Qi-Wei-Du-Qi-Wan and its major constituents exert an anti-asthmatic effect by inhibiting mast cell degranulation.

ETHNOPHARMACOLOGICAL RELEVANCE: In Asia, Qi-Wei-Du-Qi-Wan (QWDQW) is a traditional Chinese medicine that has been used to treat chest tightness, cough, shortness of breath, night sweats, frequent urination and asthma. QWDQW is recorded in Yi Zong Yi Ren Pian (Medical Physician's Compilation), which was written by Yang Cheng Liu during the Qing Dynasty. AIM OF THE STUDY: The traditional Chinese medicine QWDQW is composed of 7 ingredients and has been used in the treatment of asthma in Asia for hundreds of years. However, the mechanism through which QWDQW affects the immune system in the treatment of asthma is not known. Therefore, this study aimed to investigate whether QWDQW alleviates asthmatic symptoms in mice with chronic asthma induced by repeated stimulation with Dermatophagoides pteronyssinus (Der p) and to explore the underlying immune modulatory mechanism. MATERIALS AND METHODS: BALB/c mice were stimulated intratracheally (i.t.) with Der p (40 µl, 2.5 µg/µl) once weekly for 6 weeks. Thirty minutes prior to Der p stimulation, the mice were treated with QWDQW (0.5 g/kg and 0.17 g/kg) orally. Three days after the last stimulation, the mice were sacrificed, and infiltration of inflammatory cells, lung histological characteristics, gene expression of lung and serum total IgE were assessed. In other experiments, RBL-2H3 cells were stimulated with DNP-IgE/DNP-BSA and then treated with QWDQW, quercetin, ß-carotene, luteolin or a mixture of the three chemicals (Mix13) for 30 min, and the effects of the drugs on RBL-2H3 cell degranulation after DNP stimulation were determined. RESULTS: QWDQW significantly reduced Der p-induced airway hyperreactivity (AHR) and decreased total serum IgE and Der p-specific IgE levels. Histopathological examination showed that QWDQW reduced inflammatory cell infiltration and sputum secretion from goblet cells in the lungs. Gene expression analysis indicated that QWDQW reduced overproduction of IL-12、IFN-γ、IL-13、IL-4、RNATES、Eotaxin and MCP-1in lung. Additionally, QWDQW and Mix13 suppressed DNP induced RBL-2H3 degranulation, and the effect was maximal when quercetin, ß-carotene and luteolin were administered together. CONCLUSION: These results indicate that QWDQW plays a role in suppressing excessive airway reaction and in specific immune modulation in a mouse model of chronic asthma and that QWDQW suppresses mast cell degranulation at defined doses of quercetin, ß-carotene and luteolin.

Hapten-mediated recruitment of polyclonal antibodies to tumors engenders antitumor immunity.

Uptake of tumor antigens by tumor-infiltrating dendritic cells is limiting step in the induction of tumor immunity, which can be mediated through Fc receptor (FcR) triggering by antibody-coated tumor cells. Here we describe an approach to potentiate tumor immunity whereby hapten-specific polyclonal antibodies are recruited to tumors by coating tumor cells with the hapten. Vaccination of mice against dinitrophenol (DNP) followed by systemic administration of DNP targeted to tumors by conjugation to a VEGF or osteopontin aptamer elicits potent FcR dependent, T cell mediated, antitumor immunity. Recruitment of αGal-specific antibodies, the most abundant naturally occurring antibodies in human serum, inhibits tumor growth in mice treated with a VEGF aptamer-αGal hapten conjugate, and recruits antibodies from human serum to human tumor biopsies of distinct origin. Thus, treatment with αGal hapten conjugated to broad-spectrum tumor targeting ligands could enhance the susceptibility of a broad range of tumors to immune elimination.

Semisynthetic Bioluminescent Sensor Proteins for Direct Detection of Antibodies and Small Molecules in Solution.

Single-step immunoassays that can be performed directly in solution are ideally suited for point-of-care diagnostics. Our group recently developed a new platform of bioluminescent sensor proteins (LUMABS; LUMinescent AntiBody Sensor) that allow antibody detection in blood plasma. Thus far, LUMABS has been limited to the detection of antibodies recognizing natural peptide epitopes. Here, we report the development of semisynthetic LUMABS sensors that recognize nonpeptide epitopes. The non-natural amino acid para-azidophenylalanine was introduced at the position of the original antibody-recognition sites as a chemical handle to enable site-specific conjugation of synthetic epitope molecules coupled to a dibenzocylcooctyne moiety via strain-promoted click chemistry. The approach was successfully demonstrated by developing semisynthetic LUMABS sensors for antibodies targeting the small molecules dinitrophenol and creatinine (DNP-LUMABS and CR-LUMABS) with affinities of 5.8 pM and 1.3 nM, respectively. An important application of these semisynthetic LUMABS is the detection of small molecules using a competitive assay format, which is demonstrated here for the detection of creatinine. Using a preassembled complex of CR-LUMABS and an anti-creatinine antibody, the detection of high micromolar concentrations of creatinine was possible both in buffer and in 1:1 diluted blood plasma. The use of semisynthetic LUMABS sensors significantly expands the range of antibody targets and enables the application of LUMABS sensors for the ratiometric bioluminescent detection of small molecules using a competitive immunoassay format.

CD13/aminopeptidase N is a negative regulator of mast cell activation.

Antigen-induced mast cell (MC) activation via cross-linking of IgE-bound high-affinity receptors for IgE (FcεRI) underlies type I allergy and anaphylactic shock. Comprehensive knowledge of FcεRI regulation is thus required. We have identified a functional interaction between FcεRI and CD13 in murine MCs. Antigen-triggered activation of IgE-loaded FcεRI results in cocapping and cointernalization of CD13 and equivalent internalization rates of up to 40%. Cointernalization is not unspecific, because ligand-driven KIT internalization is not accompanied by CD13 internalization. Moreover, antibody-mediated cross-linking of CD13 causes IL-6 production in an FcεRI-dependent manner. These data are indicative of a functional interaction between FcεRI and CD13 on MCs. To determine the role of this interaction, CD13-deficient bone marrow-derived MCs (BMMCs) were analyzed. Intriguingly, antigen stimulation of CD13-deficient BMMCs results in significantly increased degranulation and proinflammatory cytokine production compared to wild-type cells. Furthermore, in a low-dose model of passive systemic anaphylaxis, antigen-dependent decrease in body temperature, reflecting the anaphylactic reaction, is substantially enhanced by the CD13 inhibitor bestatin (-5.9 ± 0.6°C) and by CD13 deficiency (-8.8 ± 0.6°C) in contrast to controls (-1.2 ± 1.97°C). Importantly, bestatin does not aggravate anaphylaxis in CD13-deficient mice. Thus, we have identified CD13 as a novel negative regulator of MC activation in vitro and in vivo-Zotz, J. S., Wölbing, F., Lassnig, C., Kauffmann, M., Schulte, U., Kolb, A., Whitelaw, B., Müller, M., Biedermann, T., Huber, M. CD13/aminopeptidase N is a negative regulator of mast cell activation.

Resveratrol Suppresses Cytokine Production Linked to FcεRI-MAPK Activation in IgE-Antigen Complex-Exposed Basophilic Mast Cells and Mice.

A complicated interplay between resident mast cells and other recruited inflammatory cells contributes to the development and progression of allergic inflammation entailing the promotion of T helper 2 (Th2) cytokine responses. The current study examined whether resveratrol suppressed the production of inflammatory Th2 cytokines in cultured rat basophilic leukemia RBL-2H3 cells. Cells pre-treated with resveratrol nontoxic at 1­25 µM were sensitized with anti-dinitrophenyl (anti-DNP), and subsequently stimulated by dinitrophenyl-human serum albumin (DNP­HSA) antigen. Resveratrol dose-dependently diminished the secretion of interleukin (IL)-3, IL-4, IL-13 as well as tumor necrosis factor (TNF)-α by the antigen stimulation from sensitized cells. It was found that resveratrol mitigated the phosphorylation of p38 MAPK, ERK, and JNK elevated in mast cells exposed to Fc epsilon receptor I (FcεRI)-mediated immunoglobulin E (IgE)-antigen complex. The FcεRI aggregation was highly enhanced on the surface of mast cells following the HSA stimulation, which was retarded by treatment with 1­25 µM resveratrol. The IgE-receptor engagement rapidly induced tyrosine phosphorylation of c-Src-related focal adhesion protein paxillin involved in the cytoskeleton rearrangement. The FcεRI-mediated rapid activation of c-Src and paxillin was attenuated in a dose-dependent manner. In addition, the paxillin activation entailed p38 MAPK and ERK-responsive signaling, but the JNK activation was less involved. Consistently, oral administration of resveratrol reduced the tissue level of phosphorylated paxillin in the dorsal skin of DNP­HSA-challenged mice. The other tyrosine kinase Tyk2-STAT1 signaling was activated in the dorsal epidermis of antigen-exposed mice, which was associated with allergic inflammation. These results showed that resveratrol inhibited Th2 cytokines- and paxillin-linked allergic responses dependent upon MAPK signaling. Therefore, resveratrol may possess the therapeutic potential of targeting mast cells in preventing the development of allergic inflammation.

Licarin A is a candidate compound for the treatment of immediate hypersensitivity via inhibition of rat mast cell line RBL-2H3 cells.

OBJECTIVES: We previously demonstrated that some phenylpropanoids are capable of inhibiting activated mast cells. This study evaluated the anti-allergic effects of licarin A, a neolignan isolated from various plants, on antigen-stimulated rat mast cell line. METHODS: The inhibitory effects of licarin A on histamine release, tumour necrosis factor-α (TNF-α) and prostaglandin D2 (PGD2) production, and cyclooxygenase-2 (COX-2) expression in dinitrophenyl-human serum albumin (DNP-HSA) rat basophilic leukemia cells (DNP-HSA-stimulated RBL-2H3 cells), were investigated by spectrofluorometry, ELISA and immunoblotting. KEY FINDINGS: Licarin A significantly and dose-dependently reduced TNF-α production (IC50 12.6 ± 0.3 µm) in DNP-HSA-stimulated RBL-2H3 cells. Furthermore, the levels of PGD2 secretion in DNP-HSA-stimulated cells pretreated with licarin A were lower than those stimulated with DNP-HSA alone (positive control). Treatment with licarin A at 20 µm produced slight suppression of DNP-HSA-induced increases in COX-2 mRNA and protein levels. We identified several signalling pathways that mediated these pharmacological effects. Licarin A treatment tended to reduce phosphorylated protein kinase C alpha/beta II (PKCα/ßII) and p38 mitogen-activated protein kinase (MAPK) protein levels. CONCLUSIONS: Our results demonstrate that licarin A reduces TNF-α and PGD2 secretion via the inhibition of PKCα/ßII and p38 MAPK pathways; this compound may be useful for attenuating immediate hypersensitivity.

Impaired expression of the mitochondrial calcium uniporter suppresses mast cell degranulation.

Calcium ion (Ca(2+)) uptake into the mitochondrial matrix influences ATP production, Ca(2+) homeostasis, and apoptosis regulation. Ca(2+) uptake across the ion-impermeable inner mitochondrial membrane is mediated by the mitochondrial Ca(2+) uniporter (MCU) complex. The MCU complex forms a pore structure composed of several proteins. MCU is a Ca(2+)-selective channel in the inner-mitochondrial membrane that allows electrophoretic Ca(2+) entry into the matrix. Mitochondrial Ca(2+) uptake 1 (MICU1) functions as a Ca(2+)-sensing regulator of the MCU complex. Previously, by microscopic analysis at the single-cell level, we found that during mast cell activation, mitochondria capture cytosolic Ca(2+) in two steps. Consequently, mitochondrial Ca(2+) uptake likely plays a role in cellular function through cytosolic Ca(2+) buffering. Here, we investigate the role of MCU and MICU1 in mitochondrial Ca(2+) uptake and mast cell degranulation using MCU- and MICU1-knockdown (KD) mast cells. Whereas MCU- and MICU1-KD mast cells show normal proliferation rates and mitochondrial membrane potential, they exhibit slow and reduced cytosolic and mitochondrial Ca(2+) elevation after antigen stimulation. Moreover, ß-hexosaminidase release induced by antigen was significantly suppressed in MCU-KD cells but not MICU1-KD cells. This suggests that both MCU and MICU1 are involved in mitochondrial Ca(2+) uptake in mast cells, while MCU plays a role in mast cell degranulation.

Formoterol synergy with des-ciclesonide inhibits IL-4 expression in IgE/antigen-induced mast cells by inhibiting JNK activation.

Inhaled corticosteroid (ICS) therapy in combination with long-acting ß-adrenergic agonists (LABA) is the most important treatment for allergic asthma, although the mechanism still remains unclear. However, mast cells play a central role in the pathogenesis of asthma. In this study, we explored the sole or synergetic effects of des-ciclesonide (ICS) and formoterol (LABA) on the cytokines IL-4 and IL-13 and on histamine release from mast cells (RBL-2H3 cells). We found that des-ciclesonide (0.1, 1 and 10nM) and formoterol (0.1, 1 and 10µM) alone attenuated DNP-BSA-induced IL-4 and IL-13 production, respectively, in a concentration-dependent manner in DNP-IgE-sensitized mast cells. Des-ciclesonide (0.2nM) and formoterol (1µM) alone also reduced histamine production. However, the combination of des-ciclesonide (0.2nM) and formoterol (1µM) had a synergistic inhibition effect on IL-4 mRNA expression and protein production but not IL-13 and histamine release. The JNK inhibitor SP600125 (10µM) inhibited antigen-induced mRNA expression and protein production of IL-4. Des-ciclesonide and formoterol alone inhibited the activation of JNK in a concentration-dependent manner, and the combination of des-ciclesonide (0.2nM) and formoterol (1µM) exhibited greater inhibition effect compared with des-ciclesonide (0.2nM) or formoterol (1µM) alone. Taken together, these synergistic effects on mast cells might provide the rationale for the development of the most recent ICS/LABA combination approved for asthma therapy.

A novel model of IgE-mediated passive pulmonary anaphylaxis in rats.

Mast cells are central effector cells in allergic asthma and are augmented in the airways of asthma patients. Attenuating mast cell degranulation and with it the early asthmatic response is an important intervention point to inhibit bronchoconstriction, plasma exudation and tissue oedema formation. To validate the efficacy of novel pharmacological interventions, appropriate and practicable in vivo models reflecting mast cell-dependent mechanisms in the lung, are missing. Thus, we developed a novel model of passive pulmonary anaphylaxis in rats. Rats were passively sensitized by concurrent intratracheal and intradermal (ear) application of an anti-DNP IgE antibody. Intravenous application of the antigen, DNP-BSA in combination with Evans blue dye, led to mast cell degranulation in both tissues. Quantification of mast cell degranulation in the lung was determined by (1) mediator release into bronchoalveolar lavage, (2) extravasation of Evans blue dye into tracheal and bronchial lung tissue and (3) invasive measurement of antigen-induced bronchoconstriction. Quantification of mast cell degranulation in the ear was determined by extravasation of Evans blue dye into ear tissue. We pharmacologically validated our model using the SYK inhibitor Fostamatinib, the H1-receptor antagonist Desloratadine, the mast cell stabilizer disodium cromoglycate (DSCG) and the ß2-adrenergic receptor agonist Formoterol. Fostamatinib was equally efficacious in lung and ear. Desloratadine effectively inhibited bronchoconstriction and ear vascular leakage, but was less effective against pulmonary vascular leakage, perhaps reflecting the differing roles for histamine receptor sub-types. DSCG attenuated both vascular leakage in the lung and bronchoconstriction, but with a very short duration of action. As an inhaled approach, Formoterol was more effective in the lung than in the ear. This model of passive pulmonary anaphylaxis provides a tissue relevant readout of early mast cell activity and pharmacological benchmarking broadly reflects responses observed in patients with asthma.

[Tc1-mediated contact sensitivity reaction, its mechanism and regulation]. / Reakcja nadwrazliwosci kontaktowej Tc1 zalezna, jej mechanizm i regulacja.

The contact hypersensitivity reaction (CHS) to haptens is a classic example of cell-mediated immune response. In the effector phase, two stages can be distinguished: an early component, that appears only 2 hours after subsequent contact with the hapten, and the late component that develops approximately 24 hours later which is mediated by TCRαß+ cells. The effector lymphocytes may be CD4+ T helper 1 (Th1) cells or CD8+ T cytotoxic 1 (Tc1) cells, which depends on the employed hapten and/or mice strain. NKT lymphocytes play the crucial role in the CHS initiation, by supporting B1 cells in the antigen-specific IgM antibodies production. The development of an early component is essential for the recruitment of T effector (Teff) cells to the side of hapten deposition and for the complete expansion of inflammatory reaction. The CHS reaction is under T regulatory (Treg) cells control, both in the induction phase as well as in the effector phase. A new view of a negative regulation of the Tc1 mediated CHS response is based on the suppression induced by epicutaneous (EC) application of protein antigen. The DNP-BSA skin application, on a gauze patch, leads to a state of immunosuppression. This maneuver results in rising the population of Treg cells with TCRαß+CD4+CD25+Foxp3+ phenotype. The mechanism of suppression requires direct contact between Treg cells and Teff cells and the participation of CTLA-4 molecule is also necessary. The described method of evoking immune tolerance via EC immunization may contribute to elaborate a new method of allergic contact dermatitis therapy. This is because of its effectiveness, ease of induction and non-invasive protein antigen application.

Mitochondrial STAT3 plays a major role in IgE-antigen-mediated mast cell exocytosis.

BACKGROUND: The involvement of mitochondrial oxidative phosphorylation (OXPHOS) in mast cell exocytosis was recently suggested by the finding that mitochondria translocate to exocytosis sites upon mast cell activation. In parallel, mitochondrial signal transducer and activator of transcription 3 (STAT3) was found to be involved in ATP production. However, the regulation of mitochondrial STAT3 function and its connection to mast cell exocytosis is unknown. OBJECTIVE: We sought to explore the role played by mitochondrial STAT3 in mast cell exocytosis. METHODS: Experiments were performed in vitro with human and mouse mast cells and rat basophilic leukemia (RBL) cells and in vivo in mice. OXPHOS activity was measured after immunologic activation. The expression of STAT3, extracellular signal-regulated kinase 1/2, and protein inhibitor of activated STAT3 in the mitochondria during mast cell activation was determined, as was the effect of STAT3 inhibition on OXPHOS activity and mast cell function. RESULTS: Here we show that mitochondrial STAT3 is essential for immunologically mediated degranulation of human and mouse mast cells and RBL cells. Additionally, in IgE-antigen-activated RBL cells, mitochondrial STAT3 was phosphorylated on serine 727 in an extracellular signal-regulated kinase 1/2-dependent manner, which was followed by induction of OXPHOS activity. Furthermore, the endogenous inhibitor of STAT3, protein inhibitor of activated STAT3, was found to inhibit OXPHOS activity in the mitochondria, resulting in inhibition of mast cell degranulation. Moreover, mice injected with Stattic, a STAT3 inhibitor, had a significant decrease in histamine secretion. CONCLUSION: These results provide the first evidence of a regulatory role for mitochondrial STAT3 in mast cell functions, and therefore mitochondrial STAT3 could serve as a new target for the manipulation of allergic diseases.

Elevated guanylate cyclase and cyclic-guanosine monophosphate-dependent protein kinase levels in nasal mucosae of antigen-challenged rats.

OBJECTIVE: In patients with severe allergic rhinitis, the most serious symptom is rhinostenosis, which is considered to be induced by a dilatation of plexus cavernosum. The vascular relaxing responses to chemical mediators are mainly mediated by the production of nitric oxide (NO). However, the exact mechanism(s) in nasal venoresponsiveness of allergic rhinitis is not fully understood. In the present study, we investigated the roles of soluble guanylate cyclase (sGC) and cyclic-guanosine monophosphate (c-GMP)-dependent protein kinase G (PKG) in venodilatation of nasal mucosae of antigen-challenged rats. METHODS: Actively sensitized rats were repeatedly challenged with aerosolized antigen (2,4-dinitrophenylated Ascaris suum). Twenty-four hours after the final antigen challenge, nasal septum mucosa was exposed surgically and observed directly in vivo under a stereoscopic microscope. The sodium nitroprusside (SNP) and 8-Br-cGMP (a PKG activator) were administered into arterial injection, and the venous diameters of nasal mucosa were observed. RESULTS: The intra-arterial injections of SNP and 8-Br-cGMP-induced venodilatation were significantly augmented in the nasal mucosae of repeatedly antigen-challenged rats. Furthermore, protein expressions of sGC and PKG were significantly increased in nasal mucosae of the antigen-challenged rats. CONCLUSION: The present findings suggest the idea that the promoted cGMP/PKG pathway may be involved in the enhanced NO-induced venodilatation in nasal mucosae of antigen-challenged rats.

Regulatory CD4+Foxp3+ T cells control the severity of anaphylaxis.

OBJECTIVE: Anaphylaxis is a life-threatening outcome of immediate-type hypersensitivity to allergen, consecutive to mast cell degranulation by allergen-specific IgE. Regulatory T cells (Treg) can control allergic sensitization and mast cell degranulation, yet their clinical benefit on anaphylactic symptoms is poorly documented. Here we investigated whether Treg action during the effector arm of the allergic response alleviates anaphylaxis. METHODS: We used a validated model of IgE-mediated passive systemic anaphylaxis, induced by intravenous challenge with DNP-HSA in mice passively sensitized with DNP-specific IgE. Anaphylaxis was monitored by the drop in body temperature as well as plasma histamine and serum mMCP1 levels. The role of Treg was analyzed using MHC class II-deficient (Aß(°/°)) mice, treatment with anti-CD25 or anti-CD4 mAbs and conditional ablation of Foxp3(+) Treg in DEREG mice. Therapeutic efficacy of Treg was also evaluated by transfer experiments using FoxP3-eGFP knock-in mice. RESULTS: Anaphylaxis did not occur in mast cell-deficient W/W(v) mutant mice and was only moderate and transient in mice deficient for histamine receptor-1. Defects in constitutive Treg, either genetic or induced by antibody or toxin treatment resulted in a more severe and/or sustained hypothermia, associated with a rise in serum mMCP1, but not histamine. Adoptive transfer of Foxp3(+) Treg from either naïve or DNP-sensitized donors similarly alleviated body temperature loss in Treg-deficient DEREG mice. CONCLUSION: Constitutive Foxp3(+) Treg can control the symptomatic phase of mast cell and IgE-dependent anaphylaxis in mice. This might open up new therapeutic avenues using constitutive rather than Ag-specific Treg for inducing tolerance in allergic patients.

Effects of magnolialide isolated from the leaves of Laurus nobilis L. (Lauraceae) on immunoglobulin E-mediated type I hypersensitivity in vitro.

ETHNOPHARMACOLOGICAL RELEVANCE: Laurus nobilis L. (Lauraceae) has been used for folk medicines in the Mediterranean area and Europe to treat various disorders including skin inflammation (dermatitis) and asthma. AIM OF THE STUDY: Our aim was to investigate the scientific evaluation of the compounds from Laurus nobilis L. on immuniglobulin E (IgE)-mediated type I hypersensitivity responses in vitro such as atopic dermatitis and asthma. METHODS AND MATERIALS: Seven compounds were isolated and examined for the mast cell stabilizing effect on IgE-sensitized RBL-2H3 mast cells by measuring the ß-hexosaminidase activity. In addition, the effects on interleukin (IL)-4 production and IL-5-dependent Y16 early B cell proliferation were investigated as well as their cytotoxic effects on RBL-2H3 cells. RESULTS: Among the seven isolated compounds, magnolialide attenuated the release of ß-hexosaminidase from RBL-2H3 cells with an IC50 value of 20.2 µM, while the other compounds revealed no significant effects at concentrations tested. Furthermore, magnolialide significantly inhibited the IL-4 release with an IC50 value of 18.1 µM and IL-4 mRNA expression with an IC50 value of 15.7 µM in IgE-sensitized RBL-2H3 cells. In addition, the inhibition of IL-5-dependent proliferation of early B cells (Y16 cells) by magnolialide was demonstrated with an IC50 value of 18.4 µM. CONCLUSION: These results suggest that the magnolialide might be a candidate for the treatment of IgE-mediated hypersensitivity responses such as atopic dermatitis and asthma by inhibiting mast cell degranulation, the IL-4 production, and IL-5-dependent early B cell proliferation, key factors in the development and amplification of type I hypersensitivity reactions.

Ethanolic extract of Alternanthera sessilis (AS-1) inhibits IgE-mediated allergic response in RBL-2H3 cells.

The present study was designed to investigate the anti-allergic effects of ethanolic extract of Alternanthera sessilis (AS-1) in rat basophilic leukemia (RBL-2H3) cells. It significantly reduced the ß-hexosaminidase release from anti-DNP-IgE sensitized RBL-2H3 cells. AS-1also inhibited the IgE antibody-induced increase in Interleukin-6 (IL-6), TNF-α, IL-13 and IL-4 production in these cells. The inhibitory effect of AS-1 on these cytokine was found to be nuclear factor-KB (NF-kB) dependent, as it attenuated the degradation of IKBa and nuclear translocation of NFkB. In addition, AS-1 significantly attenuated the DNP HAS-induced intracellular Ca(2+) release from these cells, which makes us speculate strongly that the decreased intracellular Ca(2+) is involved in the inhibitory effect of AS-1 on ß-hexoaminidase release. Taken together, anti-allergic effects of AS-1 suggest possible therapeutic application of this extract in allergic diseases.

Effect of immunization route on mucosal and systemic immune response in Atlantic salmon (Salmo salar).

This study aimed to assess systemic and mucosal immune responses of Atlantic salmon (Salmo salar) exposed to two protein-hapten antigens - dinitrophenol (DNP) and fluorescein isothiocyanate (FITC) each conjugated with keyhole limpet haemocyanin (KLH) - administered using different delivery strategies. Fish were exposed to the antigens through different routes, and were given a booster 4 weeks post initial exposure. Both systemic and mucosal antibody responses were measured for a period of 12 weeks using an enzyme-linked immunosorbent assay (ELISA). Only fish exposed to both antigens via intraperitoneal (IP) injection showed increased systemic antibody response starting 6 weeks post immunization. No treatment was able to produce a mucosal antibody response; however there was an increase in antibody levels in the tissue supernatant from skin explants obtained 12 weeks post immunization from fish injected with FITC. Western blots probed with serum and culture supernatant from skin explants showed a specific response against the antigens. In conclusion, IP injection of hapten-antigen in Atlantic salmon was the best delivery route for inducing an antibody response against these antigens in this species. Even though IP injection did not induce an increase in antibody levels in the skin mucus, there was an increased systemic antibody response and an apparent increase of antibody production in mucosal tissues as demonstrated by the increased level of specific antibody levels in supernatants from the tissue explants.

Gallic acid-grafted chitooligosaccharides suppress antigen-induced allergic reactions in RBL-2H3 mast cells.

In this study, a bioactive derivative of chitooligosaccharides (3-5 kDa) was synthesized via grafting of gallic acid onto chitooligosaccharides (G-COS) to enhance anti-allergic activity. Hence, G-COS was evaluated for its capabilities against allergic reactions in RBL-2H3 mast cells sensitized with dinitrophenyl-specific immunoglobulin E antibody and stimulated by antigen dinitrophenyl-bovine serum albumin. It was revealed that G-COS exhibited significant inhibition on histamine release and production as well as intracellular Ca(2+) elevation at the concentration of 200µg/ml. Likewise, the suppressive effects of G-COS on expression and production of interleukin (IL)-4 and tumor necrosis factor (TNF)-α were evidenced. Moreover, G-COS treatment caused a remarkable blockade on degradation of inhibitory κB-α (IκB-α) protein, translocation of nuclear factor (NF)-κB, and phosphorylation of mitogen-activated protein kinases (MAPKs). Notably, the inhibitory activities of G-COS on allergic reactions were found as a consequence of suppression of FcεRI expression in antigen-stimulated cells. Accordingly, G-COS was suggested to be a promising candidate of novel inhibitors against allergic reactions.

Enhancing effects of trichloroethylene and tetrachloroethylene on type I allergic responses in mice.

Trichloroethylene (TCE) and tetrachloroethylene (perchloroethylene; PCE) are commonly identified as environmental contaminants of groundwater. Previously, we investigated the enhancing effects of TCE and PCE on antigen-induced histamine release and inflammatory mediator production in rat mast cells. In this study, to examine the potential effect of TCE and PCE on antigen-induced histamine release from mouse mast cells, mouse bone marrow-derived mast cells (BMMC) were sensitized with anti-dinitrophenol (DNP) monoclonal IgE antibody and then stimulated with DNP-BSA containing with TCE or PCE. Both TCE and PCE significantly enhanced antigen-induced histamine release from BMMC. Next we investigated the effects of TCE and PCE on the passive cutaneous anaphylaxis (PCA) reaction in vivo using ICR mice. TCE and PCE significantly enhanced the PCA reaction in a dose-dependent manner. In addition, we examined the enhancing effects of ingesting small amount of TCE and PCE in drinking water on antigen-stimulated allergic responses. After the ICR mice had ingested TCE or PCE in their drinking water for 2 or 4 weeks, we performed the PCA reaction. Both TCE and PCE ingestion enhanced the PCA reaction in a dose-dependent manner for 4 weeks. These results suggest that exposure to TCE and PCE leads to the augmentation of type I allergic responses in many species.

Epicutaneous immunization with DNP-BSA induces CD4+ CD25+ Treg cells that inhibit Tc1-mediated CS.

As we have shown previously that protein antigen applied epicutaneously (EC) in mice inhibits TNP-specific Th1-mediated contact sensitivity (CS), we postulated that the maneuver of EC immunization might also suppress Tc1-dependent CS response. Here we showed that EC immunization of normal mice with 2,4-dinitrophenylated bovine serum albumin (DNP-BSA) applied on the skin in the form of a patch induces a state of subsequent unresponsiveness due to regulatory T cells (Treg) that inhibited sensitization and elicitation of effector T-cell responses. Suppression is transferable in vivo by TCRαß(+) CD4(+) CD25(+) lymphocytes harvested from lymph nodes (LNs) of skin-patched animals. Flow cytometry revealed that EC immunization with DNP-BSA increased TCRαß(+) CD4(+) CD25(+) FoxP3(+) lymphocytes in subcutaneous LNs, suggesting that observed suppression was mediated by Treg cells. Further, in vitro experiments showed that EC immunization with DNP-BSA prior to 1-fluoro-2,4-dinitrobenzen sensitization suppressed LN cell proliferation and inhibited production of TNF-α, IL-12 and IFN-γ. Using a transwell system or anti-CTLA-4 mAb, we found that EC induced suppression required direct Treg-effector cell contact and is CTLA-4-dependent.

Inhibitory effects of Acorus calamus extracts on mast cell-dependent anaphylactic reactions using mast cell and mouse model.

ETHNOPHARMACOLOGICAL RELEVANCE: Acorus calamus Linn. (Araceae) is a traditional herbal plant used for centuries to treat various allergic symptoms including asthma and bronchitis. AIM OF THE STUDY: The present study was focused to provide a pharmacological basis for the traditional use of Acorus calamus in allergic symptoms using the mast cell-dependent anaphylactic reactions in in vitro and in vivo models. MATERIALS AND METHODS: Cell viabilities were measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Dinitrophenyl-human serum albumin (DNP-HSA) induced ß-hexosaminidase and interleukin (IL)-4 productions in IgE-sensitized rat basophilic leukaemia (RBL-2H3) cells were measured by enzymatic assay and enzyme-linked immunosorbent assay (ELISA). Passive cutaneous anaphylaxis (PCA) reaction mouse model was implemented for in vivo studies. RESULTS: Hot water (HW), butylene glycol (BG), hexane (HE) and steam distilled (SD) extracts of Acorus calamus showed different cytoxicity levels evaluated in RBL-2H3 cells. Sub-toxic doses of HW extract suppressed the ß-hexosaminidase secretion and IL-4 production significantly and dose dependently in DNP-HSA induced IgE-sensitized RBL-2H3 cells compared to other extracts of Acorus calamus. Further, in vivo studies also revealed that the HW extract significantly inhibited the PCA reaction in mouse compared to the normal control group. CONCLUSION: HW extract of Acorus calamus most effectively inhibited degranulation and IL-4 secretion in DNP-HSA-stimulated RBL-2H3 cells and also reduced the mast cell-mediated PCA reaction in mouse, providing a therapeutic evidence for its traditional use in ameliorating allergic reactions.