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

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

Biodegradable Mesoporous Organosilica-Based Nanostabilizer Targeting Mast Cells for Long-Term Treatment of Allergic Diseases.

Allergic diseases are immune system dysfunctions mediated by mast cell (MC) activation stimulated by specific allergens. However, current small molecular MC stabilizers for allergic disease prevention often require multiple doses over a long period of time and are associated with serious side effects. Herein, we develop a diselenide-bridged mesoporous silica nanostabilizer, proving that it could specifically target sensitized MCs via the recognition of IgE aptamer and IgE. Meantime, the IgE aptamer can also mitigate allergic reactions by preventing re-exposure of allergens from the surface of sensitized MCs. Furthermore, the diselenide-bridged scaffold can be reduced by the intracellular excessive ROS, subsequently achieving redox homeostasis via ROS depletion. Finally, the precise release of small molecular MC stabilizers along with the biodegradation of nanocarrier can stabilize the membranes of MCs. In vivo assays in passive cutaneous anaphylactic (PCA) and allergic rhinitis (AR) mice indicated that our current strategy further endowed it with a high efficacy, long-term therapeutic time window, as well as negligible inflammatory side effects for allergic diseases, offering a promising therapeutic strategy for the clinical generalization of allergic diseases.

Traditional cooking methods decreased the allergenicity of egg proteins.

Egg allergy is one of the most common food allergies globally. This study aimed to assess the impact of four traditional cooking methods on the allergenicity of egg proteins using a comprehensive strategy, including simulated gastrointestinal digestion in vitro, serology experiments, a rat basophilic leukemia (RBL)-2H3 cell degranulation model, and a passive cutaneous anaphylaxis (PCA) mice model, and the structure changes were detected by circular dichroism (CD) spectra and ultraviolet (UV) spectra. The results showed that the processed egg proteins were more readily digested compared to raw egg proteins. The serological experiments revealed a significant reduction in immunoglobulin E binding of egg proteins after thermal treatments (p < 0.05), particularly after frying. Subsequently, the RBL-2H3 cell degranulation experiment demonstrated a marked decrease in the level of egg allergens-induced ß-hexosaminidase release after cooking (p < 0.05). Moreover, the results from the PCA mice model indicated that the increase in vascular permeability was effectively relieved in the treated groups, especially in frying group (p < 0.05). Additionally, the α-helix and ß-turn contents of processed egg proteins were significantly decreased (p < 0.05) compared with native egg proteins. The UV spectra findings showed that all cooking treatments caused significant alterations in the tertiary structure, and fluorescence analysis indicated that cooking decreased the surface hydrophobicity of egg proteins. In conclusion, four traditional cooking methods reduced the allergenicity of egg proteins, particularly frying, and this reduction was associated with structural changes that could contribute to the destruction or masking of epitopes of egg allergens. PRACTICAL APPLICATION: Egg allergy has a serious impact on public health, and there is no ideal treatment method at present. This study demonstrated that four traditional cooking methods (boiling, steaming, baking, and frying) reduced the allergenicity of egg proteins, especially frying, and the results will provide a basis for the development of hypoallergenic egg products.

A novel natural Syk inhibitor suppresses IgE-mediated mast cell activation and passive cutaneous anaphylaxis.

Spleen tyrosine kinase (Syk) plays a crucial role as a target for allergy treatment due to its involvement in immunoreceptor signaling. The purpose of this study was to identify natural inhibitors of Syk and assess their effects on the IgE-mediated allergic response in mast cells and ICR mice. A list of eight compounds was selected based on pharmacophore and molecular docking, showing potential inhibitory effects through virtual screening. Among these compounds, sophoraflavanone G (SFG) was found to inhibit Syk activity in an enzymatic assay, with an IC50 value of 2.2 µM. To investigate the conformational dynamics of the SYK-SFG system, we performed molecular dynamics simulations. The stability of the binding between SFG and Syk was evaluated using root mean square deviation (RMSD) and root mean square fluctuation (RMSF). In RBL-2H3 cells, SFG demonstrated a dose-dependent suppression of IgE/BSA-induced mast cell degranulation, with no significant cytotoxicity observed at concentrations below 10.0 µM within 24 h. Furthermore, SFG reduced the production of TNF-α and IL-4 in RBL-2H3 cells. Mechanistic investigations revealed that SFG inhibited downstream signaling proteins, including phospholipase Cγ1 (PLCγ1), as well as mitogen-activated protein kinases (AKT, Erk1/2, p38, and JNK), in mast cells in a dose-dependent manner. Passive cutaneous anaphylaxis (PCA) experiments demonstrated that SFG could reduce ear swelling, mast cell degranulation, and the expression of COX-2 and IL-4. Overall, our findings identify naturally occurring SFG as a direct inhibitor of Syk that effectively suppresses mast cell degranulation both in vitro and in vivo.

Fucosterol isolated from Sargassum horneri attenuates allergic responses in immunoglobulin E/bovine serum albumin-stimulated mast cells and passive cutaneous anaphylaxis in mice.

Allergic diseases have become a serious problem worldwide and occur when the immune system overreacts to stimuli. Sargassum horneri is an edible marine brown alga with pharmacological relevance in treating various allergy-related conditions. Therefore, this study aimed to investigate the effect of fucosterol (FST) isolated from S. horneri on immunoglobulin E(IgE)/bovine serum albumin (BSA)-stimulated allergic reactions in mouse bone marrow-derived cultured mast cells (BMCMCs) and passive cutaneous anaphylaxis (PCA) in BALB/c mice. The in silico analysis results revealed the binding site modulatory potential of FST on the IgE and IgE-FcεRI complex. The findings of the study revealed that FST significantly suppressed the degranulation of IgE/BSA-stimulated BMCMCs by inhibiting the release of ß-hexosaminidase and histamine in a dose-dependent manner. In addition, FST effectively decreased the expression of FcεRI on the surface of BMCMCs and its IgE binding. FST dose-dependently downregulated the expression of allergy-related cytokines (interleukin (IL)-4, -5, -6, -13, tumor necrosis factor (TNF)-α, and a chemokine (thymus and activation-regulated chemokine (TARC)) by suppressing the activation of nuclear factor-κB (NF-κB) and Syk-LAT-ERK-Gab2 signaling in IgE/BSA-stimulated BMCMCs. As per the histological analysis results of the in vivo studies with IgE-mediated PCA in BALB/c mice, FST treatment effectively attenuated the PCA reactions. These findings suggest that FST has an immunopharmacological potential as a naturally available bioactive compound for treating allergic reactions.

Leaves and pseudostems extract of Curcuma longa attenuates immunoglobulin E/bovine serum albumin-stimulated bone marrow-derived cultured mast cell activation and passive cutaneous anaphylaxis in BALB/c mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Curcuma longa, known as turmeric, is an herbaceous perennial plant belonging to the genus Curcuma. It is dispersed throughout tropical and subtropical regions worldwide. Since ancient times, turmeric has been used as an ethnomedicinal plant in the Ayurvedic system, particularly in Asian countries. Rhizomes of turmeric possess several pharmacological properties that give high value as a medicinal remedy for treating a range of conditions, including inflammation, pain, allergies, and digestive issues. Moreover, turmeric leaves and pseudostems also contain a variety of health-enhancing secondary metabolites, such as curcumin, flavonoids, and other phenolic compounds, which exhibit anti-inflammatory, antitumor, antibacterial, and antioxidant properties. AIM OF THE STUDY: Allergic diseases are a group of immune-mediated disorders mainly caused by an immunoglobulin E (IgE)-dependent immunological response to an innocuous allergen. Therefore, this study aimed to investigate the effect of leaves and pseudostems extract of turmeric (TLSWE-8510) on IgE/bovine serum albumin (BSA)-stimulated allergic responses in mouse bone marrow-derived cultured mast cells (BMCMCs) and passive cutaneous anaphylaxis (PCA) in BALB/c mice. MATERIALS AND METHODS: The effect of TLSWE-8510 on mast cell degranulation has been evaluated by investigating the release of ß-hexosaminidase and histamine in IgE/BSA-stimulated BMCMCs. Additionally, anti-allergic properties of TLSWE-8510 on IgE/BSA-stimulated BMCMCs were investigated using suppression of nuclear factor-kappa B (NF-κB), and spleen tyrosine kinase (Syk)-linker for T-cell activation (LAT)-extracellular-signal-regulated kinase (ERK)-GRB2 associated binding protein 2 (Gab2) signaling pathway and downregulation of allergy-related cytokines and chemokines expression. Furthermore, in vivo, studies were conducted using IgE-mediated PCA in BALB/c mice. RESULTS: TLSWE-8510 treatment significantly inhibited the degranulation of IgE/BSA-stimulated BMCMCs by inhibiting the release of ß-hexosaminidase and histamine dose-dependently. Additionally, TLSWE-8510 reduced the expression of high-affinity IgE receptors (Fc epsilon receptor I-FcεRI) on the surface of BMCMCs and the binding of IgE to FcεRI. Besides, the expression of cytokines and chemokines is triggered by IgE/BSA stimulation via activating the allergy-related signaling pathways. TLSWE-8510 dose-dependently downregulated the mRNA expression and the production of allergy-related cytokines (interleukin (IL)-1ß, IL-3, IL-4, IL-5, IL-6, IL-13, tumor necrosis factor (TNF)-α, and interferon (IFN)-γ), and chemokines (thymus and activation-regulated chemokine (TARC), and regulated upon activation, normal T cell expressed and secreted (RANTES)) by regulating the phosphorylation of downstream signaling molecules, NF-κB, and Syk, LAT, ERK and Gab2 in IgE/BSA-stimulated BMCMCs. Moreover, PCA reaction in IgE/BSA-stimulated BALB/c mice ears was effectively decreased by TLSWE-8510 treatment in a dose-dependent manner. CONCLUSIONS: These results collectively demonstrated that TLSWE-8510 suppressed mast cell degranulation by inhibiting the release of chemical mediators related to allergies. TLSWE-8510 downregulated the allergy-related cytokines and chemokines expression and phosphorylation of downstream signaling molecules in IgE/BSA-stimulated BMCMCs. Furthermore, in vivo studies with IgE-mediated PCA reaction in the BALB/c mice ears were attenuated by TLSWE-8510 treatment. These findings revealed that TLSWE-8510 has the potential as a therapeutic agent for the treatment of allergic diseases.

Bioactivity-boosting strategy based on combination of anti-allergic O-methylated catechin with a Citrus flavanone, hesperetin.

Many patients with allergies have anxiety about taking anti-allergic medicines due to their side effects and increased medical expenses. Thus, developing functional foods/agricultural products for allergy prevention is strongly desired. In this study, we revealed that a Citrus flavanone, hesperetin, amplified IgE/antigen-mediated degranulation-inhibitory potency of anti-allergic catechin, (-)-epigallocatechin-3-O-(3-O-methyl) gallate (EGCG3''Me), in the rat basophilic/mast cell line RBL-2H3. Hesperetin also significantly elevated the activation of acid sphingomyelinase (ASM), essential for eliciting anti-allergic effect of EGCG3''Me through the cell surficial protein, 67-kDa laminin receptor (67LR). Furthermore, oral administration of the highly absorbent hesperidin, α-glucosyl hesperidin, also enhanced the inhibitory potency of EGCG3''Me-rich 'Benifuuki' green tea (Camellia sinensis L.) on passive cutaneous anaphylaxis (PCA) reaction evoked by IgE/antigen in BALB/c mice. These observations indicate that hesperetin amplifies the ability of EGCG3''Me to inhibit the IgE/antigen-mediated degranulation through activating ASM signaling.

Chemical composition-based characterization of the anti-allergic effect of Guominkang Formula on IgE-mediated mast cells activation and passive cutaneous anaphylaxis.

Guominkang (GMK), a Chinese medicine formula, has been used to treat allergic diseases in clinical settings for many years. To evaluate the antiallergic effect and molecular mechanism of action of GMK extract, RBL-2H3 cell models and passive cutaneous anaphylaxis (PCA) mouse models were established. High performance liquid chromatography (HPLC) and ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS) analyses were performed to characterize the chemical composition of GMK. A total of 94 compounds were identified or tentatively identified from GMK. Three of them, emodin, ursolic acid, and hamaudol, were identified for the first time as potential active compounds in GMK, since they inhibited the degranulation of mast cells. The anti-allergic effect of hamaudol was the first to be discovered. GMK could markedly mitigate the shade of Evans Blue extravasation and ear incrassation in PCA mouse models. Additionally, GMK significantly inhibited the degranulation of mast cells, suppressed mast cell degranulation by reducing Ca2+ influx and the levels of TNF-α, IL-4, and histamine, and markedly inhibited the phosphorylation of Lyn, Syk, PLCγ1, IκBα, and NF-κB p65. Molecular docking results indicated that hamaudol and emodin had strong interaction with FcɛRI and NF-κB related proteins, while ursolic acid only interacted with NF-κB associated proteins. These results suggest GMK suppresses the activation of MCs both in vivo and in vitro. The underlying mechanism of its anti-allergic activity is associated with the inhibition of FcɛRI and NF-κB activation.

Vasicine alleviates 2,4-dinitrochlorobenzene-induced atopic dermatitis and passive cutaneous anaphylaxis in BALB/c mice.

Atopic dermatitis (AD), a type of skin inflammation, is associated with immune response mediated by T-helper 2 (Th2) cells, and mast cells. Vasicine is an alkaloid isolated from Adhatoda vasica, a popular Ayurvedic herbal medicine used for treating inflammatory conditions. In the present study, the anti-AD effects of vasicine were evaluated on 2,4-dinitrochlorobenzene-induced AD-like skin lesions in BALB/c mice. The potential anti-allergic effects of vasicine were also assessed using the passive cutaneous anaphylaxis (PCA) test. The results showed that the oral administration of vasicine improved the severity of AD-like lesional skin by decreasing histopathological changes and restoring epidermal thickness. Vasicine also inhibited the infiltration of mast cells in the skin and reduced the levels of pro-Th2 and Th2 cytokines as well as immunoglobulin E in the serum. Finally, vasicine inhibited the expression of pro-Th2 and Th2 cytokines in skin tissues, indicating the therapeutic potential of vasicine for AD.

Anemoside B4 protects against IgE-dependent allergic responses by suppressing the PLC/IP3 and JAK/STAT3 pathways.

Anemoside B4 (AB4) is a natural triterpenoid abundant in the roots of Pulsatilla chinensis. Although various biological activities have been widely attributed to AB4, few studies have focused on its antiallergic effects. In this study the inhibitory effects of AB4 on immunoglobulin E (IgE)-mediated allergic responses were investigated, both in vitro and in vivo, and the mechanism of its effects. IgE-mediated passive cutaneous anaphylaxis was used to elucidate the antiallergic effects of AB4 in vivo. The degranulation assay, calcium imaging, and cytokine and chemokine release in the laboratory of allergic disease 2 (LAD2) cell line were used to evaluate the antiallergic effect of AB4 in vitro. Pathological staining was performed to analyze angiectasis. Western blot analysis was performed to investigate the downstream signaling pathways. AB4 dose-dependently attenuated ovalbumin/IgE-induced paw swelling in mice, and reduced the serum concentrations of tumor necrosis factor-alpha and C-C motif chemokine 2. In addition, AB4 suppressed IgE-mediated LAD2 cell degranulation, calcium influx, and PLC/IP3 and JAK/STAT3 phosphorylation. Our results suggest that AB4 inhibits allergic reactions through the PLC/IP3 and JAK/STAT3 pathways.

Rab44 regulates murine mast cell-driven anaphylaxis through kinesin-1-dependent secretory granule translocation.

BACKGROUND: Mast cells (MCs) are key effectors of the allergic response. Following the cross-linking of IgE receptors (FcεRIs), they release crucial inflammatory mediators through degranulation. Although degranulation depends critically on secretory granule (SG) trafficking toward the plasma membrane, the molecular machinery underlying this transport has not been fully characterized. OBJECTIVES: This study analyzed the function of Rab44, a large, atypical Rab guanosine triphosphatase highly expressed in MC, in the MC degranulation process. METHODS: Murine knockout (KO) mouse models (KORab44 and DKOKif5b/Rab44) were used to perform passive cutaneous anaphylaxis experiments and analyze granule translocation in bone marrow-derived MCs during degranulation. RESULTS: This study demonstrate that mice lacking Rab44 (KORab44) in their bone marrow-derived MCs are impaired in their ability to translocate and degranulate SGs at the plasma membrane on FcεRI stimulation. Accordingly, KORab44 mice were less sensitive to IgE-mediated passive cutaneous anaphylaxis in vivo. A lack of Rab44 did not impair early FcεRI-stimulated signaling pathways, microtubule reorganization, lipid mediator release, or cytokine secretion. Mechanistically, Rab44 appears to interact with and function as part of the previously described kinesin-1-dependent transport pathway. CONCLUSIONS: These results highlight a novel role of Rab44 as a regulator of SG transport during degranulation and anaphylaxis acting through the kinesin-1-dependent microtubule transport machinery. Rab44 can thus be considered a potential target for modulating MC degranulation and inhibiting IgE-mediated allergic reactions.

Flavoring agent dihydrocoumarin alleviates IgE-mediated mast cell activation and allergic inflammation.

Mast cells (MCs) are the main effector cells in the onset of high-affinity receptor for IgE (FcεRI)-mediated allergic diseases. The aim of this study was to test whether dihydrocoumarin (DHC), a food flavoring agent derived from Melilotus officinalis, can block IgE-induced MC activation effects and to examine the potential molecular mechanisms by which DHC affects MC activation. Rat basophilic leukemia cells (RBLs) and mouse bone marrow-derived mast cells (BMMCs) were sensitized with anti-dinitrophenol (DNP) immunoglobulin (Ig)E antibodies, stimulated with DNP-human serum albumin antigen, and treated with DHC. Western blot analyses were performed to detect the expression of signaling proteins. Murine IgE-mediated passive cutaneous anaphylaxis (PCA) and ovalbumin (OVA)-induced active systemic anaphylaxis (ASA) models were used to examine DHC effects on allergic reactions in vivo. DHC inhibited MC degranulation, as evidenced by reduced ß-hexosaminidase activity and histamine levels, and reduced morphological changes associated with MC activation, namely cellular elongation and F-actin reorganization. DHC inhibited the activation of MAPK, NF-κB, and AP-1 pathways in IgE-activated MCs. Additionally, DHC could attenuate IgE/Ag-induced allergic reactions (dye extravasation and ear thickening) in PCA as well as OVA challenge-induced reactions in ASA mice (body temperature, serum histamine and IL-4 secretion changes). In conclusion, DHC suppressed MC activation. DHC may represent a new MC-suppressing treatment strategy for the treatment of IgE-mediated allergic diseases.

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 ß-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.

Sargahydroquinoic acid isolated from Sargassum serratifolium as inhibitor of cellular basophils activation and passive cutaneous anaphylaxis in mice.

Basophils and mast cells are characteristic effector cells in allergic reactions. Sargahydorquinoic acid (SHQA), a compound isolated from Sargassum serratifolium (marine alga), possesses various biochemical properties, including potent antioxidant activities. The objective of the present study was to investigate inhibitory effects of SHQA on the activation of human basophilic KU812F cells induced by phorbol myristate acetate and A23187 (PMACI), a calcium ionophore. Furthermore, we confirmed the inhibitory effects of SHQA on the activation of rat basophilic leukemia (RBL)-2H3 cells induced by compound 48/80 (com 48/80), bone marrow-derived mast cells (BMCMCs) induced by anti-dinitrophenyl(DNP)-immunoglobulin E (IgE)/DNP-bovine serum albumin (BSA), DNP/IgE and on the reaction of passive cutaneous anaphylaxis (PCA) mediated by IgE. SHQA reduced PMACI-induced intracellular reactive oxygen species (ROS) and calcium levels. Western blot analysis revealed that SHQA downregulated the activation of ERK, p38, and NF-κB in a dose-dependent manner. Moreover, SHQA suppressed the production and gene expression of various cytokines, including interleukin (IL)-1 ß, IL-4, IL-6, and IL-8 in PMACI-induced KU812F cells and IL-4 and tumor necrosis factor (TNF)- α in com 48/80-induced RBL-2H3 cells. It also determined the inhibition of PMACI, com 48/80- and IgE/DNP-induced degranulation by reducing the release of ß -hexosaminidase. Furthermore, it attenuated the IgE/DNP-induced PCA reaction in the ears of BALB/c mice. These results suggest that SHQA isolated from S. serratifolium is a potential therapeutic functional food material for inhibiting effector cell activation in allergic reactions and anaphylaxis in animal model.

Oral anaphylaxis to peanut in a mouse model is associated with gut permeability but not with Tlr4 or Dock8 mutations.

BACKGROUND: The etiology of food allergy is poorly understood; mouse models are powerful systems to discover immunologic pathways driving allergic disease. C3H/HeJ mice are a widely used model for the study of peanut allergy because, unlike C57BL/6 or BALB/c mice, they are highly susceptible to oral anaphylaxis. However, the immunologic mechanism of this strain's susceptibility is not known. OBJECTIVE: We aimed to determine the mechanism underlying the unique susceptibility to anaphylaxis in C3H/HeJ mice. We tested the role of deleterious Toll-like receptor 4 (Tlr4) or dedicator of cytokinesis 8 (Dock8) mutations in this strain because both genes have been associated with food allergy. METHODS: We generated C3H/HeJ mice with corrected Dock8 or Tlr4 alleles and sensitized and challenged them with peanut. We then characterized the antibody response to sensitization, anaphylaxis response to both oral and systemic peanut challenge, gut microbiome, and biomarkers of gut permeability. RESULTS: In contrast to C3H/HeJ mice, C57BL/6 mice were resistant to anaphylaxis after oral peanut challenge; however, both strains undergo anaphylaxis with intraperitoneal challenge. Restoring Tlr4 or Dock8 function in C3H/HeJ mice did not protect from anaphylaxis. Instead, we discovered enhanced gut permeability resulting in ingested allergens in the bloodstream in C3H/HeJ mice compared to C57BL/6 mice, which correlated with an increased number of goblet cells in the small intestine. CONCLUSIONS: Our work highlights the potential importance of gut permeability in driving anaphylaxis to ingested food allergens; it also indicates that genetic loci outside of Tlr4 and Dock8 are responsible for the oral anaphylactic susceptibility of C3H/HeJ mice.

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

BACKGROUND: Blocking the major cat allergen, Fel d 1, with mAbs was effective in preventing an acute cat allergic response. OBJECTIVES: 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. METHODS: 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. RESULTS: REGN5713, REGN5714, and REGN5715 bind with high affinity and noncompetitively to Bet v 1. A cocktail 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 ex vivo and mast cell degranulation in vivo. 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. CONCLUSIONS: 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.

Iodixanol activation of mast cells: Implications in the pathogenesis of iodixanol-induced delayed cutaneous adverse reactions.

Iodinated contrast media (ICM) is widely used in radiological examination and interventional therapy. In the commonly used ICM, iodixanol is considered to be the safer one. However, compared with other ICMs, it has a higher incidence of delayed cutaneous adverse reactions. The underlying mechanisms are unclear. In this study, mice with positive allergic reactions were selected based on the mouse clinical allergy symptom score and skin and blood samples taken 1, 6, 24, 48, and 72 h after ICMs (6 g iodine/kg) injection for histological and blood analyses. ICMs-induced pseudo-allergic reactions were investigated through in vivo intravital vascular imaging and passive cutaneous anaphylaxis (PCA) not mediated by IgE and through, calcium imaging degranulation of mast cells (MCs), and western blot assays in vitro. Results shows iodixanol-induced systemic anaphylaxis caused severe extravasation of plasma proteins and degranulation of skin MCs, and increased levels of plasma histamine, cytokines and inflammatory chemokines. Mechanistically, iodixanol increases degranulation of MCs and promotes the synthesis of inflammatory factors by activating PLC-γ and PI3K-related pathways. Trigonelline inhibit iodixanol-induced MC-related pseudo-allergic reactions in vitro and in vivo. These results suggest that mice in the iodixanol group had a higher incidence of delayed cutaneous reactions, characterized by cytokine release over time and delayed cutaneous MC degranulation. Iodixanol's delayed cutaneous adverse reactions may be due to a delayed phase of MC-related pseudo-allergic reactions. Trigonelline revealed anti-allergic activity in iodixanol-induced MC-related pseudo-allergic reactions.

Alpha-linolenic acid inhibits IgE-mediated anaphylaxis by inhibiting Lyn kinase and suppressing mast cell activation.

Excessive reactions to allergens can induce systemic, life-threatening physiological dysfunction (anaphylaxis) in humans. The surface of mast cells expresses high-affinity IgE receptors that play a vital role during anaphylaxis. Alpha-linolenic acid (ALA) is an essential non-toxic fatty acid in humans. Since it has been reported having potential to regulate pro-inflammatory reactions, we postulated that ALA could inhibit anaphylaxis by down-regulating Lyn kinase phosphorylation. We found that local and systematic inflammation induced by albumin from chicken egg white (OVA) were attenuated by ALA in vivo. Furthermore, ALA inhibited IgE-mediated Ca2+ mobilization, degranulation, and cytokine release in Laboratory of Allergic Disease 2 (LAD2) cells. The western blot results showed that ALA down-regulate the FcεRI/Lyn/Syk signaling pathway by suppressing Lyn kinase activity. Therefore, ALA could serve as a therapeutic drug candidate for preventing IgE-mediated anaphylaxis.

Quantificação de substâncias do fator de hidratação natural (NMF) do estrato córneo ex vivo em função do fototipo e idade / Ex vivo quantification of natural hydration factor (NMF) substances in the stratum corneum as a function of phototype and age

A hidratação cutânea ocorre, em parte, pelos componentes do Fator de Hidratação Natural (NMF), originados da degradação da filagrina, sendo alguns exemplos o ácido pirrolidona-carboxílico (PCA), o ácido urocânico (UCA) e a histidina (His). Estes estão presentes no estrato córneo (EC). O objetivo deste projeto de pesquisa foi determinar, por cromatografia líquida de alta eficiência (CLAE), o PCA, o UCA e a His no estrato córneo de participantes obtido por tape stripping em função do fototipo e idade do participante da pesquisa. Participantes foram selecionados em função da idade acima de 18 anos, ambos os gêneros e fototipo da pele entre I a VI, de acordo com a classificação de Fitzpatrick. As amostras do EC foram obtidas do antebraço volar por tape stripping e irradiadas artificialmente. A cromatografia líquida de alta eficiência (CLAE) foi eficaz para separação e quantificação adequada das substâncias químicas His, PCA e os isômeros de UCA (trans-UCA e cis-UCA) no estrato córneo dos participantes. O método apresentou-se seletivo e ausente de interferentes, ademais, possuiu linearidade e limites de detecção e quantificação compatíveis com os objetivos dessa investigação. No fototipo I, os níveis de His foram menores em comparação aos demais grupos. Ademais, os níveis dessa mesma substância não apresentaram diferença entre as faixas etárias. Em função da irradiação das amostras, o montante de His aumentou em todos os fototipos. Os níveis de PCA apresentaram-se menores após a irradiação em todos os fototipos de pele. Ainda, as concentrações do PCA foram mais elevadas na faixa etária de 46 a 55 anos de idade. Os níveis de concentração do isômero cis-UCA foram maiores nos participantes com fototipo III, após a irradiação UV. Os níveis de concentração do isômero trans-UCA diminuíram após a irradiação, de forma proporcional à formação de cis-UCA em todos os fototipos. A faixa etária de 46-55 anos de idade obteve níveis significativamente menores de trans-UCA e cis-UCA

Cutaneous hydration occurs, in part, by the components of the Natural Hydration Factor (MFN), originating from the degradation of filagrina, some examples being pyrrolidone-carboxylic acid (PCA), urocanic acid (UCA) and histidine (His). These are present in the stratum corneum (SC). The objective of this research project was to determine, by high efficiency liquid chromatography (HPLC), the PCA, UCA and His in the stratum corneum of participants obtained by tape stripping due to the phototype and age of the research participant. Participants were selected according to age above 18 years, both genders and skin phototype between I and VI, according to Fitzpatrick's classification. The SC samples were obtained from the volar forearm by tape stripping and artificially irradiated. High efficiency liquid chromatography (HPLC) was effective for the separation and proper quantification of the chemicals His, PCA and UCA isomers (trans-UCA and cis-UCA) in the stratum corneum of the participants. The method was selective and absent from interfering, in addition, it had linearity and limits of detection and quantification compatible with the objectives of this investigation. In phototype I, His levels were lower compared to the other groups. Moreover, the levels of this same substance showed no difference between age groups. Due to the irradiation of the samples, the amount of His increased in all phototypes. PCA levels were lower after irradiation in all skin phototypes. Furthermore, PCA concentrations were higher in the age group from 46 to 55 years of age. The concentration levels of the cis-UCA isomer were higher in participants with phototype III after UV irradiation. The concentration levels of the trans-UCA isomer decreased after irradiation, proportionally to the formation of cis-UCA in all phototypes. The age group 46-55 years of age obtained significantly lower levels of trans-UCA and cis-UCA

Biochemical Relapse in Low-risk Prostate Cancer Treated with Radical Prostatectomy and Bilateral Pelvic Lymphadenectomy / Recaída bioquímica en cáncer de próstata de bajo riesgo tratado con prostatectomía radical y linfadenectomía pélvica bilateral

Introduction For low-risk prostate cancer (PCa), curative treatment with radical prostatectomy (RP) can be performed, reporting a biochemical relapse-free survival rate (bRFS) at 5 and 7 years of 90.1% and 88.3%, respectively. Prostatic specific antigen (PSA), pathological stage (pT), and positive margins (R1) are significant predictors of biochemical relapse (BR). Even though pelvic lymphadenectomy is not recommended during RP, in the literature, it is performed in 34% of these patients, finding 0.37% of positive lymph nodes (N1). In this study, we aim to evaluate the 10-year bRFS in patients with low-risk PCa who underwent RP and extended pelvic lymph node dissection (ePLND). Methodology All low-risk patients who underwent RP plus bilateral ePLND at the National Cancer Institute of Colombia between 2006 and 2019 were reviewed. Biochemical relapse was defined as 2 consecutive increasing levels of PSA > 0.2 ng/mL. A descriptive analysis was performed using the STATA 15 software (Stata Corp., College Station, TX, USA), and the Kaplan-Meier curves and uni and multivariate Cox proportional hazard models were used for the survival outcome analysis. The related regression coefficients were used for the hazard ratio (HR), and, for all comparisons, a two-sided p-value ˂ 0.05 was used to define statistical significance. Results Two hundred and two patients met the study criteria. The 10-year bRFS for the general population was 82.5%, statistically related to stage pT3 (p = 0.047), higher Gleason grade group (GG) (p ≤ 0.001), and R1 (p ≤ 0.001), but not with N1. A total of 3.9% of the patients had N1; of these, 75% had R1, 25% GG2, and 37% GG3. Among the N0 (non-lymph node metástasis in prostate cáncer) patients, 31% of the patients had R1, 41% GG2, and 13% GG3. Conclusions Our bRFS was 82.5% in low-risk patients who underwent RP and ePLND. With higher pT, GG, and presence of R1, the probability of BR increased. Those with pN1 (pathologicaly confirmed positive lymph nodes) were not associated with bRFS, with a pN1 detection rate of 3.9%. Details: In low-risk PCa, curative treatment with RP can be performed, reporting a bRFS rate at 5 and 7 years of 90.1% and 88.3%, respectively. Despite the fact that pelvic lymphadenectomy is not recommended during RP in clinical guidelines, in the literature, it is performed in 34% of these patients, finding 0.37% of N1. In this study, we report the 10-year bRFS in patients with low-risk PCa who underwent surgery.

Introducción En el cáncer de próstata (CaP) de bajo riesgo se puede realizar un tratamiento curativo mediante prostatectomía radical (PR), con una tasa de supervivencia libre de recaída bioquímica (SLRb) a 5 y 7 años del 90,1% y el 88,3%, respectivamente. El antígeno prostático específico (PSA), el estadio patológico (pT) y los márgenes positivos (R1) son predictores significativos de recaída bioquímica (BR). Aunque la linfadenectomía pélvica no está recomendada durante la PR, en la literatura se realiza en el 34% de estos pacientes, encontrándose un 0,37% de ganglios linfáticos positivos (N1). En este estudio, nuestro objetivo es evaluar la SLB a 10 años en pacientes con CaP de bajo riesgo sometidos a PR y disección ganglionar pélvica extendida (DGLPe). Metodología Se revisaron todos los pacientes de bajo riesgo sometidos a PR más ePLND bilateral en el Instituto Nacional de Cancerología de Colombia entre 2006 y 2019. La recaída bioquímica se definió como 2 niveles crecientes consecutivos de PSA > 0,2 ng/mL. Se realizó un análisis descriptivo utilizando el software STATA 15 (Stata Corp., College Station, TX, USA), y se utilizaron las curvas de Kaplan-Meier y los modelos uni y multivariados de riesgos proporcionales de Cox para el análisis de resultados de supervivencia. Los coeficientes de regresión relacionados se utilizaron para la hazard ratio (HR), y, para todas las comparaciones, se utilizó un valor p de dos caras ˂ 0,05 para definir la significación estadística. Resultados Doscientos dos pacientes cumplieron los criterios del estudio. La bRFS a 10 años para la población general fue del 82,5%, estadísticamente relacionada con el estadio pT3 (p = 0,047), mayor grupo de grado Gleason (GG) (p ≤ 0,001), y R1 (p ≤ 0,001), pero no con N1. Un total del 3,9% de los pacientes tenían N1; de ellos, el 75% tenían R1, el 25% GG2, y el 37% GG3. Entre los pacientes N0 (metástasis no ganglionar en el cáncer de próstata), el 31% de los pacientes tenían R1, el 41% GG2 y el 13% GG3. Conclusiones Nuestra SSEb fue del 82,5% en los pacientes de bajo riesgo que se sometieron a RP y ePLND. A mayor pT, GG y presencia de R1, mayor probabilidad de RB. Aquellos con pN1 (ganglios linfáticos patológicamente confirmados como positivos) no se asociaron con la SSEb, con una tasa de detección de pN1 del 3,9%. Detalles: En el CaP de bajo riesgo se puede realizar tratamiento curativo con PR, reportando una tasa de SSEb a 5 y 7 años de 90,1% y 88,3%, respectivamente. A pesar de que la linfadenectomía pélvica no está recomendada durante la PR en las guías clínicas, en la literatura se realiza en el 34% de estos pacientes, encontrando un 0,37% de N1. En este estudio, reportamos la SLB a 10 años en pacientes con CaP de bajo riesgo sometidos a cirugía.