Mouse Model Of Anaphylaxis Research Articles

Wheatgrass-and-Aronia-Mixed Extract Suppresses Immunoglobulin E-Mediated Allergic Reactions In Vitro and In Vivo.

Mast cells are an important component of immune responses. Immunoglobulin (Ig) E-sensitized mast cells release substances within minutes of allergen exposure, triggering allergic responses. Until now, numerous pharmacological effects of wheatgrass and aronia have been verified, but the effects of wheatgrass and aronia (TAAR)-mixed extract on allergic reactions have not been identified. Therefore, the aim of this study was to demonstrate the anti-allergic effect of TAAR extract on mast cell activation and cutaneous anaphylaxis. In this study, we investigated the anti-allergic effects and related mechanisms of TAAR extract in IgE-activated mast cells in vitro. We also assessed the ameliorating effect of TAAR extract on IgE-mediated passive cutaneous anaphylaxis mice in vivo. The TAAR extract significantly reduced the expression of β-hexosaminidase, histamine, and pro-inflammatory cytokines, which are mediators related to mast cell degranulation, via the regulation of various signaling pathways. The TAAR extract also regulated oxidative-stress-related factors through the Nrf2 signaling pathway. Additionally, treatment of TAAR extract to the passive cutaneous anaphylaxis mouse model improved ear thickness and local ear pigmentation. Taken together, our results suggest that TAAR extract is a potential candidate natural product to treat overall IgE-mediated allergic inflammation and oxidative-stress-related diseases by suppressing mast cell activity.

The Hdc GC box is critical for Hdc gene transcription and histamine-mediated anaphylaxis

Histamine is a critical mediator of anaphylaxis, a neurotransmitter, and a regulator of gastric acid secretion. Histidine decarboxylase is a rate-limiting enzyme for histamine synthesis. However, invivo regulation of Hdc, the gene that encodes histidine decarboxylase, is poorly understood. We sought to investigate how enhancers regulate Hdc gene transcription and histamine synthesis in resting conditions and in a mouse model of anaphylaxis. H3K27 acetylation histone modification and chromatin accessibility were used to identify candidate enhancers. The enhancer activity of candidate enhancers was measured in a reporter gene assay, and the function enhancers were validated by CRISPR deletion. Deletion of the GC box, which binds to zinc finger transcription factors, in the proximal Hdc enhancer reduced Hdc gene transcription and histamine synthesis in mouse andhuman mast cell lines. Mast cells, basophils, brain cells, and stomach cells from GC box-deficient mice transcribed the Hdc gene much less than similar cells from wild-type mice, and Hdc GC box-deficient mice failed to develop anaphylaxis. The HDC GC box within the proximal enhancer in the mouse and human HDC gene is essential for Hdc gene transcription, histamine synthesis, and histamine-mediated anaphylaxis invitro and invivo.

Acoustic metamaterials-driven transdermal drug delivery for rapid and on-demand management of acute disease

Transdermal drug delivery provides convenient and pain-free self-administration for personalized therapy. However, challenges remain in treating acute diseases mainly due to their inability to timely administrate therapeutics and precisely regulate pharmacokinetics within a short time window. Here we report the development of active acoustic metamaterials-driven transdermal drug delivery for rapid and on-demand acute disease management. Through the integration of active acoustic metamaterials, a compact therapeutic patch is integrated for penetration of skin stratum corneum and active percutaneous transport of therapeutics with precise control of dose and rate over time. Moreover, the patch device quantitatively regulates the dosage and release kinetics of therapeutics and achieves better delivery performance in vivo than through subcutaneous injection. As a proof-of-concept application, we show our method can reverse life-threatening acute allergic reactions in a female mouse model of anaphylaxis via a multi-burst delivery of epinephrine, showing better efficacy than a fixed dosage injection of epinephrine, which is the current gold standard ‘self-injectable epinephrine’ strategy. This innovative method may provide a promising means to manage acute disease for personalized medicine.

Development of a Novel IgG1 Anaphylaxis Mouse Model with Uniquely Characteristic Skin Manifestations Induced Through the FcγRIII-Histamine Pathway

ABSTRACT Background Studies of passive anaphylaxis, in which mouse immunoglobulin G (IgG) and its antigens are administered to mice, believe that platelet-activating factor (PAF) is more important than histamine and that basophils or macrophages are primarily involved. However, the full extent of IgG-dependent anaphylaxis is still unclear; that is, little agreement has been reached about the mechanism. Methods First, we established the novel model of IgG1 anaphylaxis induced by the intravenous administration of two types of IgG1 and a fluorescent dye-labeled antigen, as IgG1 immune complex in HR-1 hairless mice. Subsequently, pharmacological analysis was used to investigate the underlying mechanisms of IgG1 anaphylaxis in this established model. Results The novel IgG1 anaphylaxis model can induce the IgG-induced Anaphylaxis-dependent Spotted Distribution of fluorescently labeled Immune complexes in the Skin, named “G-ASDIS”. Moreover, this model was triggered primarily by the FcγRIII-dependent histamine release, which is different from the conventional model in which PAF was involved in the development of IgG1 anaphylaxis. Basophils in the circulation and mast cells in the skin may participate in the development of IgG1 anaphylaxis and increased G-ASDIS. Conclusion Our results propose that the novel axis, namely the FcγRIII-basophils and/or mast cell-histamine pathway, is important for IgG1 anaphylaxis. Further analysis of our model in addition to other models will lead to a broader analysis and understanding of the IgG1 anaphylaxis mechanism.

Transmembrane stem cell factor protein therapeutics enhance revascularization in ischemia without mast cell activation.

Stem cell factor (SCF) is a cytokine that regulates hematopoiesis and other biological processes. While clinical treatments using SCF would be highly beneficial, these have been limited by toxicity related to mast cell activation. Transmembrane SCF (tmSCF) has differential activity from soluble SCF and has not been explored as a therapeutic agent. We created novel therapeutics using tmSCF embedded in proteoliposomes or lipid nanodiscs. Mouse models of anaphylaxis and ischemia revealed the tmSCF-based therapies did not activate mast cells and improved the revascularization in the ischemic hind limb. Proteoliposomal tmSCF preferentially acted on endothelial cells to induce angiogenesis while tmSCF nanodiscs had greater activity in inducing stem cell mobilization and recruitment to the site of injury. The type of lipid nanocarrier used altered the relative cellular uptake pathways and signaling in a cell type dependent manner. Overall, we found that tmSCF-based therapies can provide therapeutic benefits without off target effects.

Comparison of sensitizing and disease-eliciting capacities of salt-soluble protein extracts from diploid (einkorn) versus tetraploid (durum) wheats

Abstract Hypersensitivity reactions to wheat have reached significant levels of global public health concern for reasons that are incompletely understood. Einkorn wheat (Triticum monococcum, genome AA) is commercially available but is rarely consumed, whereas durum wheat (Triticum durum, genome AABB) is commonly used for pasta making. At present, most mouse models of wheat allergy are adjuvant-based, which fail to reveal the intrinsic allergenic potential of different wheat products. Here we compared einkorn and durum wheats in terms of their sensitizing and disease-eliciting capacities in an adjuvant-free mouse model of anaphylaxis induced by salt-soluble protein extract (SSPE). Balb/cJ female mice were sensitized transdermally with SSPE or saline for nine weeks. Later, systemic or oral anaphylactic reactions were elicited in these mice using SSPE via intraperitoneal or oral route, respectively. We did not observe major differences in sensitizing capacity as suggested by antibody responses (i.e., sIgE and tIgE levels) in SSPE-sensitized mice. However, SSPE from durum appears to be more potent than that from einkorn in eliciting systemic but not oral anaphylaxis as shown by hypothermic shock responses as well as mucosal mast cell degranulation. This is the first report on comparing the intrinsic allergenicity between tetraploid and diploid wheats in an adjuvant-free mouse model. This research was funded by: the United States Department of Agriculture (USDA)/National Institute of Food and Agriculture (NIFA); Hatch project MICL02486 (Accession Number: 1012322), Hatch project MICL01699; Agricultural and Food Research Initiative Competitive Program, grant number: 2018-67017-27876; and Project GREEEN (Michigan State University); R.J., was supported by the John Harvey Kellogg Graduate Assistantship and the Academic Achievement Graduate Assistantship from the Michigan State University.

Biotinylated peptides substituted with D-amino acids with high stability as anti-anaphylactic agents targeting platelet-activating factor.

Platelet-activating factor (PAF) is an important lipid mediator of anaphylaxis and therefore can be an anti-anaphylactic agent target. Recently, we reported that several synthetic biotinylated peptides containing a Tyr-Lys-Asp-Gly sequence markedly inhibited the bioactivities of PAF in vitro and in vivo; it also inhibited anaphylactic reactions such as hypothermia, hypotension, and vascular permeability in vivo. Here, we report the anti-anaphylactic effects of three biotinylated heptapeptides (peptide 1: H-Lys(biotinyl)-Trp-Tyr-Lys-Asp-Gly-Asp-OH, peptide 2: H-D -Lys(biotinyl)-Trp-Tyr-Lys-Asp-Gly-Asp-OH, and peptide 3: H-D -Lys(biotinyl)-Trp-Tyr-Lys-Asp-Gly-D -Asp-OH). The experiment using tryptophan fluorescence spectroscopy showed that the interaction of peptides 2 and 3 with PAF was larger than that of peptide 1. Experiments using a rat model of hind paw edema showed that peptides 1, 3, and 2 inhibited PAF-induced edema by 67.9%, 69.3%, and 79.3%, respectively. In a mouse model of anaphylaxis, both peptides 2 and 3 showed inhibitory effects on anaphylactic hypothermia, whereas peptide 1 did not. Furthermore, experiments involving in vitro rat plasma stability of peptides showed that both peptides 3 and 2 were more stable in plasma compared to peptide 1 (84.0%, 51.8%, and 0%, remained after 6h, respectively). Our results suggest that both peptides 2 and 3 may show systemic and local inhibitory effects as anti-anaphylactic agents targeting PAF.

Royal Jelly-derived Two Compounds, 10-hydroxy-2-decenoic acid and a Biotinylated Royalisin-related Peptide, Alleviate Anaphylactic Hypothermia In vivo

Platelet-activating factor (PAF) is an important mediator of allergic reactions such as anaphylaxis, and is therefore an anaphylactic drug target. Previously, we found marked inhibition of anaphylactic reactions by a synthetic N-terminally biotinylated peptide (BP21) capable of specifically binding PAF. In this study, we investigated the effects of 10-hydroxy-2-decenoic acid (10-HDA) and various peptides derived from royalisin, both of which are widely known to be major components of royal jelly from honeybees, on PAF/histamine-induced rat hind-paw edema and mouse anaphylactic hypothermia. In a rat model of hind paw edema, both 10-HDA (50 nmol/paw) and a royalisin-related N-terminally biotinylated peptide, Biotin-RL-Y11, (10 nmol/paw) markedly inhibited PAF-induced paw edema by 95.5±5.0% and 71.9±9.9%, respectively. 10-HDA also inhibited histamine-induced paw edema by 42.7±19.2%. In contrast, three peptides with N-terminal HDA modifications, namely 10-HDA-RL-Y11, 10-HDA-RL-R11, and 10-HDA-P21 only weakly inhibited PAF-induced paw edema. In a mouse model of anaphylaxis, the administration of 10-HDA (100-1000 nmol/mouse) and/or Biotin-RL-Y11 (100 nmol/mouse) showed the inhibitory effects on anaphylactic hypothermia. These results suggest that both 10-HDA and Biotin-RL-Y11 may exert an anti-anaphylactic effect targeting PAF.

Anti-Allergic Effect of 3,4-Dihydroxybenzaldehyde Isolated from Polysiphonia morrowii in IgE/BSA-Stimulated Mast Cells and a Passive Cutaneous Anaphylaxis Mouse Model.

In this study, we investigated the anti-allergic effects of 3,4-dihydroxybenzaldehyde (DHB) isolated from the marine red alga, Polysiphonia morrowii, in mouse bone-marrow-derived cultured mast cells (BMCMCs) and passive cutaneous anaphylaxis (PCA) in anti-dinitrophenyl (DNP) immunoglobulin E (IgE)-sensitized mice. DHB inhibited IgE/bovine serum albumin (BSA)-induced BMCMCs degranulation by reducing the release of β-hexosaminidase without inducing cytotoxicity. Further, DHB dose-dependently decreased the IgE binding and high-affinity IgE receptor (FcεRI) expression and FcεRI-IgE binding on the surface of BMCMCs. Moreover, DHB suppressed the secretion and/or the expression of the allergic cytokines, interleukin (IL)-4, IL-5, IL-6, IL-13, and tumor necrosis factor (TNF)-α, and the chemokine, thymus activation-regulated chemokine (TARC), by regulating the phosphorylation of IκBα and the translocation of cytoplasmic NF-κB into the nucleus. Furthermore, DHB attenuated the passive cutaneous anaphylactic (PCA) reaction reducing the exuded Evans blue amount in the mouse ear stimulated by IgE/BSA. These results suggest that DHB is a potential therapeutic candidate for the prevention and treatment of type I allergic disorders.

Anti-Inflammatory Effects of Novel Glycyrrhiza Variety Wongam In Vivo and In Vitro

Licorice is the common name of Glycyrrhiza species, which is an important plant for edible and medicinal purposes; however, Glycyrrhiza resources have become limited because of desertification, depletion of natural resources, and environmental restrictions. For this reason, a novel Glycyrrhiza variety named Wongam, a hybrid of G. glabra and G. uralensis, was developed by the Korea Rural Development Administration. To elucidate the antiallergic inflammatory effects of Wongam, we investigated its effects using a compound-48/80-induced anaphylaxis in vivo model and PMA/A23187-stimulated HMC-1 cells and immunoglobulin E (IgE)/DNP-stimulated RBL-2H3 cells in in vitro models. Wongam treatment reduced mortality and serum IgE levels and downregulated proinflammatory cytokines and chemokines in a compound-48/80-induced anaphylaxis mouse model. Wongam decreased histamine release and the expression of proinflammatory cytokines in HMC-1 and RBL-2H3 cells. Wongam treatment downregulated the expression of chemokines, T helper 2 cytokines, and cell surface antigens in PMA/A23187-stimulated HMC-1 cells. We confirmed that these effects were associated with the inhibition of the MAPK and NF-κB signaling pathways by Wongam. The present study suggests that Wongam ameliorates mast-cell-mediated allergic inflammatory responses by reducing mast cell activation and may serve as an effective agent for the prevention and treatment of allergic inflammatory responses.

REGN1908-1909 monoclonal antibodies block Fel d 1 in cat allergic subjects: Translational pharmacokinetics and pharmacodynamics.

REGN1908‐1909, a 1:1 cocktail of two fully human IgG4 monoclonal antibodies (mAbs), REGN1908 and REGN1909, is being evaluated for treatment of cat allergy. Both REGN1908 and REGN1909 bind to the dominant cat allergen, Fel d 1. Adults with cat allergy confirmed by skin prick test (SPT) were randomized to single subcutaneous administration of placebo (n = 6) or REGN1908‐1909 at doses of 150 (n = 6), 300 (n = 6), or 600 mg (n = 6). Blood samples were taken at prespecified time points for pharmacokinetic (PK) analysis and exploratory evaluation of biomarkers (IgE and SPT). Safety was assessed. Drug concentration‐time profiles in serum for ascending doses of REGN1908‐1909 were consistent with linear PKs. Noncompartmental analysis showed that maximum concentration (Cmax) and exposure increased proportionately with dose, with similar time to maximum concentration (Tmax) for REGN1908 and REGN1909 (6.2 to 8.2 days across doses), and a longer terminal half‐life for REGN1908 (~ 30 days) relative to REGN1909 (~ 21 days). Adverse events were not dose dependent; there were no dose‐limiting toxicities. REGN1908‐1909 is characterized by linear and dose‐proportional kinetics of the two individual mAb components. A single 600 mg dose maintains total mAb mean concentrations in serum above the target (mean of ~ 10 mg/L) for 8–12 weeks. Maintaining this mean target concentration resulted in translational pharmacodynamic effects: maximal mast cell degranulation in a passive cutaneous anaphylaxis mouse model, and maintenance of clinical efficacy measured by Total Nasal Symptom Score in a previous proof‐of‐mechanism study.

Targeting immunodominant Bet v 1 epitopes with monoclonal antibodies prevents the birch allergic response

Blocking the major cat allergen, Fel d 1, with mAbs was effective in preventing an acute cat allergic response. This study sought to extend the allergen-specific antibody approach and demonstrate that a combination of mAbs targeting Bet v 1, the immunodominant and most abundant allergenic protein in birch pollen, can prevent the birch allergic response. Bet v 1-specific mAbs, REGN5713, REGN5714, and REGN5715, were isolated using the VelocImmune platform. Surface plasmon resonance, x-ray crystallography, and cryo-electron microscopy determined binding kinetics and structural data. Inhibition of IgE-binding, basophil activation, and mast cell degranulation were assessed via blocking ELISA, flow cytometry, and the passive cutaneous anaphylaxis mouse model. REGN5713, REGN5714, and REGN5715 bind with high affinity and noncompetitively to Bet v 1. Acocktail of all 3 antibodies, REGN5713/14/15, blocks IgE binding to Bet v 1 and inhibits Bet v 1- and birch pollen extract-induced basophil activation exvivo and mast cell degranulation invivo. Crystal structures of the complex of Bet v 1 with immunoglobulin antigen-binding fragments of REGN5713 or REGN5715 show distinct interaction sites on Bet v 1. Cryo-electron microscopy reveals a planar and roughly symmetrical complex formed by REGN5713/14/15 bound to Bet v 1. These data confirm the immunodominance of Bet v 1 in birch allergy and demonstrate blockade of the birch allergic response with REGN5713/14/15. Structural analyses show simultaneous binding of REGN5713, REGN5714, and REGN5715 with substantial areas of Bet v 1 exposed, suggesting that targeting specific epitopes is sufficient to block the allergic response.

Nanoparticles Displaying Allergen and Siglec-8 Ligands Suppress IgE-FcεRI-Mediated Anaphylaxis and Desensitize Mast Cells to Subsequent Antigen Challenge.

Siglec-8 is an inhibitory receptor expressed on eosinophils and mast cells. In this study, we took advantage of a novel Siglec-8 transgenic mouse model to assess the impact of modulating IgE-dependent mast cell degranulation and anaphylaxis using a liposomal platform to display an allergen with or without a synthetic glycan ligand for Siglec-8 (Sig8L). The hypothesis is that recruitment of Siglec-8 to the IgE-FcεRI receptor complex will inhibit allergen-induced mast cell degranulation. Codisplay of both allergen and Sig8L on liposomes profoundly suppresses IgE-mediated degranulation of mouse bone marrow-derived mast cells or rat basophilic leukemia cells expressing Siglec-8. In contrast, liposomes displaying only Sig8L have no significant suppression of antigenic liposome-induced degranulation, demonstrating that the inhibitory activity by Siglec-8 occurs only when Ag and Sig8L are on the same particle. In mouse models of anaphylaxis, display of Sig8L on antigenic liposomes completely suppresses IgE-mediated anaphylaxis in transgenic mice with mast cells expressing Siglec-8 but has no protection in mice that do not express Siglec-8. Furthermore, mice protected from anaphylaxis remain desensitized to subsequent allergen challenge because of loss of Ag-specific IgE from the cell surface and accelerated clearance of IgE from the blood. Thus, although expression of human Siglec-8 on murine mast cells does not by itself modulate IgE-FcεRI-mediated cell activation, the enforced recruitment of Siglec-8 to the FcεRI receptor by Sig8L-decorated antigenic liposomes results in inhibition of degranulation and desensitization to subsequent Ag exposure.

Inhibitory effect of paeoniflorin on IgE-dependent and IgE-independent mast cell degranulation in vitro and vivo.

The incidence of allergic diseases has increased to such a point that they have become common and have reached epidemic levels. However, their pathogenesis is not fully understood. Paeoniae Radix Rubra is a traditional Chinese medicine that is also used as a dietary supplement. Its main active ingredient is paeoniflorin. Paeoniflorin has good anti-inflammatory, immunomodulation, and antitumor effects. It is utilized in the treatment of various diseases in clinical settings. However, its effects on type I allergies and pseudoallergic reactions have not been comprehensively studied. In this study, we aimed to use DNP-IgE/DNP-BSA and C48/80 to simulate type I allergies and pseudoallergic reactions to evaluate the therapeutic effects of paeoniflorin to these diseases and identify its molecular mechanisms in cell degranulation both in vivo and in vitro. Results showed that paeoniflorin inhibited the degranulation of RBL-2H3 cells induced by these two stimuli (IgE-dependent and IgE-independent stimuli) in a dose-dependent manner. Moreover, qPCR and western blot analyses indicated that paeoniflorin may regulate the IgE/FcεR I, MRGPRB3, and downstream signal transduction pathways to exert its therapeutic effects on type I allergies and pseudoallergic reactions. In addition, DNP-IgE/DNP-BSA and compound 48/80 were used to induce the establishment of a passive cutaneous anaphylaxis mouse model. Paeoniflorin was found to suppress the extravasation of Evans Blue and tissue edema in the ears, back skin, and paws of the mice. This result further confirmed that paeoniflorin has a notable therapeutic effect on type I allergies and pseudoallergic reactions. Therefore, paeoniflorin could potentially be used as a drug for the treatment of type I allergies and pseudoallergic reactions. This study provides new insights into expanding the treatment range of paeoniflorin and its pharmacological mechanism.

Abstract 15720: Transmembrane Stem Cell Factor Delivered in Nanocarriers Promotes Angiogenesis in Ischemia Without Mast Cell Activation

Introduction: The current standard cares for peripheral artery disease (PAD) include surgical revascularizations with bypass grafting or percutaneous interventions. However, these interventions cannot be performed in a significant portion of patients, and many do not respond to these surgical procedures. Protein therapy to stimulate the body to create new vasculature is another alternative, which is minimally invasive to patients. Stem cell factor (SCF) is a candidate protein for treating PAD, but clinical use of SCF has been limited due to toxicity related to mast cell activation. SCF also exists in a transmembrane form (tmSCF), possessing differential activities from soluble SCF and has not been explored as a therapeutic agent. Results: To develop tmSCF as a therapeutic we created tmSCF embedded in liposome or lipid nanodisc (Fig. A) . Hindlimb ischemia model on WT and ob/ob mice showed that tmSCF proteliposome (tmSCFPL) and nanodisc (tmSCFND) improved blood flow recovery significantly more than control (Fig. B, C) . Mouse model of anaphylaxis revealed that tmSCF-based therapies did not activate mast cells (Fig. D, E) . Colocalization assay of c-Kit and clathrin/caveolin revealed that mast cells preferentially use clathrin-mediated pathways to internalize SCF and caveolin-mediated pathways for tmSCF-based therapies (Fig. F, G) . Surface c-Kit internalization study on mast cells showed faster uptake of SCF in comparison to tmSCF-based therapies (Fig. H) . Previous study indicates that clathrin-mediated internalization causes increased activation of mast cells. Our studies together with the previous finding suggest that mast cell activation does not occur for tmSCF-based therapies because of the slower uptake, greater utilization of the caveolin internalization pathway and weaker activation of mast cells. Conclusions: TmSCF-based therapies can provide therapeutic benefits without off-target effects on mast cells by tuning activation with nanocarriers.

Bruton's tyrosine kinase inhibition effectively protects against human IgE-mediated anaphylaxis.

No known therapies can prevent anaphylaxis. Bruton's tyrosine kinase (BTK) is an enzyme thought to be essential for high-affinity IgE receptor (FcεRI) signaling in human cells. We tested the hypothesis that FDA-approved BTK inhibitors (BTKis) would prevent IgE-mediated responses including anaphylaxis. We showed that irreversible BTKis broadly prevented IgE-mediated degranulation and cytokine production in primary human mast cells and blocked allergen-induced contraction of isolated human bronchi. To address their efficacy in vivo, we created and used what we believe to be a novel humanized mouse model of anaphylaxis that does not require marrow ablation or human tissue implantation. After a single intravenous injection of human CD34+ cells, NSG-SGM3 mice supported the population of mature human tissue-resident mast cells and basophils. These mice showed excellent responses during passive systemic anaphylaxis using human IgE to selectively evoke human mast cell and basophil activation, and response severity was controllable by alteration of the amount of allergen used for challenge. Remarkably, pretreatment with just 2 oral doses of the BTKi acalabrutinib completely prevented moderate IgE-mediated anaphylaxis in these mice and also significantly protected against death during severe anaphylaxis. Our data suggest that BTKis may be able to prevent anaphylaxis in humans by inhibiting FcεRI-mediated signaling.

Anti-allergic effect of the Src family kinase inhibitor saracatinib.

The aim of this study was to evaluate the anti-anaphylactic and anti-allergic potentials of saracatinib, a Src family kinase inhibitor that was already shown to be safe in clinical trials when it was used as an anti-cancer drug. Using in vitro mast cell models, we found that saracatinib inhibited the degranulation response and cytokine production in RBL2H3 cells that were stimulated with IgE and antigen without affecting cell viability. Phosphorylation of Lyn, Akt, a PI3K substrate, and MAPKs including ERK, JNK, and p38, as well as the intracellular Ca2+ increase induced by this stimulation were also suppressed by saracatinib. This drug also inhibited symptoms in our established anaphylaxis mouse model, anaphylaxis-dependent spotted distribution of immune complex in skin (ASDIS). The intravenous injection of the mixture of IgE and antigen induced acute spotted distribution of immune complex in skin in hairless HR-1 mice, and its inhibition by intradermal injection of saracatinib was observed. Moreover, toluidine blue-stained skin sections indicated that the degranulation ratio of dermal mast cells was reduced in saracatinib-treated skin compared with vehicle-treated skin. Because only a few signaling inhibitors are used as anti-anaphylaxis and anti-allergic drugs, these results indicated the valuable suggestion that saracatinib and the Src family kinase inhibitors are good candidates for anti-anaphylaxis and anti-allergic drugs.

Eckol from Ecklonia cava Suppresses Immunoglobulin E-mediated Mast Cell Activation and Passive Cutaneous Anaphylaxis in Mice.

Eckol, a precursor compound belonging to the dibenzo-1,4-dioxin class of phlorotannins, is a phloroglucinol derivative that exerts various activities. In the present study, we investigated the antiallergic effects of eckol isolated from the marine brown algae, Ecklonia cava using immunoglobulin E (IgE)/bovine serum albumin (BSA)-stimulated mouse bone marrow-derived cultured mast cells (BMCMC) and a mouse model of anaphylaxis. Eckol inhibited IgE/BSA-induced BMCMC degranulation by reducing β-hexosaminidase release. A flow cytometric analysis revealed that eckol decreases FcεRI expression on cell surface and IgE binding to the FcεRI in BMCMC. Moreover, eckol suppressed the production of the cytokines, interleukin (IL)-4, IL-5, IL-6, and IL-13 and the chemokine, thymus activation-regulated chemokine (TARC) by downregulating, IκB-α degradation and NF-κB nuclear translocation. Furthermore, it attenuated the passive cutaneous anaphylactic reaction induced by IgE/BSA-stimulation in the ear of BALB/c mice. These results suggest that eckol is a potential therapeutic candidate for the prevention and treatment of allergic disorders.

Transmembrane stem cell factor protein therapeutics enhance revascularization in ischemia without mast cell activation

Stem cell factor (SCF) is a cytokine that regulates hematopoiesis and other biological processes. While clinical treatments using SCF would be highly beneficial, these have been limited by toxicity related to mast cell activation. Transmembrane SCF (tmSCF) has differential activity from soluble SCF and has not been explored as a therapeutic agent. We created novel therapeutics using tmSCF embedded in proteoliposomes or lipid nanodiscs. Mouse models of anaphylaxis and ischemia revealed the tmSCF-based therapies did not activate mast cells and improved the revascularization in the ischemic hind limb. Proteoliposomal tmSCF preferentially acted on endothelial cells to induce angiogenesis while tmSCF nanodiscs had greater activity in inducing stem cell mobilization and recruitment to the site of injury. The type of lipid nanocarrier used altered the relative cellular uptake pathways and signaling in a cell type dependent manner. Overall, we found that tmSCF-based therapies can provide therapeutic benefits without off target effects.

A biotinylated peptide, BP21, alleviates hypotension in anaphylactic mice.

Platelet-activating factor (PAF) is known as an important mediator of anaphylaxis and, therefore, may possibly serve as a direct target for anti-anaphylactic drugs. We recently reported that a synthetic N-terminally biotinylated peptide, BP21, alleviates hypothermia and vascular hyperpermeability during anaphylactic reactions in a mouse model of anaphylaxis via the direct binding of a Tyr-Lys-Asp-Gly sequence in the peptide to PAF. In this study, we investigated the effect of BP21 on in vivo anaphylactic hypotension. Results showed that BP21 significantly inhibited anaphylactic hypotension in a dose-dependent manner, with peak severity being reached within 20minutes. Adrenaline, which is the recommended first line treatment for anaphylactic patients, did not inhibit anaphylactic hypothermia. The combined administration of BP21 with adrenaline inhibited both hypotension and hypothermia, even at both low doses, more effectively compared with solo administration of BP21 or adrenaline. These results suggested that BP21 could potentially be a novel anti-anaphylactic agent for targeting PAF in vivo.