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.

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.

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.

Sequential engagement of adhesion molecules and cytokine receptors impacts both piecemeal and anaphylactic degranulation of human basophils.

Basophils are rare granulocytes in circulation which home to tissues in a process depending on rolling, adhesion and cytokine exposure. However, it is still unclear how these steps affect basophil degranulation. Our aim was to imitate these processes associated with homing by sequential crosslinking of adhesion molecules and cytokine exposure and evaluate the effect on basophil piecemeal (PMD) and anaphylactic degranulation (AND). Blood donors with or without allergic asthma were recruited from an ongoing cohort study. Basophils were subjected to CD62L-, CD49d- or CD11b crosslinking and IL-3 or IL-33 stimulation in different orders followed by anti-IgE and fMLP stimulation. Basophil CD203c and CD63 expression were analysed by flow cytometry to determine PMD and AND, respectively. IL-3 induced PMD in basophils and combined with CD62L- or CD11b crosslinking, IL-3 potentiated the degranulation regardless of sequential order. IL-3 priming followed by adhesion molecule crosslinking induced AND and potentiated the effect of anti-IgE. CD62L- and CD11b crosslinking did not further potentiate this effect. CD49d crosslinking followed by IL-3 increased CD63 expression following anti-IgE. IL-3 potentiated the effect of fMLP on AND while adhesion molecule crosslinking did not. IL-33 had impact on PMD only when followed by adhesion molecule crosslinking but did not potentiate neither IgE-dependent nor IgE-independent degranulation. Our data indicate that sequential interactions between basophils, cytokines and adhesion molecule ligands have a decisive effect on basophil degranulation and that these interactions are operational for fine-tuning the activity of tissue dwelling basophils. These data should be considered when the effect of different pharmaceutical on basophil function is studied.

Circulating Basophils as a Prognostic Marker for Response to Bacillus Calmette-Guérin.

PURPOSE: To predict recurrence and progression in non-muscle-invasive bladder cancer (NMIBC) patients receiving bacillus Calmette-Guérin (BCG), we evaluated circulating basophils as a biomarker that could be detected from the complete blood count. PATIENTS AND METHODS: We use a pooled cohort of patients from the Centre Hospitalier Universitaire de Québec-Université Laval (2016-2020) and the Vancouver General Hospital (2010-2018) where a complete blood count was available before transurethral resection of bladder tumor (TURBT) of a high-grade NMIBC and subsequent BCG. Descriptive statistics described the cohort based on the dichotomous presence or absence of basophils on the complete blood count. Kaplan-Meier estimates and a log-rank test compared recurrence-free survival (RFS) and progression-free survival (PFS), with multivariable cox regression analysis used to estimate proportional hazard ratios. RESULTS: The study cohort included 261 patients, with a median follow-up of 31.5 months (interquartile range 18.1-45.0 months). The median age was 74.0 years and 16.8% were female. Circulating basophils were detectable in 49 (18.9%) patients. Both RFS and PFS were significantly lower in patients with detectable basophils. Multivariable analysis demonstrated detectable basophils were an independent predictor of both recurrence (HR = 1.85; 95% confidence interval [CI] 1.20-2.85; P = .01) and progression (HR = 2.29; 95% CI 1.14-4.60; P = .02). CONCLUSION: Our results confirm that baseline levels of circulating basophils are an immunological biomarker to predict recurrence and progression of NMIBC.

[Recent advances in understanding of basophil function and differentiation].

Basophils are the rarest granulocytes representing less than 1% of peripheral blood leukocytes. Even though basophils have been discovered more than 140 years ago, their roles in immune reactions had long been an enigma, partly because of their rarity and the similarity to tissue-resident mast cells. However, recent development of the analytical tools for basophil research, such as basophil-depletion antibody and basophil-related engineered mice, has uncovered the unique roles of basophils in various immune reactions. Basophils are now appreciated as a critical immune cell in various type 2-immune responses including the induction of chronic allergic inflammation and protective immunity against parasites. In this review, we summarize the recent understandings in the roles of basophils in allergic inflammation with especial focus on skin inflammation. We then focus on our recent findings in the differentiation and maturation pathways of basophils.

[New developments in mast cell/basophil research].

Mast cells and basophils share many characteristics, such as surface expression of the high affinity receptor of IgE, FcεRI, granule storage of histamine, which is released during their activation, and potentials to produce pro-/anti-inflammatory cytokines. These similar leukocytes, however, were found to have their own process of differentiation. Indeed, accumulating evidence suggests that these cells should play critical roles in type I allergy including anaphylaxis and in urticaria. Various inflammatory mediators derived from mast cells/basophils, such as histamine, platelet-activating factor, prostanoids, and leukotrienes, have been paid a particular attention to as the therapeutic targets for type I allergy and inflammatory diseases. Recent progress in the field of mast cell/basophil research has shed light on their physiological roles in bacterial infection, energy metabolism, and cutaneous/intestinal inflammation. This review makes a brief introduction of these recent studies, which are expected to provide novel therapeutic approaches for infectious and chronic inflammatory diseases.

Coronavirus Receptor Expression Profiles in Human Mast Cells, Basophils, and Eosinophils.

A major problem in SARS-CoV-2-infected patients is the massive tissue inflammation in certain target organs, including the lungs. Mast cells (MC), basophils (BA), and eosinophils (EO) are key effector cells in inflammatory processes. These cells have recently been implicated in the pathogenesis of SARS-CoV-2 infections. We explored coronavirus receptor (CoV-R) expression profiles in primary human MC, BA, and EO, and in related cell lines (HMC-1, ROSA, MCPV-1, KU812, and EOL-1). As determined using flow cytometry, primary MC, BA, and EO, and their corresponding cell lines, displayed the CoV-R CD13 and CD147. Primary skin MC and BA, as well as EOL-1 cells, also displayed CD26, whereas primary EO and the MC and BA cell lines failed to express CD26. As assessed using qPCR, most cell lines expressed transcripts for CD13, CD147, and ABL2, whereas ACE2 mRNA was not detectable, and CD26 mRNA was only identified in EOL-1 cells. We also screened for drug effects on CoV-R expression. However, dexamethasone, vitamin D, and hydroxychloroquine did not exert substantial effects on the expression of CD13, CD26, or CD147 in the cells. Together, MC, BA, and EO express distinct CoV-R profiles. Whether these receptors mediate virus-cell interactions and thereby virus-induced inflammation remains unknown at present.

Comparison of the passive mast cell activation test with the basophil activation test for diagnosis of perioperative rocuronium hypersensitivity.

BACKGROUND: Rocuronium is a major cause of perioperative hypersensitivity (POH). Skin tests (STs) and quantification of specific immunoglobulin E antibodies (sIgEs) can yield incongruent results. In such difficult cases, the basophil activation test (BAT) can be helpful. Here, we evaluated the passive mast cell activation test (pMAT) as a substitute of BAT as part of the diagnostic tests for rocuronium allergy. METHODS: Sera from patients with a suspected POH reaction potentially related to rocuronium were included. All patients had a complete diagnostic investigation, including STs, quantification of sIgEs to morphine and rocuronium, and BAT. For execution of pMAT, human mast cells were generated from healthy donor peripheral blood CD34+ progenitor cells and sensitised overnight with patient sera. RESULTS: In total, 90 sera were studied: 41 from ST+sIgE+ patients, 13 from ST-sIgE- patients, 20 from ST+sIgE- patients, and 16 from ST-sIgE+ patients. According to BAT results, patients were further allocated into subgroups. Of the 38 BAT+ patients, 25 (66%) showed a positive pMAT as well. Of the 44 BAT- patients, 43 (98%) also showed a negative pMAT. Mast cells that were not passively sensitised did not respond to rocuronium. CONCLUSIONS: We show that the pMAT, in many cases, can substitute for BAT in the diagnosis of rocuronium hypersensitivity and advance diagnosis in difficult cases with uncertain ST or sIgE results when BAT is not locally available.

Decreased Basophil Activation against House Dust Mite after Japanese Cedar Pollen Subcutaneous Immunotherapy: A Retrospective Study.

BACKGROUND: Allergen-specific immunotherapy (AIT), an established treatment for allergic diseases, prevents the development of other allergic manifestations. Although the mechanisms remain unclear, AIT has been shown to reduce basophil activation (BA) against nontarget allergens. OBJECTIVES: The aim of this study was to assess immunological changes in Dermatophagoides farinae (Der f) after Japanese cedar pollen (JCP)-based subcutaneous immunotherapy (SCIT) monotherapy. METHOD: The data of 16 patients (age: 6-37 years) with JCP-induced allergic rhinitis who were sensitive to Der f (serum Der f-specific immunoglobulin E [IgE] level >0.34 kUA/L) and received JCP-based SCIT for 5 years were reviewed retrospectively. BA by Der f and JCP extracts and serum-specific IgE and immunoglobulin G4 (IgG4) levels against these allergens were evaluated before and after completing 5 years of JCP-based SCIT monotherapy. RESULTS: The areas under the dose-response curves of BA by Der f and JCP extracts were significantly reduced (p = 0.02 and p = 0.002, respectively). JCP-specific IgE levels decreased and JCP-specific IgG4 levels increased significantly (p < 0.001 for both), whereas Der f-specific IgE and IgG4 levels did not change significantly. CONCLUSIONS: JCP-based SCIT monotherapy reduced Der f-specific BA. These findings suggest that JCP-based SCIT has the potential to modulate immune response toward nontarget allergens.

Combining avidin with CD63 improves basophil activation test accuracy in classifying peanut allergy.

BACKGROUND: Conventional basophil activation tests (BATs) measure basophil activation by the increased expression of CD63. Previously, fluorophore-labeled avidin, a positively-charged molecule, was found to bind to activated basophils, which tend to expose negatively charged granule constituents during degranulation. This study further compares avidin versus CD63 as basophil activation biomarkers in classifying peanut allergy. METHODS: Seventy subjects with either a peanut allergy (N = 47), a food allergy other than peanut (N = 6), or no food allergy (N = 17) were evaluated. We conducted BATs in response to seven peanut extract (PE) concentrations (0.01-10,000 ng/mL) and four control conditions (no stimulant, anti-IgE, fMLP (N-formylmethionine-leucyl-phenylalanine), and anti-FcεRI). We measured avidin binding and CD63 expression on basophils with flow cytometry. We evaluated logistic regression and XGBoost models for peanut allergy classification and feature identification. RESULTS: Avidin binding was correlated with CD63 expression. Both markers discriminated between subjects with and without a peanut allergy. Although small by percentage, an avidin+ /CD63- cell subset was found in all allergic subjects tested, indicating that the combination of avidin and CD63 could allow a more comprehensive identification of activated basophils. Indeed, we obtained the best classification accuracy (97.8% sensitivity, 96.7% specificity) by combining avidin and CD63 across seven PE doses. Similar accuracy was obtained by combining PE dose of 10,000 ng/mL for avidin and PE doses of 10 and 100 ng/mL for CD63. CONCLUSIONS: Avidin and CD63 are reliable BAT activation markers associated with degranulation. Their combination enhances the identification of activated basophils and improves the classification accuracy of peanut allergy.