and interleukin-5 in mouse splenocytes, whereas significantly increased the level of interferon gamma. Furthermore, Chondria crassicaulis ethanol extract alleviated skin lesions induced by atopic dermatitis without causing toxicity to mouse splenocytes. Therefore, the present study findings suggest that Chondria crassicaulis ethanol extract can relieve atopic dermatitis by regulating the activity of type 1 T helper and type 2 T helper cells that mediate cellular immune response and it can be used as an effective alternative therapy for atopic dermatitis.

A Traditional Medicine-Based Combination of Bamboo Salt and Egg White-Chalcanthite Ameliorates Atopic Dermatitis

Bamboo salt and egg white-chalcanthite mixture has been prescribed for relieving the inflammation of atopic dermatitis in Hamyang, Gyeongsangnam-do, Korea. This study was conducted to demonstrate the effect of atopic dermatitis on cell and atopic animal models using a combination of bamboo salt with egg white-chalcanthite, which have been used as folk remedies for inflammatory conditions. Based on the in vitro experiment, the 1:20 mixture of egg white-chalcanthite and bamboo salt was shown to inhibit the release of beta-hexosaminidase and histamine in rat basophilic leukemia cell line RBL-2H3 by sensitizing immunoglobulin E and inducing allergic responses by dinitrophenyl as well as the expression of inflammatory cytokines and inflammatory mediators in a dose-dependent manner in lipopolysaccharide-treated mouse macrophage cell line RAW 264.7 cells. Furthermore, egg white-chalcanthite with bamboo salt and its combination at different mixing ratios showed a decrease in inflammatory mediators. Dinitrochlorbenzene was applied to NC/Nga mice for 6 w to induce the atopic in vivo model. After 4 w administration of the test substance, the dermatitis decreased visually and dermal hyperplasia/inflammation, mast cell/eosinophils counts and degree of nerve fiber infiltration were decreased according to histopathological examination. In addition, the secretion rate of interferon gamma in high-dose treated group was lower than that in atopic dermatitis control group. These results show that bamboo salt with egg white-chalcanthite effectively relieves skin inflammation in atopic dermatitis and it might be a promising mineral agent for relieving the symptoms of atopic dermatitis.

In vitro anti-allergic activity of Moringa oleifera Lam. extracts and their isolated compounds

BackgroundMoringa oleifera Lam. is a commonly used plant in herbal medicine and has various reported bioactivities such as antioxidant, antimicrobial, anticancer and antidiabetes. It is rich in nutrients and polyphenols. The plant also has been traditionally used for alleviating allergic conditions. This study was aimed to examine the anti-allergic activity of M. oleifera extracts and its isolated compounds.MethodM. oleifera leaves, seeds and pods were extracted with 80% of ethanol. Individual compounds were isolated using a column chromatographic technique and elucidated based on the nuclear magnetic resonance (NMR) and electrospray ionisation mass spectrometry (ESIMS) spectral data. The anti-allergic activity of the extracts, isolated compounds and ketotifen fumarate as a positive control was evaluated using rat basophilic leukaemia (RBL-2H3) cells for early and late phases of allergic reactions. The early phase was determined based on the inhibition of beta-hexosaminidase and histamine release; while the late phase was based on the inhibition of interleukin (IL-4) and tumour necrosis factor (TNF-α) release.ResultsTwo new compounds; ethyl-(E)–undec-6-enoate (1) and 3,5,6-trihydroxy-2-(2,3,4,5,6-pentahydroxyphenyl)-4H-chromen-4-one (2) together with six known compounds; quercetin (3), kaempferol (4), β-sitosterol-3-O-glucoside (5), oleic acid (6), glucomoringin (7), 2,3,4-trihydroxybenzaldehyde (8) and stigmasterol (9) were isolated from M. oleifera extracts. All extracts and the isolated compounds inhibited mast cell degranulation by inhibiting beta-hexosaminidase and histamine release, as well as the release of IL-4 and TNF-α at varying levels compared with ketotifen fumarate.ConclusionThe study suggested that M. oleifera and its isolated compounds potentially have an anti-allergic activity by inhibiting both early and late phases of allergic reactions.

Description and Characterization of a Novel Human Mast Cell Line for Scientific Study.

Background: Laboratory of allergic diseases 2 (LAD2) human mast cells were developed over 15 years ago and have been distributed worldwide for studying mast cell proliferation, receptor expression, mediator release/inhibition, and signaling. LAD2 cells were derived from CD34+ cells following marrow aspiration of a patient with aggressive mastocytosis with no identified mutations in KIT. Another aspiration gave rise to a second cell line which has recently been re-established (LADR). We queried whether LADR had unique properties for the preclinical study of human mast cell biology. Methods: LADR and LAD2 cells were cultured under identical conditions. Experiments examined proliferation, beta-hexosaminidase (β-hex) release, surface receptor and granular protease expression, infectivity with HIV, and gene expression. Results: LADR cells were larger and more granulated as seen with Wright–Giemsa staining and flow cytometry, with cell numbers doubling in 4 weeks, in contrast to LAD2 cells, which doubled every 2 weeks. Both LADR and LAD2 cells released granular contents following aggregation of FcεRI. LADR cells showed log-fold increases in FcεRI/CD117 and expressed CD13, CD33, CD34, CD63, CD117, CD123, CD133, CD184, CD193, and CD195, while LAD2 cells expressed CD33, CD34, CD63, CD117, CD133, CD193 but not CD13, CD123, CD184, or CD195. LADR tryptase expression was one-log-fold increased. LADR cell and LAD2 cell chymase expression were similar. Both cell lines could be infected with T-tropic, M-tropic, and dual tropic HIV. Following monomeric human IgE stimulation, LADR cells showed greater surface receptor and mRNA expression for CD184 and CD195. Expression arrays revealed differences in gene upregulation, especially for the suppressor of cytokine signaling (SOCS) family of genes with their role in JAK2/STAT3 signaling and cellular myelocytomatosis oncogene (c-MYC) in cell growth and regulation. Conclusions: LADR cells are thus unique in that they exhibit a slower proliferation rate, are more advanced in development, have increased FcεRI/CD117 and tryptase expression, have a different profile of gene expression, and show earlier infectivity with HIV-BAL, LAV, and TYBE when compared to LAD2 cells. This new cell line is thus a valuable addition to the few FcεRI+ human mast cell lines previously described and available for scientific inquiry.

Siglec-8 Engagement Selectively Inhibits Allergen-Dependent Degranulation of Human Skin Mast Cells In Vitro

Mast cells (MCs) are well-known for their role in allergic responses. They can be activated through cross-linking of their high affinity IgE receptors (FcεRI) by allergen, leading to the release of many pro-inflammatory mediators. Sialic acid-binding immunoglobulin-like (Siglec)-8 is a CD33-related receptor expressed on human MCs, though not yet described on skin MCs. A key feature of Siglec-8 is its inhibitory (ITIM) motif located within the cytoplasmic domain, suggesting that it may inhibit activating (ITAM) signaling. We hypothesized that Siglec-8 would be expressed on human skin MCs and that its engagement would prevent MC degranulation induced by some secretagogues but not others. Primary MCs were obtained by enzymatic dispersion of discarded human skin. MC were assessed for purity and evaluated for Siglec-8 expression using flow cytometry. Once MCs achieved greater than 95% purity, around 4 weeks post-isolation, the function of Siglec-8 was evaluated. MCs were stimulated with various concentrations of anti-FcεRI in the presence or absence of mouse anti-Siglec-8 (1 μg/ml) and degranulation was assessed via colorimetric assay of beta-hexosaminidase. Siglec-8 inhibitory effects were also evaluated following stimulation with complement factor 5a (C5a) or compound 48/80 which signal through C5aR and MrgX2, respectively, on MCs. All human skin MCs expressed Siglec-8 on their cell surface throughout the culture period. Siglec-8 engagement inhibited beta-hexosaminidase release triggered via FcεRI, but not via C5aR or MrgX2. Siglec-8 engagement inhibits ITAM, but not seven-spanner receptor-mediated MC activation in vitro.

Ameliorative effects of green tea extract from tannase digests on house dust mite antigen-induced atopic dermatitis-like lesions in NC/Nga mice

Atopic dermatitis (AD) is one of the most common chronic inflammatory skin diseases, which is affected by several factors. Anti-histamines, steroids, and immunosuppressive agents have been used for the treatment of AD. However, many studies have reported that long-term use and abuse of these drugs causes many side effects. This study was performed to evaluate the ameliorative effect of green tea extract on AD-like lesions in NC/Nga mice. Green tea extract from tannase digest (GTT), beta-hexosaminidase, and histamine were measured in IgE-antigen complex-stimulated RBL-2H3 cells. Dorsal skin application of house dust mite-ointment induced AD-like symptoms in NC/Nga mice. Dermatitis scores, skin moisture, transepidermal waterloss (TEWL), thickness of skin and ear, T-cell proliferation, levels of immunoglobulins and cytokines, and infiltration of mast cell were measured to assess the degree of AD induction. Skin moisture and TEWL were measured using probes, and ELISA was performed to measure the immunoglobulin and cytokine levels in blood. GTT was selected based on its ability to inhibit the release of beta-hexosaminidase and histamine in IgE-antigen complex-stimulated RBL-2H3 cells. Oral administration of GTT significantly suppressed the skin inflammation and symptoms of AD-like skin lesions in NC/Nga mice. GTT may have a potential therapeutic effect in the treatment of AD.

Stimulation of TLR3 triggers release of lysosomal ATP in astrocytes and epithelial cells that requires TRPML1 channels

Cross-reactions between innate immunity, lysosomal function, and purinergic pathways may link signaling systems in cellular pathologies. We found activation of toll-like receptor 3 (TLR3) triggers lysosomal ATP release from both astrocytes and retinal pigmented epithelial (RPE) cells. ATP efflux was accompanied by lysosomal acid phosphatase and beta hexosaminidase release. Poly(I:C) alkalinized lysosomes, and lysosomal alkalization with bafilomycin or chloroquine triggered ATP release. Lysosomal rupture with glycyl-L-phenylalanine-2-naphthylamide (GPN) eliminated both ATP and acid phosphatase release. Secretory lysosome marker LAMP3 colocalized with VNUT, while MANT-ATP colocalized with LysoTracker. Unmodified membrane-impermeant 21-nt and “non-targeting” scrambled 21-nt siRNA triggered ATP and acid phosphatase release, while smaller 16-nt RNA was ineffective. Poly(I:C)-dependent ATP release was reduced by TBK-1 block and in TRPML1−/− cells, while TRPML activation with ML-SA1 was sufficient to release both ATP and acid phosphatase. The ability of poly(I:C) to raise cytoplasmic Ca2+ was abolished by removing extracellular ATP with apyrase, suggesting ATP release by poly(I:C) increased cellular signaling. Starvation but not rapamycin prevented lysosomal ATP release. In summary, stimulation of TLR3 triggers lysosomal alkalization and release of lysosomal ATP through activation of TRPML1; this links innate immunity to purinergic signaling via lysosomal physiology, and suggests even scrambled siRNA can influence these pathways.

EFFECT OF TOFACITINIB ON IGE-MEDIATED DEGRANULATION OF LAD2 HUMAN MAST CELLS AND PGD2 PRODUCTION

Tofacitinib is an inhibitor of the enzymes JAK1 and JAK3, and can interfere with the JAK-STAT signaling pathway, transmission of extracellular information into the cell nucleus and DNA transcription. Tofacitinib is approved for use in rheumatoid arthritis and is being explored for use in psoriasis, colitis, atopic dermatitis and ankylosing spondylitis. Recently, tofacitinib was said to lead to symptomatic improvement in two patients diagnosed with mast cell activation syndrome. This prompted us to examine the effect of tofacitinib on the biologic reactivity of the human mast cell line, LAD2. In preliminary experiments, cultured LAD2 cells were incubated overnight with biotinylated human IgE. Cells were washed and then incubated with 1, 10, 100, 1000 nM tofacitinib for 1 hour. FcepsilonRI crosslinking was next performed with streptavidin, and degranulation monitored by the release of beta-hexosaminidase (b-Hex). LAD2 cells were also incubated with tofacitinib followed by stimulation with PMA, degranulation and release of b-Hex. The effect on PgD2 production was measured 30 minutes following IgE mediated mast cell activation. In LAD2 cells, b-Hex release following FcepsilonRI crosslinking or PMA stimulation remained consistently high (50-60%) in the presence of tofacitinib with no dose-response reduction in degranulation. Similarly, tofacitinib had no effect on PgD2 release. The JAK kinase inhibitor tofacitinib had no effect on LAD2 human mast cell degranulation or PgD2 production. Additional experiments are underway including an examination of the effects of tofacitinib on LAD2 cytokine production, adhesion and proliferation.

Analysis of TRPV channel activation by stimulation of FCεRI and MRGPR receptors in mouse peritoneal mast cells.

The activation of mast cells (MC) is part of the innate and adaptive immune responses and depends on Ca2+ entry across the plasma membrane, leading to the release of preformed inflammatory mediators by degranulation or by de novo synthesis. The calcium conducting channels of the TRPV family, known by their thermo and osmotic sensitivity, have been proposed to be involved in the MC activation in murine, rat, and human mast cell models. So far, immortalized mast cell lines and nonspecific TRPV blockers have been employed to characterize the role of TRPV channels in MC. The aim of this work was to elucidate the physiological role of TRPV channels by using primary peritoneal mast cells (PMCs), a model of connective tissue type mast cells. Our RT-PCR and NanoString analysis identified the expression of TRPV1, TRPV2, and TRPV4 channels in PMCs. For determination of the functional role of the expressed TRPV channels we performed measurements of intracellular free Ca2+ concentrations and beta-hexosaminidase release in PMCs obtained from wild type and mice deficient for corresponding TRPV1, TRPV2 and TRPV4 in response to various receptor-mediated and physical stimuli. Furthermore, substances known as activators of corresponding TRPV-channels were also tested using these assays. Our results demonstrate that TRPV1, TRPV2, and TRPV4 do not participate in activation pathways triggered by activation of the high-affinity receptors for IgE (FcεRI), Mrgprb2 receptor, or Endothelin-1 receptor nor by heat or osmotic stimulation in mouse PMCs.

Effect of plant extracts on rat basophilic leukemia (RBL-2H3) cells sensitized with IgE

In this study, the inhibitory activities of one hundred plant extracts were analyzed against the IgE-induced allergy reaction in the rat basophilic leukemia (RBL-2H3) cells. Release of IL-4 and beta-hexosaminidase from the IgE-sensitized RBL-2H3 cells treated with the plant extracts were measured. Additionally, the effects of the plant extracts on the cell viability were tested. From the analysis, 17 extracts including Rhamnus davurica Pall. (branch) significantly inhibited beta-hexosaminidase release at 20 µg/mL. And 41 plants such as Artemisia absinthium (leaf and stem) showed high suppressive activities on IL-4 release. All of the extracts proliferated the sensitized cells above 80% compared with control, which was not treated with plant extract samples but treated only with the buffer used for sample preparation. In conclusion, the result suggests that the plants with the inhibitory activities on allergy reaction at cell level are candidates for developing useful anti-allergy materials and need to be study more.

RPbPga1 from Paracoccidioides brasiliensis Activates Mast Cells and Macrophages via NFkB.

BackgroundThe fungus Paracoccidioides brasiliensis is the leading etiological agent of paracoccidioidomycosis (PCM), a systemic granulomatous disease that typically affects the lungs. Cell wall components of P. brasiliensis interact with host cells and influence the pathogenesis of PCM. In yeast, many glycosylphosphatidylinositol (GPI)-anchored proteins are important in the initial contact with the host, mediating host-yeast interactions that culminate with the disease. PbPga1 is a GPI anchored protein located on the surface of the yeast P. brasiliensis that is recognized by sera from PCM patients.Methodology/Principal FindingsEndogenous PbPga1 was localized to the surface of P. brasiliensis yeast cells in the lungs of infected mice using a polyclonal anti-rPbPga1 antibody. Furthermore, macrophages stained with anti-CD38 were associated with P. brasiliensis containing granulomas. Additionally, rPbPga1 activated the transcription factor NFkB in the macrophage cell line Raw 264.7 Luc cells, containing the luciferase gene downstream of the NFkB promoter. After 24 h of incubation with rPbPga1, alveolar macrophages from BALB/c mice were stimulated to release TNF-α, IL-4 and NO. Mast cells, identified by toluidine blue staining, were also associated with P. brasiliensis containing granulomas. Co-culture of P. Brasiliensis yeast cells with RBL-2H3 mast cells induced morphological changes on the surface of the mast cells. Furthermore, RBL-2H3 mast cells were degranulated by P. brasiliensis yeast cells, but not by rPbPga1, as determined by the release of beta-hexosaminidase. However, RBL-2H3 cells activated by rPbPga1 released the inflammatory interleukin IL-6 and also activated the transcription factor NFkB in GFP-reporter mast cells. The transcription factor NFAT was not activated when the mast cells were incubated with rPbPga1.Conclusions/SignificanceThe results indicate that PbPga1 may act as a modulator protein in PCM pathogenesis and serve as a useful target for additional studies on the pathogenesis of P. brasiliensis.

Damnacanthal inhibits IgE receptor-mediated activation of mast cells

Damnacanthal, an anthraquinone obtained from the noni fruit (Morinda citrifolia L.), has been described to possess anti-cancer and anti-inflammatory properties. Since mast cells are key players in various inflammatory conditions as well as in cancer, we considered the possibility that the biological actions of damnacanthal, at least partly, could be due to effects on mast cells. Many of the biological activities of mast cells are mediated by IgE receptor cross-linking, which results in degranulation with release of preformed granule mediators, as well as de novo synthesis and release of additional compounds. Here we show that damnacanthal has profound inhibitory activity on mast cell activation through this pathway. The release of the granule compounds beta-hexosaminidase and tryptase release was completely abrogated by damnacanthal at doses that were non-toxic to mast cells. In addition, damnacanthal inhibited activation-dependent pro-inflammatory gene induction, as well as cytokine/chemokine release in response to mast cell stimulation. The mechanism underlying damnacanthal inhibition was linked to impaired phosphorylation of Syk and Akt. Furthermore, damnacanthal inhibited mast cell activation in response to calcium ionophore A23187. Altogether, the data presented here demonstrate that damnacanthal inhibits mast cell activation induced by different stimuli and open a new window for the use of this compound as a mast cell stabilizer.

IgE로 감작된 RBL-2H3 세포에서 버섯자원의 항알레르기 활성 탐색

For evaluating the anti-allergy activity of mushrooms, forty two mushroom extracts were studied their inhibitory activities on the IgE-mediated degranulation in rat basophilic leukemia cell line (RBL-2H3 cell). Effects of mushroom extracts on the release of interleukin-4 (IL-4) and beta-hexosaminidase, and the cell viability of the IgE-sensitized were measured. From the analysis, five mushroom extracts such as the water extract from Flammulina velutipes (Curt.:Fr.) Sing showed the suppressive activities on IL-4 release as 20%. Eight extracts including the water extract of Ganoserma lucidum showed the suppressive activities on β-hexosaminidase release as 20%. Almost all of the extracts stimulated the proliferation of RBL-2H3 cell. The water extracts of Flammulina velutipes and Phellinus linteus were examined against the inhibitory activity in the production of IL-4 and β-hexosaminidase. Additionally, the extracts from Ganoserma lucidum, Isaria japonica, Phellinus linteus and Pleurotus ostreatus inhibited dose-dependently on β-hexosaminidase production. In conclusion, the result suggests that the mushrooms with the potent inhibitory efficacies on the degranulation of the mast cell would be candidate resources for the anti-allergy resources, and thus need to study for their utilization.

Prostaglandin E2 prevents hyperosmolar-induced human mast cell activation through prostanoid receptors EP2 and EP4.

BackgroundMast cells play a critical role in allergic and inflammatory diseases, including exercise-induced bronchoconstriction (EIB) in asthma. The mechanism underlying EIB is probably related to increased airway fluid osmolarity that activates mast cells to the release inflammatory mediators. These mediators then act on bronchial smooth muscle to cause bronchoconstriction. In parallel, protective substances such as prostaglandin E2 (PGE2) are probably also released and could explain the refractory period observed in patients with EIB.ObjectiveThis study aimed to evaluate the protective effect of PGE2 on osmotically activated mast cells, as a model of exercise-induced bronchoconstriction.MethodsWe used LAD2, HMC-1, CD34-positive, and human lung mast cell lines. Cells underwent a mannitol challenge, and the effects of PGE2 and prostanoid receptor (EP) antagonists for EP1–4 were assayed on the activated mast cells. Beta-hexosaminidase release, protein phosphorylation, and calcium mobilization were assessed.ResultsMannitol both induced mast cell degranulation and activated phosphatidyl inositide 3-kinase and mitogen-activated protein kinase (MAPK) pathways, thereby causing de novo eicosanoid and cytokine synthesis. The addition of PGE2 significantly reduced mannitol-induced degranulation through EP2 and EP4 receptors, as measured by beta-hexosaminidase release, and consequently calcium influx. Extracellular-signal-regulated kinase 1/2, c-Jun N-terminal kinase, and p38 phosphorylation were diminished when compared with mannitol activation alone.ConclusionsOur data show a protective role for the PGE2 receptors EP2 and EP4 following osmotic changes, through the reduction of human mast cell activity caused by calcium influx impairment and MAP kinase inhibition.

Systemic and localized seminal plasma hypersensitivity patients exhibit divergent immunologic characteristics

Systemic and localized seminal plasma hypersensitivity patients exhibit divergent immunologic characteristics