Inhibition Of Mast Cell Activation Research Articles

The Ribonucleotide Reductase Inhibitor, Didox, Reduces the In Vivo Vascular Inflammation and Oxidative Stress Induced By Acute Hemolysis

Introduction: Several diseases and disorders, including hereditary and acquired hemolytic anemia, blood transfusion reactions, preeclampsia and some infections, incur intravascular hemolysis to varying degrees. The destruction of red blood cells and the release of hemoglobin (Hb) and heme into the circulation results in vascular inflammation, characterized by the recruitment of leukocytes to the vascular endothelium, reduced nitric oxide bioavailability and oxidative stress, all of which may contribute to complications seen in hemolytic diseases, such as pulmonary hypertension, cutaneous leg ulcers and priapism. Didox (3,4-dihydroxybenzohydroxamic acid), a ribonucleotide reductase inhibitor, has been shown to have anti-inflammatory and anti-oxidative effects. This molecule has potential as a cancer therapy and has demonstrated beneficial effects in numerous pathologies, diminishing vaso-occlusive processes in mice with sickle cell disease, and suppressing mast cell activation and degranulation, amongst other effects. Aims: The aim of this study was to evaluate the effects of didox on the vascular inflammatory effects of in vivo acute hemolysis. Methods: C57BL/6 mice received i.v. didox (10 mg/animal) or the same volume of saline vehicle immediately before receiving the hemolytic stimulus. In some mice, didox (10 mg) was given intraperitonally (i.p.) 30 min before hemolysis. Acute hemolysis (HEM) was induced by injecting mice (i.v.) with 100 µl sterile water; for non-hemolysis controls (CON), the same quantity of saline was administered i.v.. At 15 min post-hemolysis, blood was obtained by cardiac puncture for biochemical and flow cytometry analyses; other animals were submitted to cremaster muscle exposure followed by intravital microscopy. Results: Intravascular H2O administration successfully induced hemolysis within 15 min, as demonstrated by elevated plasma cell-free Hb and heme levels (plasma Hb: 0.20±0.05 and 0.66±0.16 mg/ml, P<0.01; total plasma heme: 26.3±4.9 and 87.1±18.4 µM, for CON and HEM, respectively, N=≥6, P<0.001). Interestingly, pre-administration of mice with didox slightly, but significantly, decreased plasma Hb and Heme (plasma Hb: 0.43±0.07 and 0.54±0.07 mg/ml, P<0.05; total plasma heme: 62.7.3±9.5 and 71.5±6.7 µM for i.v. and i.p. didox, respectively, N=6, P<0.05). Concomitantly, acute hemolysis significantly augmented leukocyte (WBC) recruitment and extravasation in the cremaster microcirculation (WBC adhesion: 3.07±0.28 and 13.41±1.02 WBC µm-1, P<0.001; WBC extravasation: 0.72±0.16 and 2.93±0.45 WBC (100x50 µm-2) for CON and HEM, respectively, N=≥5, P<0.001). Didox significantly abrogated this effect of hemolysis, decreasing WBC adhesion to 6.12±0.62 and 2.10±0.30 WBC µm-1 and WBC extravasation to 1.32±0.24 and 1.14±0.21 WBC (100x50 µm-2) (for i.v. and i.p. didox, respectively, N≥5, P<0.001). Flow cytometry demonstrated that hemolysis elevated both the generation of reactive oxygen species (ROS) in the granulocytes of mice and their expression of the Mac-1 integrin subunit, CD11b (ROS: 590±58 and 1140±187 MFI, P<0.01; CD11b: 3028±387 and 4534±416 MFI, for CON and HEM, respect. N=3-6, P<0.01). Importantly, the inhibition of vascular inflammation by didox was associated with a significant decrease in both ROS generation and CD11b expression by granulocytes (ROS: 591±53 and 699±120 MFI, P<0.01; CD11b 2399±126 and 2379±207 MFI, for i.v. and i.p. didox, respectively. N=3-6, P<0.01). Conclusion: Didox, when administrated both intravascularly and intraperitoneally in mice, can inhibit the significant elevations in leukocyte integrin expression and cellular recruitment in the microcirculation that are induced by acute hemolysis. This improvement in vascular inflammation is accompanied by the prevention of oxidative stress in the leukocytes. While didox was found to slightly reduce hemolysis in response to osmotic shock, an interesting observation in itself, it is probable that the beneficial effects of this molecule on vascular inflammation are mediated largely by its effect on oxidative stress parameters. As such, this molecule may provide a novel therapeutic approach to reduce the oxidative stress and vascular inflammation caused by hemolytic events. This includes potential clinical use to treat sickle cell disease and other disorders in which hemolysis is a contributing factor to the pathophysiology. DisclosuresElford:Molecules for Health, Inc.: Equity Ownership, Patents & Royalties: Didox.

AK002, A NOVEL SIGLEC-8 ANTIBODY DEPLETES HUMAN EOSINOPHILS AND INHIBITS ANAPHYLAXIS IN HUMANIZED MICE

Introduction Pathologic accumulation and over-activation of mast cells and eosinophils have been implicated in certain allergic and inflammatory diseases. Siglec-8 is an inhibitory receptor selectively expressed on human eosinophils, mast cells, and, to a significantly lesser extent, basophils. Antibodies to Siglec-8 have the potential to be broadly anti-inflammatory by inducing apoptosis of eosinophils and inhibiting mast cell activation. AK002 is a novel, humanized, non-fucosylated IgG1 monoclonal antibody to Siglec-8. This study examines the ex vivo activity of AK002 on blood and tissue eosinophils and in vivo activity against human mast cells. Methods The apoptosis and antibody-dependent cell-mediated cytotoxicity (ADCC) activity of AK002 or an isotype-matched control antibody was assessed using human blood and tissue eosinophils by flow cytometry. In addition, the activity of a mouse AK002 precursor (mAK002) was tested in a model of IgE-induced systemic and cutaneous anaphylaxis in humanized mice. Results In the presence of peripheral blood NK cells, AK002 demonstrated potent ADCC activity against blood eosinophils. AK002 also directly induced apoptosis of cytokine-activated blood eosinophils. In ex vivo human lung tissue, Siglec-8 expression was found on all tissue eosinophils and AK002 significantly reduced these cells compared to an isotype control. Lastly, mAK002 inhibited passive systemic and cutaneous anaphylaxis in humanized mice, consistent with mast cell inhibition (Figure 1). Conclusions AK002 selectively depletes blood and tissue eosinophils and inhibits IgE-mediated anaphylaxis. AK002 may represent a novel therapeutic agent for patients with eosinophil and mast cell driven inflammatory diseases.

Ursodeoxycholate inhibits mast cell activation and reverses biliary injury and fibrosis in Mdr2−/− mice and human primary sclerosing cholangitis

Ursodeoxycholic acid (UDCA) is used to treat biliary disorders; and, bile acids alter mast cell (MC) histamine release. MCs infiltrate Mdr2−/− mice liver (model of primary sclerosing cholangitis (PSC)). MC-derived histamine increases inflammation, hepatic stellate cell (HSC) activation and fibrosis. The objective was to determine the effects of UDCA treatment on MC infiltration, biliary damage, inflammation and fibrosis in Mdr2−/− mice and human PSC. Wild-type and Mdr2−/− mice were fed bile acid control diet or UDCA (0.5% wt/wt). Human samples were collected from control and PSC patients treated with placebo or UDCA (15 mg/kg/BW). MC infiltration was measured by immunhistochemistry and quantitative polymerase chain reaction (qPCR) for c-Kit, chymase, and tryptase. The HDC/histamine/histamine receptor (HR)-axis was evaluated by EIA and qPCR. Intrahepatic bile duct mass (IBDM) and biliary proliferation was evaluated by CK-19 and Ki-67 staining. Fibrosis was detected by immunostaining and qPCR for fibrotic markers. Inflammatory components were measured by qPCR. HSC activation was measured by SYP-9 staining. Inflammation was detected by qPCR for CD68. In vitro, MCs were treated with UDCA (40 μM) prior to HA secretion evaluation and coculturing with cholangiocytes or HSCs. BrDU incorporation and fibrosis by qPCR was performed. UDCA reduced MC number, the HDC/histamine/HR-axis, IBDM, HSC activation, inflammation, and fibrosis in Mdr2−/− mice and PSC patients. In vitro, UDCA decreases MC-histamine release, which was restored by blocking ASBT and FXRβ. Proliferation and fibrosis decreased after treatment with UDCA-treated MCs. We conclude that UDCA acts on MCs reducing histamine levels and decreases the inflammatory/hyperplastic/fibrotic reaction seen in PSC. Ursodeoxycholic acid (UDCA) is used to treat biliary disorders; and, bile acids alter mast cell (MC) histamine release. Following liver injury like primary sclerosing cholangitis in mice and humans, MCs infiltrate. MC-derived histamine increases biliary damage, fibrosis, and inflammation. UDCA treatment decreases these parameters via reduced MC activation.The bile acid rsodeoxycholic acid (UDCA) is the choice of treatment for primary biliary cholangitis patients with abnormal liver enzymes; however, its mechanism is not clear, and is the focus of this investigation. Bile acids alter mast cell (MC) histamine release. Following liver injury, such as that seen in primary sclerosing cholangitis, MCs infiltrate the liver. MC-derived histamine increases biliary damage, fibrosis, and inflammation. UDCA treatment decreases these parameters via reduced MC activation.

Glucocorticoids decrease the numbers and activation of mast cells by inducing the transactivation receptors of AGEs.

Glucocorticoids (GCs) are potent anti-allergic compounds that function, at least in part, by inhibiting signaling pathways in mast cells. We hypothesized that the GC-induced mastocytopenia and suppression of mast cell activation are mediated by the advanced glycation end products (AGEs)/receptors of AGEs (RAGEs) signaling axis. We evaluated the role of AGEs in GC-mediated mastocytopenia and impaired mast cell degranulation in male Wistar rats and Swiss-Webster mice subcutaneously injected with dexamethasone or prednisolone (0.1mg/kg) once a day for 21 consecutive days. The animals were treated with either the AGE inhibitor aminoguanidine (250mg/kg), the RAGE antagonist FPS-ZM1 (1mg/kg) or the galectin-3 antagonist GSC-100 (1mg/kg) daily for 18 days, starting 3 days following GC treatment. Aminoguanidine inhibited GC-induced mast cell apoptosis and restored mast cell numbers in the pleural cavity of GC-treated rats. Aminoguanidine also reversed the GC-induced reduction in histamine release triggered by allergens or compound 48/80 in vitro. GC treatment induced RAGE and galectin expression in mast cells, and blocking these agents by FPS-ZM1 or GSC-100 significantly reversed mast cell numbers in the peritoneal cavity and mesenteric tissue of GC-treated mice. In addition, the combination of GC and AGE-induced mast cell apoptosis in vitro was inhibited by both FPS-ZM1 and GSC-100. We concluded that the GC-induced mastocytopenia and suppression of mast cell stimulation are associated with the gene transactivation of RAGE and galectin-3.

Adenosine activates Gαs proteins and inhibits C3a-induced activation of human mast cells

Anaphylatoxin C3a and adenosine receptors (AR) are implicated in the inflammatory process associated with allergic rhinitis and asthma by modifying mast cell (MC) responses. Possible interactions between these G-protein coupled receptor (GPCR) pathways in MCs have not yet been demonstrated. LAD2 human MC were stimulated with C3a in the presence or absence of AR agonists and antagonists and their adhesion, chemotaxis and mediator release were measured. The pan-specific AR agonist, 5′-N-Ethylcarboxamidoadenosine (NECA) inhibited C3a-induced LAD2 cell migration, adhesion, degranulation, production of CCL2, and ERK1/2 phosphorylation. The selective A2A receptor agonist CGS 21680 inhibited C3a-mediated degranulation, while the A2B and A3 receptor agonists BAY 60-6583 and IB-MECA, respectively, had no effect. Moreover, an A2A receptor antagonist SCH 58261 blocked the inhibitory effect of NECA on C3a-induced degranulation, suggesting that inhibition of degranulation was mediated through the A2A receptor. NECA increased intracellular cAMP in C3a-activated mast cells, suggesting that Gαs protein signals are required for adenosine-induced inhibition of C3a-mediated human mast cell activation. The adenylyl cyclase inhibitor SQ 22536 attenuated the inhibitory effect of NECA on C3a-activated degranulation, and the A2A agonist CSG 21680 potentiated the inhibition of mast cell activation mediated by the A2A receptor. Our results suggest that adenosine inhibits C3a-mediated activation of human mast cells, possibly through a Gαs protein-dependent pathway.

The Allergen Der p3 from House Dust Mite Stimulates Store-Operated Ca2+ Channels and Mast Cell Migration through PAR4 Receptors

The house dust mite is the principal source of perennial aeroallergens in man. How these allergens activate innate and adaptive immunity is unclear, and therefore, there are no therapies targeting mite allergens. Here, we show that house dust mite extract activates store-operated Ca2+ channels, a common signaling module in numerous cell types in the lung. Activation of channel pore-forming Orai1 subunits by mite extract requires gating by STIM1 proteins. Although mite extract stimulates both protease-activated receptor type 2 (PAR2) and PAR4 receptors, Ca2+ influx is more tightly coupled to the PAR4 pathway. We identify a major role for the serine protease allergen Der p3 in stimulating Orai1 channels and show that a therapy involving sub-maximal inhibition of both Der p3 and Orai1 channels suppresses mast cell activation to house dust mite. Our results reveal Der p3 as an important aeroallergen that activates Ca2+ channels and suggest a therapeutic strategy for treating mite-induced asthma.

Benzoxazole derivatives suppress lipopolysaccharide-induced mast cell activation.

Mast cells are central regulators of allergic inflammation that function by releasing various proallergic inflammatory mediators, including histamine, eicosanoids and proinflammatory cytokines. Occasionally, bacterial infections may initiate or worsen allergic inflammation. A number of studies have indicated that activation of lipoxygenase in mast cells positive regulates allergic inflammatory responses by generating leukotrienes and proinflammatory cytokines. In the present study, the effects of benzoxazole derivatives on the lipopolysaccharide (LPS)‑induced expression of proinflammatory cytokines, production of histamine and surface expression of co‑stimulatory molecules on bone marrow-derived mast cells (BMMCs) were studied. The benzoxazole derivatives significantly reduced the expression of interleukin (IL)‑1β, IL‑6, IL‑13, tumor necrosis factor‑α, perilipin (PLIN) 2, and PLIN3 in BMMCs treated with LPS. Furthermore, histamine production was suppressed in BMMCs treated with LPS, or treated with phorbol-12-myristate-13-acetate/ionomycin. Benzoxazole derivatives marginally affected the surface expression of cluster of differentiation (CD)80 and CD86 on BMMCs in the presence of LPS, although LPS alone did not increase the expression of those proteins. Therefore, benzoxazole derivatives inhibited the secretion of proinflammatory cytokines in mast cells and may be potential candidate anti‑allergic agents to suppress mast cell activation.

An unexpected protective role of low-affinity allergen-specific IgG through the inhibitory receptor FcγRIIb

Induction of allergen-specific IgG antibodies is a critical parameter for successful allergen-specific immunotherapy. IgG antibodies can inhibit IgE-mediated mast cell activation through direct allergen neutralization or through the inhibitory receptor FcγRIIb. The affinity of IgE antibodies to the allergen has been shown to be critical for cellular activation. Here we addressed the question of affinity thresholds of allergen-specific IgG antibodies for inhibition of mast cell activation using 2 different mAbs against the major cat allergen Fel d 1 both invitro and invivo in mice. Sequences of the 2 high-affinity mAbs were back-mutated to germline, resulting in low-affinity (10-7mol/L) antibodies of the exact same specificity. Using these newly generated recombinant antibodies, we demonstrate that low-affinity antibodies are still able to inhibit mast cell activation through FcγRIIb but do not neutralize the allergen. Antibody affinity dictates the mechanism of mast cell inhibition, and IgG antibodies triggering the inhibitory FcγRIIb pathway can show a broader cross-reactivity pattern than previously thought. This indicates that allergen-specific immunotherapy generates a larger protective umbrella of inhibitory IgG antibodies than previously appreciated.

Mast Cell Activation in Brain Injury, Stress, and Post-traumatic Stress Disorder and Alzheimer's Disease Pathogenesis.

Mast cells are localized throughout the body and mediate allergic, immune, and inflammatory reactions. They are heterogeneous, tissue-resident, long-lived, and granulated cells. Mast cells increase their numbers in specific site in the body by proliferation, increased recruitment, increased survival, and increased rate of maturation from its progenitors. Mast cells are implicated in brain injuries, neuropsychiatric disorders, stress, neuroinflammation, and neurodegeneration. Brain mast cells are the first responders before microglia in the brain injuries since mast cells can release prestored mediators. Mast cells also can detect amyloid plaque formation during Alzheimer's disease (AD) pathogenesis. Stress conditions activate mast cells to release prestored and newly synthesized inflammatory mediators and induce increased blood-brain barrier permeability, recruitment of immune and inflammatory cells into the brain and neuroinflammation. Stress induces the release of corticotropin-releasing hormone (CRH) from paraventricular nucleus of hypothalamus and mast cells. CRH activates glial cells and mast cells through CRH receptors and releases neuroinflammatory mediators. Stress also increases proinflammatory mediator release in the peripheral systems that can induce and augment neuroinflammation. Post-traumatic stress disorder (PTSD) is a traumatic-chronic stress related mental dysfunction. Currently there is no specific therapy to treat PTSD since its disease mechanisms are not yet clearly understood. Moreover, recent reports indicate that PTSD could induce and augment neuroinflammation and neurodegeneration in the pathogenesis of neurodegenerative diseases. Mast cells play a crucial role in the peripheral inflammation as well as in neuroinflammation due to brain injuries, stress, depression, and PTSD. Therefore, mast cells activation in brain injury, stress, and PTSD may accelerate the pathogenesis of neuroinflammatory and neurodegenerative diseases including AD. This review focusses on how mast cells in brain injuries, stress, and PTSD may promote the pathogenesis of AD. We suggest that inhibition of mast cells activation and brain cells associated inflammatory pathways in the brain injuries, stress, and PTSD can be explored as a new therapeutic target to delay or prevent the pathogenesis and severity of AD.

Sodium butyrate enhances intestinal integrity, inhibits mast cell activation, inflammatory mediator production and JNK signaling pathway in weaned pigs.

The present study aimed to investigate the effects of sodium butyrate on the intestinal barrier and mast cell activation, as well as inflammatory mediator production, and determine whether mitogen-activated protein kinase signaling pathways are involved in these processes. A total of 72 piglets, weaned at 28 ± 1 d age, were allotted to two dietary treatments (control vs. 450 mg/kg sodium butyrate) for 2 wk. The results showed that supplemental sodium butyrate increased daily gain, improved intestinal morphology, as indicated by greater villus height and villus height:crypt depth ratio, and intestinal barrier function reflected by increased transepithelial electrical resistance and decreased paracellular flux of dextran (4 kDa). Moreover, sodium butyrate reduced the percentage of degranulated mast cells and its inflammatory mediator content (histamine, tryptase, TNF-α and IL-6) in the jejunum mucosa. Sodium butyrate also decreased the expression of mast cell-specific tryptase, TNF-α and IL-6 mRNA. Sodium butyrate significantly decreased the phosphorylated ratio of JNK whereas not affecting the phosphorylated ratios of ERK and p38. The results indicated that the protective effects of sodium butyrate on intestinal integrity were closely related to inhibition of mast cell activation and inflammatory mediator production, and that the JNK signaling pathway was likely involved in this process.

Management of Inducible Urticarias

Purpose of review This review aims to update the information and latest advances in chronic inducible urticarias. Recent findings In contrast to chronic spontaneous/idiopathic uriticarias, chronic inducible urticarias are associated with a specific trigger and factor. One must accurately identify and characterize the specific trigger for distinguishing chronic spontaneous/idiopathic uriticaria and chronic inducible urticarias. The latter are diagnosed based on patient history and results of provocation testing which contribute to confirm the exact diagnosis, assess the threshold, and determine the severity of the disease. The general management of chronic inducible urticarias includes the identification and avoidance of the specific trigger, and symptomatic treatment with the goal of reaching complete symptom control. Symptomatic treatment for chronic inducible urticarias targets the blocking of mast-cell mediators (H1-antihistamines, H2-antihistamies, leukotriene antagonists) and inhibition of mast-cell activation (omalizumab, cyclosporine, phototherapy). H1-antihistamines are fundamental treatments for chronic inducible urticarias, but H1-antihistamine-resistant cases are not rare and additional treatment is often necessary. Also, therapeutic reactivity varies according to each subtype and patient. Summary Notably, some subtypes of chronic inducible urticaria can be treated by desensitization and/or tolerance induction to triggers. In contrast, the underlying pathogenesis of chronic inducible urticaria is not known and further studies are needed to better clarify its etiology.

Resveratrol improves prostate fibrosis during progression of urinary dysfunction in chronic prostatitis by mast cell suppression.

Voiding dysfunction is the primary clinical manifestation of chronic prostatitis (CP), which is a common urological disease. The present study investigated whether prostate fibrosis was associated with urinary dysfunction in CP and if resveratrol improved urinary dysfunction, and the underlying molecular mechanism. A rat model of CP was established via subcutaneous injections of the pertussis-diphtheria-tetanus vaccine, which was followed by treatment with resveratrol. Bladder pressure and volume tests were performed to investigate the effect of resveratrol on urinary dysfunction in CP rats. Western blotting and immunohistochemical staining examined the expression levels of tryptase, chymase, transforming growth factor (TGF)-β, Wnt and α-smooth muscle actin (α-SMA). The results demonstrated that the maximum capacity of the bladder, residual urine volume and maximum voiding pressure were increased significantly in the CP group compared with the control group. Mast cell (MC) activation, the activity of TGF-β/Wnt/β-catenin pathways, and the expression levels of tryptase and α-SMA in the CP group were increased significantly compared with the control group. Resveratrol treatment significantly reversed these factors. Therefore, the results indicate that MC infiltration may induce prostate fibrosis, which exhibits a close association with urinary dysfunction in CP. Resveratrol may improve fibrosis via the suppression of MC activation and TGF-β/Wnt/β-catenin pathway activities.

Rifampicin Alleviates Atopic Dermatitis-Like Response in vivo and in vitro.

Atopic dermatitis (AD) is a common inflammatory skin disorder mediated by inflammatory cells, such as macrophages and mast cells. Rifampicin is mainly used for the treatment of tuberculosis. Recently, it was reported that rifampicin has anti-inflammatory and immune-suppressive activities. In this study, we investigated the effect of rifampicin on atopic dermatitis in vivo and in vitro. AD was induced by treatment with 2, 4-dinitrochlorobenzene (DNCB) in NC/Nga mice. A subset of mice was then treated with rifampicin by oral administration. The severity score and scratching behavior were alleviated in the rifampicin-treated group. Serum immunoglobulin E (IgE) and interleukin-4 (IL-4) levels were also ameliorated in mice treated with rifampicin. We next examined whether rifampicin has anti-atopic activity via suppression of mast cell activation. Rifampicin suppressed the release of β-hexosaminidase and histamine from human mast cell (HMC)-1 cultures stimulated with compound 48/80. Treatment with rifampicin also inhibited secretion of inflammatory mediators, such tumor necrosis factor-α (TNF-α) and prostaglandin D2 (PGD2), in mast cells activated by compound 48/80. The mRNA expression of cyclooxygenase 2 (COX-2) was reduced in the cells treated with rifampicin in a concentration-dependent manner. These results suggest that rifampicin can be used to treat atopic dermatitis.

Mast cell degranulation and calcium influx are inhibited by an Echinacea purpurea extract and the alkylamide dodeca-2E,4E-dienoic acid isobutylamide

Ethnopharmacological relevanceNative Americans used plants from the genus Echinacea to treat a variety of different inflammatory conditions including swollen gums, sore throats, skin inflammation, and gastrointestinal disorders. Today, various Echinacea spp. preparations are used primarily to treat upper respiratory infections. Aim of the studyThe goal of this study was to evaluate the effects of an ethanolic E. purpurea (L) Moench root extract and the alkylamide dodeca-2E,4E-dienoic acid isobutylamide (A15) on mast cells, which are important mediators of allergic and inflammatory responses. Inhibition of mast cell activation may help explain the traditional use of Echinacea. Materials and methodsA15 was evaluated for its effects on degranulation, calcium influx, cytokine and lipid mediator production using bone marrow derived mast cells (BMMCs) and the transformed rat basophilic leukemia mast cell line RBL-2H3. Methods included enzymatic assays, fluorimetry, ELISAs, and microscopy. A root extract of E. purpurea, and low and high alkylamide-containing fractions prepared from this extract, were also tested for effects on mast cell function. Finally, we tested A15 for effects on calcium responses in RAW 264.7 macrophage and Jurkat T cell lines. ResultsA15 inhibited ß-hexosaminidase release from BMMCs and RBL-2H3 cells after treatment with the calcium ionophore A23187 by 83.5% and 48.4% at 100µM, respectively. Inhibition also occurred following stimulation with IgE anti-DNP/DNP-HSA. In addition, A15 inhibited 47% of histamine release from A23187-treated RBL-2H3 cells. A15 prevented the rapid rise in intracellular calcium following FcεRI crosslinking and A23187 treatment suggesting it acts on the signals controlling granule release. An E. purpurea root extract and a fraction with high alkylamide content derived from this extract also displayed these activities while fractions with little to no detectable amounts of alkylamide did not. A15 mediated inhibition of calcium influx was not limited to mast cells as A23187-stimulated calcium influx was blocked in both RAW 264.7 and Jurkat cell lines with 60.2% and 43.6% inhibition at 1min post-stimulation, respectively. A15 also inhibited the release of TNF-α, and PGE2 to a lesser degree, following A23187 stimulation indicating its broad activity on mast cell mediator production. ConclusionsThese findings suggest that Echinacea extracts and alkylamides may be useful for treating allergic and inflammatory responses mediated by mast cells. More broadly, since calcium is a critical second messenger, the inhibitory effects of alkylamides on calcium uptake would be predicted to dampen a variety of pathological responses, suggesting new uses for this plant and its constituents.

Long-term effects: Galectin-1 and specific immunotherapy for allergic responses in the intestine.

Mast cell activation interferes with the effects of allergen-specific immunotherapy (SIT). Galectin-1 (Gal-1) is capable of regulating immune cells' functions. This study tests the hypothesis that administration of Gal-1 promotes and prolongs the efficacy of SIT via suppressing mast cell activation. An intestinal allergy mouse model was developed. The coadministration of SIT and Gal-1 on suppression of the allergic responses, prevention of mast cell activation, and generation of antigen-specific regulatory T cells (Treg) in the intestine was observed in sensitized mice. The coadministration of Gal-1 and SIT markedly suppressed the allergic responses in the mouse intestine vs the use of either SIT alone or Gal-1 alone. The Gal-1 binds to the IgE/FcɛRI complexes on the surface of mast cells to prevent mast cell activation during SIT. Gal-1 promoted the SIT-generated allergen-specific Tregs in the intestine of sensitized mice. Coadministration of Gal-1 and SIT significantly enhanced the efficacy of immunotherapy in suppressing allergic responses in the intestine, which lasted for at least for 12months. Long-term effects of specific immunotherapy on intestinal allergy can be achieved with Gal-1/SIT therapy by inhibiting mast cell activation and facilitating Treg development.

Suppression of mast cell activation by exopolysaccharide produced by Bacillus subtilis.

Abstract The incidence of allergies is on the rise, and to prevent or treat allergic diseases, we need a means of inhibiting immune pathways leading to allergic responses. Reduced exposure to microbes, increased use of antibiotics and changes in diet all lead to an increase in allergies. The consequences of these changes in lifestyle include an alteration of intestinal bacteria that are important for development of the immune system. Our work seeks to utilize the beneficial effects of gut microbiota to manipulate allergic responses. A key cell responsible for the symptoms of allergies is the mast cell. We are using the probiotic bacterium Bacillus subtilis which produces an exopolysaccharide (EPS) with anti-inflammatory activity. We found that EPS treatment alone was unable to alter mast cell degranulation following IgE-mediated activation, however treatment with peritoneal cells from EPS-injected mice prevented mast cell degranulation. We hypothesize that EPS induces a suppressive cell in the peritoneal cavity that leads to the suppression of mast cell activation and could be used to suppress the immune response against common food or environmental factors.

Treatment with rifampicin ameliorates atopic dermatitis-like skin disease in NC/Nga mice through mast cell inactivation

Abstract Atopic dermatitis (AD) is a common inflammatory skin disorder mediated by inflammatory cells such as macrophages and mast cells. Rifampicin is a major drug for treatment of tuberculosis. Recently it was reported that rifampicin has anti-inflammatory and immune suppressive activity. We herein investigated the effect of rifampicin on atopic dermatitis in NC/Nga mouse model. AD was induced by treatment of 1-chloro 2, 4-dinitrobenzene (DNCB). And then mice were treated with rifampicin by oral administration and were observed to relieve the diverse symptoms. The severity score and scratching behavior were alleviated in rifampicin-treated groups. Serum IgE and IL-4 levels were also decreased in rifampicin-treated groups. We next examined if rifampicin has anti-atopic activity via the suppression of mast cell activation. Rifampicin suppressed the release of β-hexosaminidase and histamine from human mast cell (HMC)-1 cells stimulated with compound 48/80. Treatment of rifampicin inhibited secretion of inflammatory mediators, such TNF-α and PGD2, in mast cells activated by compound 48/80. The mRNA expression of COX-2 was reduced in the cell treated with rifampicin at a dose-dependent manner. These results suggest that rifampicin can be used to treat atopic dermatitis.

Activation‑induced upregulation of MMP9 in mast cells is a positive feedback mediator for mast cell activation.

Activated mast cells are involved in the pathogenesis of allergic rhinitis (AR). As a member of the matrix metalloproteinase (MMP) family, MMP9 has been previously demonstrated act in a pro‑inflammatory manner. Mast cells regulate the activity of MMP9, and mast cells themselves have been reported to produce MMP9. However, to the best of our knowledge, the involvement of MMP9 in mast cell activation remains to be elucidated. The present study demonstrated an upregulation of MMP9 protein and mRNA expression levels in mast cells activated by phorbol ester and ionomycin. Phosphorylated ERK and AKT protein levels also markedly increased in activated mast cells, and inhibition of the ERK and AKT signaling pathways prevented the increase of MMP9 in activated mast cells. MMP9 was demonstrated to be involved in mast cell activation, since inhibition of MMP9 activity or expression inhibited mast cell activation. Furthermore, IL‑4 treatment reduced MMP9 upregulation in activated mast cells, and interference with IL‑4 signaling with an IL‑4 neutralizing antibody promoted MMP9 upregulation in activated mast cells. These results revealed a novel MMP9‑mediated mechanism underlying mast cell activation, thus providing novel ideas for AR therapy.

Use of Marijuana in Patients with Sickle Cell Disease Increased the Frequency of Hospitalization for Acute Painful Vaso-Occlusive Crises

Introduction : Recently, there has been general interest in using marijuana as an analgesic for various types of pain. Cannabis is known to relieve severe nausea and vomiting. Anecdotally, medical marijuana has analgesic effects in patients with chronic and neuropathic pain other than sickle cell pain, decreases the frequency of migraine headache, has anti-spastic effect in multiple sclerosis, and may relieve some of the signs and symptoms of amyotrophic lateral sclerosis and Crohn's disease. Its role in epilepsy is controversial. On the negative side long-term cannabis use is associated with periodontal disease, maternal marijuana use at 20 weeks gestation was strongly associated with spontaneous pre-term birth independent of maternal cigarette smoking and illegal synthetic cannabinoids (K2/spice) were associated with catatonia and myocardial infarction in adolescents. Studies in the transgenic sickle cell mouse showed that cannabinoid receptor-specific mechanisms ameliorate pain via inhibition of mast cell activation and neurogenic inflammation. A questionnaire study from the United Kingdom showed that 36% of the patients with sickle cell disease (SCD) have used cannabis in the previous 12 months to relieve symptoms. Another longitudinal questionnaire study in Jamaica found that marijuana was not related to the clinical severity of SCD. The objective of this study is to report the epidemiological and clinical features of the patients with SCD who were found to be taking marijuana by using random urine drug screening from 1994 to 2009. The frequency of vaso-occlusive crises (VOCs) in these patients was compared to patents with SCD whose urine drug screening showed no marijuana. The terms marijuana and cannabinoid will be used interchangeably in this Abstract. Patients &amp; Methods: A total of 270 random urine drug screen tests were done on 72 patients representing 10% of our patients with SCD during the period of the study. Written consent was obtained from the patients for enrollment in the study that was approved by the IRB. Types of SCD were determined by routine lab tests. Urine samples, collected randomly, were analyzed for the presence of amphetamine, benzodiazepines, opiates, barbiturate, cannabinoid, propoxyphene, methadone and phencyclidine. Samples were classified as either positive or negative for cannabinoid. Statistical analysis included the two-tailed student's t, Fisher exact and the Chi Square tests. Results: Thirty-seven of the 72 patients tested positive for cannabinoid in their urine and 35 patients tested negative. Table 1 shows the major epidemiological and clinical feature of the patients studied. There was no significant difference in ages of men and women who used or did not use marijuana (p &gt; 0.05). However, males who used marijuana were significantly younger (p &lt; 0.001) than males who did not (Table 1). Moreover, the ages of females who tested positive for cannabinoid were not significantly different from women who tested negative (Table 1). The patients who tested positive for cannabinoids also tested positive for other illicit drugs more often than patients who tested negative. Positivity for benzodiazepines, cocaine and phencyclidine were significantly higher in patients who used marijuana than the nonusers. Notably, there was no significant difference in the use of opioids consumed by users and non-users of marijuana. Similarly, there was no significant difference in the frequency of complications of SCD between users and non-users of marijuana (Table 1). The most important aspect of the study is the effect of marijuana use on the frequency of utilization of medical facilities. Patients in the marijuana cohort were admitted to the hospital for VOCs more frequently (Table 1) than the control group: 2,443 versus1,602 admissions respectively (p &lt; 0.05). Conclusion: Together these data suggest that patients in the marijuana cohort were more often in severe pain that required treatment in the hospital for VOCs than non-users. This difference could be due to the quality of the marijuana used, the neuronal effects of marijuana and the severity of pain in the users of marijuana. The role of marijuana in the treatment of sickle cell pain seems more complex than thought. Further controlled trials are needed to clarify this issue. Table 1 Table 1. Disclosures No relevant conflicts of interest to declare.

The Role of the Mast Cell in Skin Aging

Skin is the external organ of human body that provides protection against environmental stress. Direct and indirect damage causes skin aging. Scientists have been struggling to explore the mechanisms of skin aging and to think of ways to delay this course. The role of mast cell in regulating skin aging has attracted much attention recently. The granules of mast cell contain a great diversity of highly toxic compounds and active mediators. Degranulation of mast cell in papillary dermis may lead to extra cellular matrix reconstruction, inflammation and angiogenesis which result in accelerated skin aging. Essential factors implicated in aging and tissue damage in skin are possibly related to mast cells. Mast cell is becoming an important target for anti-aging therapy. Many natural products capable of inhibiting mast cell activation are considered functional ingredients for anti-skin aging.