Basophil allergen threshold sensitivity to casein (casein-specific CD-sens) predicts allergic reactions at a milk challenge in most but not all patients.

BACKGROUND: The basophil activation test is an emerging clinical tool in the diagnosis of cow's milk allergy (CMA). The aim was to assess the association between the basophil allergen threshold sensitivity to the major milk protein casein (casein-specific CD-sens), the levels of milk- and casein-specific Immunoglobulin E antibodies (IgE-ab), and the severity of allergic reactions at milk challenges. METHODS: We enrolled 34 patients aged 5-15 (median 9) years who underwent a double-blind placebo-controlled milk-challenge (DBPCMC) as screening before inclusion in an oral immunotherapy study for CMA. The severity of the allergic reaction at the DBPCMC was graded using Sampson's severity score. Venous blood was drawn before the DBPCMC. Milk- and casein-specific IgE-ab were analyzed. Following in vitro stimulation of basophils with casein, casein-specific CD-sens, was determined. RESULTS: Thirty-three patients completed the DBPCMC. There were strong correlations between casein-specific CD-sens and IgE-ab to milk (rs = 0.682, p < .001), and between casein-specific CD-sens and IgE-ab to casein (rs = 0.823, p < .001). There was a correlation between the severity of the allergic reaction and casein-specific CD-sens level (rs = 0.395, p = .041) and an inverse correlation between casein-specific CD-sens level and the cumulative dose of milk protein to which the patient reacted at the DBPCMC (rs = -0.418, p = .027). Among the 30 patients with an allergic reaction at the DBPCMC, 67% had positive casein-specific CD-sens, 23% had negative casein-specific CD-sens, and 10% were declared non-responders. CONCLUSION: Two thirds of those reacting at the DBPMC had positive casein-specific CD-sens, but reactions also occurred despite negative casein-specific CD-sens. The association between casein-specific CD-sens and the severity of the allergic reaction and cumulative dose of milk protein, respectively, was moderate.

Light microscopic morphology of blood cells of non-descript indigenous Zoar chicken of Mizoram, India.

Identifying and analysing distinct blood cells is crucial for the diagnosis and treatment of diseases in the field of biomedicine. The present study was undertaken to study the cytomorphological and cytochemical characteristics of the blood cells of Zoar, a non-descript indigenous breed of chicken extensively reared under backyard poultry farming in Mizoram, India. For this study, 2 mL of blood samples were aseptically collected from the wings veins of 12 chickens and were processed for light microscopic study under standard protocols. The matured erythrocytes were elliptical, while the immature erythrocytes appeared oval. The heterophils were positive for SBB (SBB), Periodic Acid Schiff (PAS), acid phosphatase, alkaline phosphatase and Arylsulphatase while the eosinophils were positive for SBB, PAS, alkaline phosphatase, cytochrome oxidase and peroxidase. The basophils of were positive for toluidine blue while the thrombocytes were positive for PAS. These cytochemical and cytoenzymatic staining properties plays a very important role in diagnosis, differentiation, and classification of leukaemias.

PD-L1- and IL-4-expressing basophils promote pathogenic accumulation of T follicular helper cells in lupus.

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by anti-nuclear autoantibodies whose production is promoted by autoreactive T follicular helper (TFH) cells. During SLE pathogenesis, basophils accumulate in secondary lymphoid organs (SLO), amplify autoantibody production and disease progression through mechanisms that remain to be defined. Here, we provide evidence for a direct functional relationship between TFH cells and basophils during lupus pathogenesis, both in humans and mice. PD-L1 upregulation on basophils and IL-4 production are associated with TFH and TFH2 cell expansions and with disease activity. Pathogenic TFH cell accumulation, maintenance, and function in SLO were dependent on PD-L1 and IL-4 in basophils, which induced a transcriptional program allowing TFH2 cell differentiation and function. Our study establishes a direct mechanistic link between basophils and TFH cells in SLE that promotes autoantibody production and lupus nephritis.

The association between expression of CD200 on B lymphocytes and the count of eosinophils and basophils in atopic dermatitis patients with and without dupilumab therapy - Pilot study.

BACKGROUND: Eosinophils and basophils are implicated in allergic reactions, and the molecule CD200 on B cells may have regulatory functions. Assessing the associations between the expression of CD200 on B lymphocytes and eosinophils and basophils helps unravel the complex immune interactions in atopic dermatitis, aiding in targeted therapeutic approaches. OBJECTIVE: The aim of our study is to evaluate the association between the count of eosinophils, basophils, CD16+ eosinophils, CD203+ basophils, the expression of activation marker CD200 on B cells and on their subsets in patients suffering from atopic dermatitis with and without dupilumab and in control group. MATERIALS AND METHODS: Altogether we examined 75 subjects: 45 patients suffering from atopic dermatitis -32 patients without dupilumab treatment, 13 patients with dupilumab treatment and 30 subjects as a control group. Immunophenotype was examined by flow cytometry in which monoclonal antibodies with fluorescent molecules were used. For statistical analysis we used non-parametric Kruskal-Wallis one-factor analysis of variance with post-hoc by Dunn's test with Bonferroni modification and the Spearman's rank correlation coefficient with calculation of R2 (%, percent of Variation Explained). RESULTS: In patients with dupilumab therapy we confirmed the association between absolute eosinophils and expression of molecule CD200 on total B lymphocytes (in 23.9 %), non-switched (in 27.2 %), naive (in 25 %) and memory (in 20.3 %) B lymphocytes and between relative eosinophils and expression of CD200 on total B lymphocytes (in 22.8 % %), non-switched (in 29 %), naive (in 21.3 %) and memory (in 22.3 %) B lymphocytes. This association is low in AD patients without dupilumab and even non linear in control healthy subjects. CONCLUSION: The higher association between eosinophils and expression of CD200 molecule on memory, naive and non switched B lymphocytes in AD patients under dupilumab therapy suggests that activation of B lymphocytes is caused by IL-4, whose production involves eosinophils and the CD200 molecule on B lymphocytes.

Basophil activation in insect venom allergy: comparison of an established test using liquid reagents with a test using 5-color tubes with dried antibody reagents.

BACKGROUND: Flow cytometry-based basophil activation tests (BAT) have been performed with various modifications, differing in the use of distinct identification and activation markers. Established tests use liquid reagents while a new development involves the use of tubes with dried antibody reagents. The aim of this pilot study was to compare these two techniques in patients with insect venom allergy. METHODS: Seventeen patients with an insect venom allergy were included in the study. The established "BAT 1" utilizes conventional antibody solutions of anti-CCR3 for basophil identification and anti-CD63 to assess basophil activation, whereas "BAT 2" uses dried anti-CD45, anti-CD3, anti-CRTH2, anti-203c and anti-CD63 for identification and activation measurement of basophils. Negative and positive controls as well as incubations with honey bee venom and yellow jacket venom at three concentrations were performed. RESULTS: Seven patients had to be excluded due to low basophil counts, high values in negative controls or negative positive controls. For the remaining 10 patients the overall mean (± SD) difference in activated basophils between the two tests was 0.2 (± 12.2) %P. In a Bland-Altman plot, the limit of agreement (LoA) ranged from 24.0 to -23.7. In the qualitative evaluation (value below/above cut-off) Cohen's kappa was 0.77 indicating substantial agreement. BAT 2 took longer to perform than BAT 1 and was more expensive. CONCLUSION: The BAT 2 technique represents an interesting innovation, however, it was found to be less suitable compared to an established BAT for the routine diagnosis of insect venom allergies.

Asparaginase-specific basophil recognition and activation predict Asparaginase hypersensitivity in mice.

Background: Asparaginase (ASNase) is a crucial part of acute leukemia treatment, but immune responses to the agent can reduce its effectiveness and increase the risk of relapse. Currently, no reliable and validated biomarker predicts ASNase-induced hypersensitivity reactions during therapy. We aimed to identify predictive biomarkers and determine immune cells responsible for anaphylaxis using a murine model of ASNase hypersensitivity. Methods: Our preclinical study uses a murine model to investigate predictive biomarkers of ASNase anaphylaxis, including anti-ASNase antibody responses, immune complex (IC) levels, ASNase-specific binding to leukocytes or basophils, and basophil activation. Results: Our results indicate that mice immunized to ASNase exhibited dynamic IgM, IgG, and IgE antibody responses. The severity of ASNase-induced anaphylaxis was found to be correlated with levels of IgG and IgE, but not IgM. Basophils from immunized mice were able to recognize and activate in response to ASNase ex vivo, and the extent of recognition and activation also correlated with the severity of anaphylaxis observed. Using a multivariable model that included all biomarkers significantly associated with anaphylaxis, independent predictors of ASNase-induced hypersensitivity reactions were found to be ASNase IC levels and ASNase-specific binding to leukocytes or basophils. Consistent with our multivariable analysis, we found that basophil depletion significantly protected mice from ASNase-induced hypersensitivity reactions, supporting that basophils are essential and can be used as a predictive marker of ASNase-induced anaphylaxis. Conclusions: Our study demonstrates the need for using tools that can detect both IC- and IgE-mediated hypersensitivity reactions to mitigate the risk of ASNase-induced hypersensitivity reactions during treatment.

White blood cells and type 2 diabetes: A Mendelian randomization study.

BACKGROUND: Observational studies have demonstrated an association between white blood cells (WBC) subtypes and type 2 diabetes (T2D) risk. However, it is unknown whether this relationship is causal. We used Mendelian randomization (MR) to investigate the causal effect of WBC subtypes on T2D and glycemic traits. METHODS: The summary data for neutrophil, lymphocyte, monocyte, eosinophil, and basophil counts were extracted from a recent genome-wide association study (n = 173,480). The DIAGRAM and MAGIC consortia offered summary data pertaining to T2D and glycemic characteristics, including fasting glucose (FG) (n = 133,010), glycosylated hemoglobin (HbA1c) (n = 46,368), and homeostatic model assessment-estimated insulin resistance (HOMA-IR) (n = 37,037). A series of MR analyses (univariable MR, multivariable MR, and reverse MR) were used to investigate the causal association of different WBC subtypes with T2D and glycemic traits. RESULTS: Using the inverse-variance weighted method, we found one standard deviation increases in genetically determined neutrophil [odd ratio (OR): 1.086, 95% confidence interval (CI): 0.877-1.345], lymphocyte [0.878 (0.766-1.006)], monocyte [1.010 (0.906-1.127)], eosinophil [0.995 (0.867-1.142)], and basophil [0.960 (0.763-1.207)] were not causally associated with T2D risk. These findings were consistent with the results of three pleiotropy robust methods (MR-Egger, weighted median, and mode-based estimator) and multivariable MR analyses. Reverse MR analysis provided no evidence for the reverse causation of T2D on WBC subtypes. The null causal effects of WBC subtypes on FG, HbA1c, and HOMA-IR were also identified. CONCLUSIONS: WBCs play no causal role in the development of insulin resistance and T2D. The observed association between these factors may be explained by residual confounding.

Basophils are important for development of allergic skin inflammation.

BACKGROUND: Atopic dermatitis skin lesions exhibit increased infiltration by basophils. Basophils produce IL-4, which plays an important role in the pathogenesis of atopic dermatitis. OBJECTIVE: We sought to determine the role of basophils in a mouse model of antigen-driven allergic skin inflammation. METHODS: Wild-type mice, mice with selective and inducible depletion of basophils, and mice expressing Il4-driven enhanced green fluorescent protein were subjected to epicutaneous sensitization with ovalbumin or saline. Sensitized skin was examined by histology for epidermal thickening. Cells were analyzed for surface markers and intracellular expression of enhanced green fluorescent protein by flow cytometry. Gene expression was evaluated by real-time reverse transcription-quantitative PCR. RESULTS: Basophils were important for epidermal hyperplasia, dermal infiltration by CD4+ T cells, mast cells, and eosinophils in ovalbumin-sensitized mouse skin and for the local and systemic TH2 response to epicutaneous sensitization. Moreover, basophils were the major source of IL-4 in epicutaneous-sensitized mouse skin and promote the ability of dendritic cells to drive TH2 polarization of naive T cells. CONCLUSION: Basophils play an important role in the development of allergic skin inflammation induced by cutaneous exposure to antigen in mice.

Allergy to PEG (polyethylene glycol) - sensitivity of basophil activation test with COVID-19 mRNA-vaccine BNT162B2.

Basophil activation test (BAT) with COVID-19 mRNA vaccine seems particularly suitable for detecting sensitization to polyethylene glycol (PEG) in patients with PEG allergy. It was the aim of this study to determine the cutoffs for BAT using BNT162B2 (Comirnaty®) in a larger group of PEG allergic patients and controls. 10 PEG allergic patients and 10 controls were studied. BAT was performed using anti-CCR3 for basophil identification and anti-CD63 to assess basophil activation. Incubations with BNT162B2 at four different concentrations were performed. Basophil activation was significantly higher in PEG allergic patients compared to controls at the higher concentrations used. ROC curves showed best results with a sensitivity of 60% and specificity of 100% with a cutoff of 5% CD63+ basophils at a concentration of 4.5 µg/ml. Controls showed no positive results. In our group of PEG allergic patients, a concentration of 4.5 µg/ml BNT162B2 with a cutoff of 5% CD63+ basophils was the most suitable condition for identifying patients with a sensitization to PEG. Allergological work-up of PEG allergic patients including BAT with PEGylated lipid nanoparticles might play a role in the future when these substances will be used for other vaccines and cancer immunotherapies.

Accurate determination of house dust mite sensitization in asthma and allergic rhinitis through cytometric detection of Der p 1 and Der p 2 binding on basophils (CytoBas).

BACKGROUND: House dust mite (HDM) is the most common allergen trigger globally for allergic rhinitis and atopic asthma. OBJECTIVES: To expedite accurate confirmation of allergen sensitization, we designed fluorescent allergen tetramers to directly stain specific IgE on basophils to detect specific allergen sensitization using the flow cytometric CytoBas assay. METHODS: Recombinant proteins of major HDM allergens (component), Der f 1, Der p 1, and Der p 2 were biotinylated and conjugated with fluorochrome streptavidins as tetramers. Blood samples from 64 patients who are HDM-allergic and 26 controls that are non-HDM-sensitized were incubated with allergen tetramers for evaluation of basophil binding (CytoBas) and activation (BAT) with flow cytometry. RESULTS: The tetramers effectively bound and activated basophils from patients who are allergic but not from controls who are nonsensitized. CytoBas with Der p 1 as a single allergen had comparable sensitivity and specificity (92% and 100%) to BAT (91% and 100%) in detecting allergen sensitization, as did CytoBas with Der p 2 (95% and 96%) to BAT (95% and 87%). A positive staining for Der p 1 and/or Der p 2 in CytoBas was 100% sensitive and 96% specific for HDM allergy. CONCLUSIONS: CytoBas has diagnostic accuracy for group 1 and group 2 HDM allergens that is comparable to BAT, but with additional advantages of multiple allergen components in a single tube and no requirement for in vitro basophil activation. These findings endorse a single, multiplex CytoBas assay for accurate and component-resolved diagnosis of aeroallergen sensitization in patients with allergic asthma and/or rhinitis.

Non-IgE-Mediated Immediate Drug-Induced Hypersensitivity Reactions.

Immediate drug-induced hypersensitivity reactions (IDHSRs) have conventionally been attributed to an immunoglobulin E (IgE)-mediated mechanism. Nevertheless, it has now been acknowledged that IDHSRs can also occur independently of IgE involvement. Non-IgE-mediated IDHSRs encompass the activation of effector cells, both mast cell-dependent and -independent and the initiation of inflammatory pathways through immunogenic and nonimmunogenic mechanisms. The IDHSRs involve inflammatory mediators beyond histamine, including the platelet-activating factor, which activates multiple cell types, including smooth muscle, endothelium, and MC, and evidence supports its importance in IgE-mediated reactions in humans. Clinically, distinguishing IgE from non-IgE mechanisms is crucial for future treatment strategies, including drug(s) restriction, readministration approaches, and pretreatment considerations. However, this presents significant challenges because certain drugs can trigger both mechanisms, and their presentations can appear similarly, ranging from mild to life-threatening symptoms. Thus, history alone is often inadequate for differentiation, and skin tests lack a standardized approach. Moreover, drug-specific IgE immunoassays have favorable specificity but low sensitivity, and the usefulness of the basophil activation test remains debatable. Lastly, no biomarker reliably differentiates between both mechanisms. Whereas non-IgE-mediated mechanisms likely predominate in IDHSRs, reclassifying most drug-related IDHSRs as non-IgE-mediated, with suggested prevention through dose administration adjustments, is premature and risky. Therefore, continued research and validated diagnostic tests are crucial to improving our capacity to distinguish between these mechanisms, ultimately enhancing patient care.

Density Gradient Centrifugation-Independent Purification of Human Basophils.

Basophils represent the rarest type of granulocyte in human peripheral blood. Thus, researching basophils has historically been challenging and has often been reliant on enrichment protocols using density gradient centrifugation. This article describes a novel, fast, and cost-effective method to purify highly viable human basophils from peripheral blood through negative immunomagnetic selection, foregoing the density centrifugation step in the Basic Protocol. The technique is easy to use and consistently produces purities >96%. Furthermore, the Support Protocols describe procedures to determine basophil yield, purity, and viability, and how to investigate functional activity of the purified basophils through flow cytometry and visualize the basophils through microscopy. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Gradient centrifugation-independent basophil isolation Support Protocol 1: Flow cytometry staining to assess basophil yield, purity, and viability Support Protocol 2: Giemsa staining Support Protocol 3: Calcium flux analysis Support Protocol 4: Basophil activation test.