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.

Serum CD203c+ Extracellular Vesicle Serves as a Novel Diagnostic and Prognostic Biomarker for Succinylated Gelatin Induced Perioperative Hypersensitive Reaction.

Background: Perioperative hypersensitivity reaction (HR) is an IgE-FcϵRI-mediated hypersensitivity reaction with degranulation and activation of mast cells and basophils. Several studies have focused on assessing the degranulation and activation of mast cells and basophils to diagnose and predict the prognosis of drug induced HR. However, it is challenging to isolate sufficiently pure mast cells and basophils from human sources to investigate. Effective biomarkers to assess mast cells and basophils activation in vivo could potentially have high diagnostic and prognostic values. In the present study, we investigated EVs pelleted from serum in patients with succinylated gelatin induced HR. Methods: Extracellular vesicles (EVs) were isolated using a total exosome isolation kit and ultracentrifugation, characterized by Western blot, transmission electron microscopy, and nanoparticle tracking analysis. Basophils were isolated from fresh peripheral blood by negative selection using Basophil Isolation Kit II. Human mast cell line was stimulated with IL4. The expression levels of proteins related to the hypersensitive response were evaluated by Western blotting and flow Cytometer. Histamine and tryptase levels were tested using a commercial ELISA kit, and gene expression of inflammatory mediators was evaluated by qRT-PCR. The receiver operating characteristic (ROC) curve was used to evaluate the specificity and sensitivity of biomarker in predicting HR. Results: The concentration of EVs and protein expression level of CD63, FcϵRI, CD203c and tryptase were significantly (p< 0.05) increased in HR samples. The expression level of mast cell/basophil specific CD203c were significantly increased in EVs derived from serum and basophils of HR patients, and the CD203c+-EVs production in mast cells is dramatically increased in the presence of IL4, which positively correlated with histamine, tryptase and inflammatory mediators. Moreover, the ROC curve of EVs concentration and CD203c expression indicated that CD203c+-EVs had a strong diagnostic ability for HR. Conclusion: Serum CD203c+-EVs serves as a novel diagnostic and prognostic biomarker for HR.

Detection of drug-specific immunoglobulin E (IgE) and acute mediator release for the diagnosis of immediate drug hypersensitivity reactions.

The diagnosis of a drug hypersensitivity reaction (DHR) is complex. The first step after taking the clinical history is to look for a sensitization to confirm or exclude the diagnosis and to identify the culprit drug. Skin tests are the primary means of detecting sensitization in DHR, but are associated with a risk for a severe reaction and may be contraindicated. In vitro tests offer the potential to support or confirm a diagnosis of DHR and influence medical decision making. For immediate-type DHR, a few validated assays for measurement of specific IgE (sIgE) are commercially available to a limited number of drugs. In addition, several home-made sIgE radioimmunoassays have been used in other studies. The sensitivity of the sIgE assay is drug-dependant and generally low (0-85%) for betalactams and reported heterogeneous for other drugs ranging from 26% for chlorhexidine and 44% for suxamethonium to 92% for chlorhexidine. However, as all these studies included patients, in whom DHR was confirmed only by skin tests and not by provocation, the results have to be interpreted carefully and may be unreliable. Determination of mediators during an acute phase of a reaction may indirectly support the diagnosis of a DHR by demonstrating mast cell and basophil mediator release. Negative in vitro tests do not exclude a DHR or imputability of a drug, but a positive result may support causality and eliminate the necessity for a drug provocation test. Unfortunately, evidence is limited with a lack of well-controlled studies in larger numbers of well-phenotyped patients, which results in susceptibility for bias and a need for future multicenter studies.

Fel d1 Blocking Antibodies: A Novel Method to Reduce IgE-Mediated Allergy to Cats.

Fel d1 is an important allergen produced by cats that causes IgE reactions in up to 95% of cat-allergic adults. Immunotherapy to reduce human allergy to cats has demonstrated that people have the capacity to produce allergen-specific neutralizing antibodies that block IgE-mediated allergic responses. We wished to determine if "blocking" antibodies could be used to reduce the IgE binding ability of cat allergens prior to their exposure to humans. Here, we describe the characterization of Fel d1-specific antibodies. We demonstrated the efficacy of a rabbit polyclonal and an allergen-specific chicken IgY to bind to Fel d1 in cat saliva and block Fel d1-IgE binding and IgE-mediated basophil degranulation. Fel d1 blocking antibodies offer a new and exciting approach to the neutralization of cat allergens.

IL-37 Targets TSLP-Primed Basophils to Alleviate Atopic Dermatitis.

Atopic dermatitis (AD) represents a severe global burden on physical, physiological and mental health. Innate immune cell basophils are essential for provoking allergic inflammation in AD. However, the roles of novel immunoregulatory cytokine IL-37 in basophils remain elusive. We employed in vitro co-culture of human basophils and human keratinocyte HaCaT cells and an in vivo MC903-induced AD murine model to investigate the anti-inflammatory mechanism of IL-37. In the in vitro model, IL-37b significantly decreased Der p1-induced thymic stromal lymphopoietin (TSLP) overexpression in HaCaT cells and decreased the expression of TSLP receptor as well as basophil activation marker CD203c on basophils. IL-37 could also reduce Th2 cytokine IL-4 release from TSLP-primed basophils ex vivo. In the in vivo model, alternative depletion of basophils ameliorated AD symptoms and significantly lowered the Th2 cell and eosinophil populations in the ear and spleen of the mice. Blocking TSLP alleviated the AD-like symptoms and reduced the infiltration of basophils in the spleen. In CRISPR/Cas9 human IL-37b knock-in mice or mice with direct treatment by human IL-37b antibody, AD symptoms including ear swelling and itching were significantly alleviated upon MC903 challenge. Notably, IL-37b presence significantly reduced the basophil infiltration in ear lesions. In summary, IL-37b could regulate the TSLP-mediated activation of basophils and reduce the release of IL-4. The results, therefore, suggest that IL-37 may target TSLP-primed basophils to alleviate AD.

Structure-guided design of ultrapotent disruptive IgE inhibitors to rapidly terminate acute allergic reactions.

BACKGROUND: Anaphylaxis represents one of the most severe and fatal forms of allergic reactions. Like most other allergies, it is caused by activation of basophils and mast cells by allergen-mediated cross-linking of IgE bound to its high-affinity receptor, FcεRI, on the cell surface. The systemic release of soluble mediators induces an inflammatory cascade, rapidly causing symptoms with peak severity in minutes to hours after allergen exposure. Primary treatment for anaphylaxis consists of immediate intramuscular administration of adrenaline. OBJECTIVE: While adrenaline alleviates life-threatening symptoms of an anaphylactic reaction, there are currently no disease-modifying interventions available. We sought to develop potent and fast-acting IgE inhibitors with the potential to rapidly terminate acute allergic reactions. METHODS: Using affinity maturation by yeast display and structure-guided molecular engineering, we generated 3 optimized disruptive IgE inhibitors based on designed ankyrin repeat proteins and assessed their ability to actively remove IgE from allergic effector cells in vitro as well as in vivo in mice. RESULTS: The engineered IgE inhibitors rapidly dissociate preformed IgE:FcεRI complexes, terminate IgE-mediated signaling in preactivated human blood basophils in vitro, and shut down preinitiated allergic reactions and anaphylaxis in mice in vivo. CONCLUSIONS: Fast-acting disruptive IgE inhibitors demonstrate the feasibility of developing kinetically optimized inhibitors for the treatment of anaphylaxis and the rapid desensitization of allergic individuals.

Peripheral blood basophils are the main source for early interleukin-4 secretion upon in vitro stimulation with Culicoides allergen in allergic horses.

Interleukin-4 (IL-4) is a key cytokine secreted by type 2 T helper (Th2) cells that orchestrates immune responses during allergic reactions. Human and mouse studies additionally suggest that basophils have a unique role in the regulation of allergic diseases by providing initial IL-4 to drive T cell development towards the Th2 phenotype. Equine Culicoides hypersensitivity (CH) is a seasonal immunoglobulin E (IgE)-mediated allergic dermatitis in horses in response to salivary allergens from Culicoides (Cul) midges. Here, we analyzed IL-4 production in peripheral blood mononuclear cells (PBMC) of CH affected (n = 8) and healthy horses (n = 8) living together in an environment with natural Cul exposure. During Cul exposure when allergic horses had clinical allergy, IL-4 secretion from PBMC after stimulation with Cul extract was similar between healthy and CH affected horses. In contrast, allergic horses had higher IL-4 secretion from PBMC than healthy horses during months without allergen exposure. In addition, allergic horses had increased percentages of IL-4+ cells after Cul stimulation compared to healthy horses, while both groups had similar percentages of IL-4+ cells following IgE crosslinking. The IL-4+ cells were subsequently characterized using different cell surface markers as basophils, while very few allergen-specific CD4+ cells were detected in PBMC after Cul extract stimulation. Similarly, IgE crosslinking by anti-IgE triggered basophils to produce IL-4 in all horses. PMA/ionomycin consistently induced high percentages of IL-4+ Th2 cells in both groups confirming that T cells of all horses studied were capable of IL-4 production. In conclusion, peripheral blood basophils produced high amounts of IL-4 in allergic horses after stimulation with Cul allergens, and allergic horses also maintained higher basophil percentages throughout the year than healthy horses. These new findings suggest that peripheral blood basophils may play a yet underestimated role in innate IL-4 production upon allergen activation in horses with CH. Basophil-derived IL-4 might be a crucial early signal for immune induction, modulating of immune responses towards Th2 immunity and IgE production.

Basophil and mast cell activation tests by flow cytometry in immediate drug hypersensitivity: Diagnosis and beyond.

Immediate drug hypersensitivity reactions (IDHRs) constitute a significant health issue with serious consequences of diagnostic error. The primary diagnostics to document IDHRs usually consists of quantification of drug-specific IgE (sIgE) antibodies and skin tests. Unfortunately, the positive predictive value (PPV) and negative predictive value (NPV) of these tests are not absolutely, which leaves room for new tests. Over the last two decades, the basophil activation test (BAT), in which ex vivo activation of individual basophils is quantified by flow cytometry, has emerged as a reliable complementary diagnostic to document IDHRs, to explore allergenic recognition, to study cross-reactivity and to monitor therapy. However, the BAT is technically challenging requiring specialized personnel and equipment, fresh samples and the technique is lost as a diagnostic in patients showing a non-responder status of their cells. By consequence, the BAT has still not entered mainstream application. In contrast, mast cell activation tests (MATs) use serum samples that can be frozen, stored, and shipped to a recognized reference centre experienced in mast cell (MC) lines and/or cultures and capable of offering batch testing with necessary quality controls. This review does not only highlight the use of the BAT and MAT as diagnostics in IDHRs, but also outlines the potential of both techniques in further exploring and unveiling the mechanisms that govern drug-induced basophil and MC activation and degranulation.

Induction of the apoptosis, degranulation and IL-13 production of human basophils by butyrate and propionate via suppression of histone deacetylation.

Allergic diseases are caused by dysregulated Th2 immune responses involving multiple effector cells including basophils. Short chain fatty acids (SCFAs), mainly acetate, propionate and butyrate, exert immunomodulatory functions via activation of its receptors GPR41 and GPR43, and inhibition of the histone deacetylases (HDACs) activity. In allergic diseases, SCFAs suppress the activity of mast cells, eosinophils and type 2 innate lymphoid cells (ILC2) but enhance the function of Th2 cells. Here, we aimed to elucidate the function of SCFAs on human basophils. Human basophils were purified from healthy donors by flow cytometric sorting. The surface proteins, apoptosis and degranulation of basophils were analyzed by flow cytometric analysis. The mRNA expression was assayed using real-time PCR. Interleukin 4 (IL-4) and IL-13 were measured by ELISA. Histone acetylation was examined by western blot. GPR41 was expressed by basophils and was enhanced by IL-3. Acetate induced intracellular calcium influx in basophils which was suppressed by blocking GPR41. Propionate and butyrate, but not acetate, induced the expression of CD69 and IL-13. In addition, propionate and butyrate enhanced IgE-mediated basophil degranulation but inhibited basophil survival and IL-4 secretion. Propionate and butyrate induced histone acetylation of basophils and suppression of HDACs activity mimicked the effects of propionate and butyrate on human basophils. Our findings demonstrate that propionate and butyrate may play a complex role in regulating basophil apoptosis, activation and degranulation via inhibiting HDACs activity. The in vivo effects of SCFAs on the regulation of basophil-associated allergic diseases need to be further explored.

Anti-Food Allergic Compounds from Penicillium griseofulvum MCCC 3A00225, a Deep-Sea-Derived Fungus.

Ten new (1-10) and 26 known (11-36) compounds were isolated from Penicillium griseofulvum MCCC 3A00225, a deep sea-derived fungus. The structures of the new compounds were determined by detailed analysis of the NMR and HRESIMS spectroscopic data. The absolute configurations were established by X-ray crystallography, Marfey's method, and the ICD method. All isolates were tested for in vitro anti-food allergic bioactivities in immunoglobulin (Ig) E-mediated rat basophilic leukemia (RBL)-2H3 cells. Compound 13 significantly decreased the degranulation release with an IC50 value of 60.3 µM, compared to that of 91.6 µM of the positive control, loratadine.

Diverse innate stimuli activate basophils through pathways involving Syk and IκB kinases.

Mature basophils play critical inflammatory roles during helminthic, autoimmune, and allergic diseases through their secretion of histamine and the type 2 cytokines interleukin 4 (IL-4) and IL-13. Basophils are activated typically by allergen-mediated IgE cross-linking but also by endogenous "innate" factors. The aim of this study was to identify the innate stimuli (cytokines, chemokines, growth factors, hormones, neuropeptides, metabolites, and bacterial products) and signaling pathways inducing primary basophil activation. Basophils from naïve mice or helminth-infected mice were cultured with up to 96 distinct stimuli and their influence on basophil survival, activation, degranulation, and IL-4 or IL-13 expression were investigated. Activated basophils show a heterogeneous phenotype and segregate into distinct subsets expressing IL-4, IL-13, activation, or degranulation markers. We find that several innate stimuli including epithelial derived inflammatory cytokines (IL-33, IL-18, TSLP, and GM-CSF), growth factors (IL-3, IL-7, TGFß, and VEGF), eicosanoids, metabolites, TLR ligands, and type I IFN exert significant direct effects on basophils. Basophil activation mediated by distinct upstream signaling pathways is always sensitive to Syk and IκB kinases-specific inhibitors but not necessarily to NFAT, STAT5, adenylate cyclase, or c-fos/AP-1 inhibitors. Thus, basophils are activated by very diverse mediators, but their activation seem controlled by a core checkpoint involving Syk and IκB kinases.

A Phase I, Randomized, Double-Blind, Placebo-Controlled, Single-Dose and Multiple-Rising-Dose Study of the BTK Inhibitor TAK-020 in Healthy Subjects.

Bruton's tyrosine kinase (BTK) is a target for treatment of hematologic malignancies and autoimmune diseases. TAK-020 is a highly selective covalent BTK inhibitor that inhibits both B cell receptor and fragment crystallizable receptor signaling. We assessed the safety/tolerability and pharmacokinetics/pharmacodynamics (PDs) of TAK-020 in healthy subjects. Each cohort of the single-rising dose (n = 72; 9 cohorts) and the multiple-rising dose (n = 48; 6 cohorts) portions of the study comprised six TAK-020-treated and two placebo-treated, subjects aged 18-55 years (inclusive). The PD effects were assessed by measuring BTK occupancy and the inhibition of fragment crystallizable epsilon receptor 1 (FcεRI)-mediated activation of basophils. Overall, treatment-emergent adverse events (TEAEs) were similar to placebo; there were no serious TEAEs or no TEAEs leading to discontinuation. TAK-020 was rapidly absorbed (median time to maximum plasma concentration (Tmax ) 45-60 minutes) with a half-life of ~ 3-9 hours at doses ≥ 2.5 mg. TAK-020 exposure was generally dose proportional for single doses ≤ 70 mg and after multiple doses of ≤ 60 mg once daily. Target occupancy was dose dependent, with doses ≥ 2.5 mg yielding maximum and sustained occupancy > 70% for > 96 hours. Single doses ≥ 4.4 mg reduced FcεRI-mediated activation of basophils by > 80% and comparable inhibition was observed with daily dosing ≥3.75 mg for 9 days. Inhibition persisted for 24-72 hours postdose and the duration generally increased with dose. TAK-020 was generally well-tolerated in healthy subjects after single and multiple doses and demonstrated target engagement and pathway modulation. The PD effects outlasted drug exposures, as expected for covalent inhibition of BTK.

Response of peripheral blood basophils in subjects with chronic spontaneous urticaria during treatment with omalizumab.

BACKGROUND: Treatment of patients with asthma or food allergy with omalizumab results in several consistent changes in circulating basophils. The multiple basophil phenotypes observed in patients with chronic spontaneous urticaria (CSU) present some unique attributes that may not respond in a similar fashion to patients with asthma or food allergy. As part of a clinical study on the therapeutic outcomes of omalizumab treatment in CSU, the basophil compartment was examined for changes in characteristics predicted by prior studies. OBJECTIVE: This study sought to examine the changes in basophil function and its relationship to auto-antibodies in serum during treatment with omalizumab. METHODS: At multiple time points before and during omalizumab treatment of patients with CSU, basophil surface IgE and FcεRI expression, cellular spleen tyrosine kinase (SYK) expression, IgE-mediated histamine release (HR), and the presence of auto-antibodies in serum were determined. RESULTS: Three basophil phenotypes were enumerated in the clinical study and used to group results in this basophil study: subjects with (1) basopenia, (2) normal basophil numbers with normal IgE-mediated HR, and (3) normal basophil numbers with poor HR. Basopenia was highly associated with the presence of auto-antibodies to unoccupied FcεRI and basophil numbers did not change during treatment. Likewise, subjects who are basopenic showed no changes in SYK expression or HR during treatment. In basophils of subjects who are nonbasopenic, increases in SYK expression and HR showed the expected inverse relationship to starting SYK and HR levels. Treatment with omalizumab resulted in similar kinetics for decreases in surface FcεRI and IgE in all 3 groups. CONCLUSIONS: A unifying interpretation of the results revolves around the presence of auto-antibodies to FcεRI in CSU. If present, basopenia and an absence of changes in basophils during omalizumab treatment are observed. If auto-antibodies are absent, the changes in the basophil compartment are consistent with prior studies of asthma and food allergy. These group differences also are related to efficacy of the treatment for clinical outcomes, as found in the parent clinical study.

Effects of thermal processing on the allergenicity, structure, and critical epitope amino acids of crab tropomyosin.

Food processing can change the structure and immunoreactivity of purified allergens, but the effect of food processing on the immunoreactivity of the processed and purified allergen is still poorly understood. In this study, tropomyosin (TM) was obtained from Scylla paramamosain and purified after different treatments. A basophil activation test was employed to detect the allergenicity of allergens. The protein structure was detected by mass spectrometry, circular dichroism spectroscopy and surface hydrophobicity. Critical amino acids were identified by Dot blot. Heating obviously affects the biochemical characteristics of TM. The allergenicity of TM was decreased in high temperature-pressure-processed crabs, due to alteration in the protein structure (e.g. denaturation). Seven critical amino acids, namely, R21, E103, E104, E115, A116, E122 and E156, related to the maintenance of the IgE-binding activity of TM were identified. This research of thermal processing helps to accurately reduce or eliminate the immunoreactivity of crabs by food processing.

Digestion and Transport across the Intestinal Epithelium Affects the Allergenicity of Ara h 1 and 3 but Not of Ara h 2 and 6.

SCOPE: No accepted and validated methods are currently available which can accurately predict protein allergenicity. In this study, the role of digestion and transport on protein allergenicity is investigated. METHODS AND RESULTS: Peanut allergens (Ara h 1, 2, 3, and 6) and a milk allergen (ß-lactoglobulin) are transported across pig intestinal epithelium using the InTESTine model and afterward basophil activation is measured to assess the (remaining) functional properties. Additionally, allergens are digested by pepsin prior to epithelial transport and their allergenicity is assessed in a human mast cell activation assay. Remarkably, transported Ara h 1 and 3 are not able to activate basophils, in contrast to Ara h 2 and 6. Digestion prior to transport results in a significant increase in mast cell activation of Ara h 1 and 3 dependent on the length of digestion time. Activation of mast cells by Ara h 2 and 6 is unaffected by digestion prior to transport. CONCLUSIONS: Digestion and transport influences the allergenicity of Ara h 1 and 3, but not of Ara h 2 and 6. The influence of digestion and transport on protein allergenicity may explain why current in vitro assays are not predictive for allergenicity.

Propolis suppresses cytokine production in activated basophils and basophil-mediated skin and intestinal allergic inflammation in mice.

BACKGROUND: Propolis is a resinous mixture produced by honey bees that contains cinnamic acid derivatives and flavonoids. Although propolis has been reported to inhibit mast cell functions and mast cell-dependent allergic responses, the effect of propolis on basophil biology remains unknown. This study aimed to investigate the inhibitory effect of propolis on FcεRI-mediated basophil activation. METHODS: To determine the inhibitory effect of propolis on basophil activation in vitro, cytokine production and FcεRI signal transduction were analyzed by ELISA and western blotting, respectively. To investigate the inhibitory effect of propolis in vivo, IgE-CAI and a food allergy mouse model were employed. RESULTS: Propolis treatment resulted in the suppression of IgE/antigen-induced production of IL-4, IL-6 and IL-13 in basophils. Phosphorylation of FcεRI signaling molecules Lyn, Akt and ERK was inhibited in basophils treated with propolis. While propolis did not affect the basophil population in the treated mice, propolis did inhibit IgE-CAI. Finally, ovalbumin-induced intestinal anaphylaxis, which involves basophils and basophil-derived IL-4, was attenuated in mice prophylactically treated with propolis. CONCLUSIONS: Taken together, these results demonstrate the ability of propolis to suppress IgE-dependent basophil activation and basophil-dependent allergic inflammation. Therefore, prophylactic treatment with propolis may be useful for protection against food allergic reactions in sensitive individuals.

Effects of omalizumab on basophils: Potential biomarkers in asthma and chronic spontaneous urticaria.

Omalizumab is an anti-IgE humanized monoclonal antibody approved for the treatment of severe asthma and chronic spontaneous urticaria. Omalizumab binds free serum IgE and antagonizes its interaction with FcεRI, which is considered the main pharmacodynamic mechanism responsible for the clinical response to the treatment. The reduction of IgE serum concentration down-regulates the cellular expression of FcεRI on basophils. However, the biological events occurring on basophils during the therapy with omalizumab are multiple and complex. Here we review the current evidence regarding the specific biological effects of omalizumab on basophils in patients with asthma and chronic spontaneous urticaria. In addition to the modulation of IgE receptors, omalizumab may affect basophils homeostasis, intra-cellular signaling, cellular responsiveness/activation and cytokine release. These effects may be partially responsible for the clinical success of omalizumab and potentially provide useful biological markers for future assessment of the clinical response to the treatment. However, further investigation is required to better elucidate the role of basophils during the treatment with omalizumab.