Mast cell versus basophil activation test in allergy: Current status.

In the past two decades, we witnessed the evolution of the basophil activation test (BAT) from mainly research applications to a potential complementary diagnostic tool to document IgE-dependent allergies. However, BAT presents some technical weaknesses. Around 10%-15% of tested patients are non-responders, BAT can be negative immediately post-reaction and the use of fresh basophils, ideally analysed within 4 h of collection, restricts the number of tests that can be performed per sample. The need for fresh basophils is especially limiting when conducting batch analyses and interlaboratory comparisons to harmonize BAT methodology. These limitations significantly hinder the wider application of BAT and urge the development of alternative testing, such as the mast cell activation test (MAT). The essential difference between BAT and MAT is the heterogeneity of the starting material used to perform the assays. Mast cells are tissue-resident, so cannot be easily accessed. Current alternative sources for functional studies are generating primary human mast cells, differentiated from donor progenitor cells, or using immortalized mast cell lines. Hence, the methodological approaches for MAT are not only vastly different from BAT, but also different among MAT protocols. This review summarizes the advantages and disadvantages of BAT and MAT assays, dedicating special attention to elucidating the key differences between the cellular sources used and provides an overview of studies hitherto performed comparing BAT and MAT in the diagnosis of IgE-mediated food and drug allergies.

P2X7 Receptor-Induced Human Mast Cell Degranulation Is Enhanced by Interleukin 33.

MCs are tissue-resident immune cells that strategically reside in barrier organs and respond effectively to a wide range of stimuli, such as IL-33, a mediator released upon epithelial damage. Adenosine triphosphate (ATP) accumulates at sites of tissue injury and is known to modulate MC activities. This study investigated how an inflammatory tissue environment rich in IL-33 modulates the ATP-mediated activation of MCs. Human primary MCs primed with IL-33 displayed a strongly increased response to ATP but not ADP. This resulted in increased degranulation, IL-8 release, and pERK1/2 signalling. Such effects are unique to IL-33 stimulation and not shared by the epithelial alarmin, TSLP. MC exposure to IL-33 also increased membrane expression of purinergic and ATP-binding P2X receptors. The use of selective P2X receptor inhibitors identified P2X7 receptor as the key mediator of the enhanced ATP-induced ERK1/2 signalling and degranulation in IL-33-primed MCs. Whilst the inhibition of P2X1 and P2X4 receptors had no effect on MC degranulation, inhibiting these receptors together with P2X7 resulted in further decreased MC-mediated degranulation. These data therefore point toward the potential mechanisms by which IL-33 contributes to the modulation of ATP-mediated activation in human MCs.

Performance Characteristics of Basophil Activation Tests for Diagnosing Penicillin Allergy: A Meta-Analysis.

BACKGROUND: Approximately 10% of the global population identify themselves as penicillin allergic, yet 90% are not truly allergic and could safely tolerate penicillin. There is no simple way to identify these people. Current in vitro diagnostics include specific immunoglobulin E (with a sensitivity of 19% and specificity of 97%) and a basophil activation test (BAT) with undefined sensitivity and specificity. OBJECTIVE: To define the sensitivity and specificity of BAT in the diagnosis of penicillin allergy METHODS: We searched PubMed and EMBASE from inception to April 2, 2023, for original studies evaluating the performance characteristics of BAT for penicillin allergy in adults. Study selection, data extraction, risk of bias, assessment with QUADAS-2 tool, certainty assessment with Grading of Recommendations, Assessment, Development, and Evaluation (GRADE) methodology were performed independently, in duplicate. Meta-analysis was performed using Reitsma methodology. RESULTS: Twenty-two studies fulfilled the inclusion criteria. Twelve used the same positive threshold giving a summary point sensitivity 51% (95% confidence interval [95% CI]46%-56%) and specificity 89% (95% CI 85%-93%). Significant risk of bias was identified owing to patient selection. GRADE certainty of evidence rated sensitivity very low due to imprecision and specificity as low. There was great heterogeneity in methods used. Use of 1,000 basophils per test did not improve performance above 500 basophils. CONCLUSIONS: BAT sensitivity is highly variable across studies and remains too low to be considered as a routine element of clinical practice. BAT specificity is not as good as specific immunoglobulin E in penicillin allergy diagnosis. Significant further work is required in this field before clinical application of BAT in routine practice.

Human Mast Cells Upregulate Cathepsin B, a Novel Marker of Itch in Psoriasis.

Mast cells (MCs) contribute to skin inflammation. In psoriasis, the activation of cutaneous neuroimmune networks commonly leads to itch. To dissect the unique contribution of MCs to the cutaneous neuroinflammatory response in psoriasis, we examined their density, distribution, relation to nerve fibres and disease severity, and molecular signature by comparing RNA-seq analysis of MCs isolated from the skin of psoriasis patients and healthy volunteers. In involved psoriasis skin, MCs and Calcitonin Gene-Related Peptide (CGRP)-positive nerve fibres were spatially associated, and the increase of both MC and nerve fibre density correlated with disease severity. Gene set enrichment analysis of differentially expressed genes in involved psoriasis skin showed significant representation of neuron-related pathways (i.e., regulation of neuron projection along with dendrite and dendritic spine morphogenesis), indicating MC engagement in neuronal development and supporting the evidence of close MC-nerve fibre interaction. Furthermore, the analysis of 208 identified itch-associated genes revealed that CTSB, TLR4, and TACR1 were upregulated in MCs in involved skin. In both whole-skin published datasets and isolated MCs, CTSB was found to be a reliable indicator of the psoriasis condition. Furthermore, cathepsin B+ cells were increased in psoriasis skin and cathepsin B+ MC density correlated with disease severity. Therefore, our study provides evidence that cathepsin B could serve as a common indicator of the MC-dependent itch signature in psoriasis.

Bacillus Calmette-Guérin-Induced Human Mast Cell Activation Relies on IL-33 Priming.

Bacillus Calmette-Guérin (BCG) vaccine is an attenuated strain of Mycobacterium bovis that provides weak protection against tuberculosis (TB). Mast cells (MCs) are tissue-resident immune cells strategically that serve as the first line of defence against pathogenic threats. In this study, we investigated the response of human MCs (hMCs) to BCG. We found that naïve hMCs exposed to BCG did not secrete cytokines, degranulate, or support the uptake and intracellular growth of bacteria. Since we could show that in hMCs IL-33 promotes the transcription of host-pathogen interaction, cell adhesion and activation genes, we used IL-33 for cell priming. The treatment of hMCs with IL-33, but not IFN-γ, before BCG stimulation increased IL-8, MCP-1 and IL-13 secretion, and induced an enhanced expression of the mycobacteria-binding receptor CD48. These effects were comparable to those caused by the recombinant Mycobacterium tuberculosis (Mtb) 19-KDa lipoprotein. Finally, stimulation of hMCs with IL-33 incremented MC-BCG interactions. Thus, we propose that IL-33 may improve the immunogenicity of BCG vaccine by sensitising hMCs.

Novel Approaches in the Inhibition of IgE-Induced Mast Cell Reactivity in Food Allergy.

Allergy is an IgE-dependent type-I hypersensitivity reaction that can lead to life-threatening systemic symptoms such as anaphylaxis. In the pathogenesis of the allergic response, the common upstream event is the binding of allergens to specific IgE, inducing cross-linking of the high-affinity FcεRI on mast cells, triggering cellular degranulation and the release of histamine, proteases, lipids mediators, cytokines and chemokines with inflammatory activity. A number of novel therapeutic options to curb mast cell activation are in the pipeline for the treatment of severe allergies. In addition to anti-IgE therapy and allergen-specific immunotherapy, monoclonal antibodies targeted against several key Th2/alarmin cytokines (i.e. IL-4Rα, IL-33, TSLP), active modification of allergen-specific IgE (i.e. inhibitory compounds, monoclonal antibodies, de-sialylation), engagement of inhibitory receptors on mast cells and allergen-specific adjuvant vaccines, are new promising options to inhibit the uncontrolled release of mast cell mediators upon allergen exposure. In this review, we critically discuss the novel approaches targeting mast cells limiting allergic responses and the immunological mechanisms involved, with special interest on food allergy treatment.

Use of Toll-Like Receptor (TLR) Ligation to Characterize Human Regulatory B-Cells Subsets.

Toll-like receptors (TLRs) are pattern recognition receptors (PRRs), which constitute key components in the recognition of pathogens, thereby initiating innate immune responses and promoting adaptive immune responses. In B cells, TLR ligation is important for their activation and, together with CD40, for their differentiation. TLR ligands are also strong promoters of regulatory B (Breg)-cell development, by enhancing the production of IL-10 and their capacity to induce tolerance. In inflammatory diseases, such as autoimmunity or allergies, Breg-cell function is often impaired, while in chronic infections, such as with helminths, or cancer, Breg-cell function is boosted. Following pathogen exposure, B cells can respond directly by producing cytokines and/or IgM (innate response) and develop into various memory B (Bmem)-cell subsets with class-switched immunoglobulin receptors. Depending on the disease state or chronic infection conditions, various Breg subsets can be recognized as well. Currently, a large array of surface markers is known to distinguish between these large range of B-cell subsets. In recent years, the development of mass cytometers and spectral flow cytometry has allowed for high-dimensional detection of up to 48 markers, including both surface and intracellular/intranuclear markers. Therefore, this novel technology is highly suitable to provide a comprehensive overview of Bmem/Breg-cell subsets in different disease states and/or in clinical intervention trials. Here, we provide detailed instructions of the steps necessary to obtain high-quality data for high-dimensional analysis of multiple human Breg-cell subsets using various TLR ligands.

Idiopathic anaphylaxis.

Idiopathic anaphylaxis (IA) or spontaneous anaphylaxis is a diagnosis of exclusion when no cause can be identified. The exact incidence and prevalence of IA are not known. The clinical manifestations of IA are similar to other known causes of anaphylaxis. A typical attack is usually acute in onset and can worsen over minutes to a few hours. The pathophysiology of IA has not yet been fully elucidated, although an IgE-mediated pathway by hitherto unidentified trigger/s might be the main underlying mechanism. Elevated concentrations of urinary histamine and its metabolite, methylimidazole acetic acid, plasma histamine and serum tryptase have been reported, consistent with mast cell activation. There is some evidence that corticosteroids reduce the frequency and severity of episodes of IA, consistent with a steroid-responsive condition. Important differential diagnoses of IA include galactose alpha-1,3 galactose (a carbohydrate contained in red meat) allergy, pigeon tick bite (Argax reflexus), wheat-dependent exercise-induced anaphylaxis, Anisakis simplex allergy and mast cell disorders. Other differential diagnoses include "allergy-mimics" such as asthma masquerading as anaphylaxis, undifferentiated somatoform disorder, panic attacks, globus hystericus, vocal cord dysfunction, scombroid poisoning, vasoactive amine intolerance, carcinoid syndrome and phaeochromocytoma. Acute treatment of IA is the same as for other forms of anaphylaxis. Long-term management is individualized and dictated by frequency and severity of symptoms and involves treatment with H1 and H2 receptor blockers, leukotriene receptor antagonist and consideration for prolonged reducing courses of oral corticosteroids. Patients should possess an epinephrine autoinjector with an anaphylaxis self-management plan. There are anecdotal reports regarding the use of omalizumab. For reasons that remain unclear, the prognosis of IA is generally favourable with appropriate treatment and patient education. If remission cannot be achieved, the diagnosis should be reconsidered.

Day-On, Day-Off emtricitabine, tenofovir disoproxil fumarate and efavirenz single tablet regimen (DODO) as maintenance therapy in HIV-infected patients.

Reduced dose schedules may be feasible options to simplify antiretroviral therapy (ART) in selected HIV-1-infected individuals. Efficacy and safety of a Day-on, Day-off (DODO) schedule of tenofovir disoproxil fumarate, emtricitabine and efavirenz (FTC/TDF/EFV) single tablet regimen (STR) was assessed. Twenty-seven patients were prescribed the DODO schedule and were monitored for 48 weeks. Switching criteria were: no previous ART failure, no AIDS-defining illnesses, T CD4 cell nadir >200/mmc, and HIV-RNA below detection limit (40 copies/mL) for at least six months. Clinical and laboratory data, including plasma HIV-RNA levels, T CD4 and CD8 counts, liver and kidney function, lipid levels and ultrasensitive C-reactive protein (us-CRP) were assessed at baseline, week 4, 12, 24, and 48. Statistical analysis was performed by paired Student's T-test for comparison between baseline and each time point and Chi square test for CD4/CD8 ratio comparison. In all, 26 out of 27 patients maintained plasma HIV-RNA levels below the detection limit through the entire follow-up. One patient experienced low level plasma HIV-RNA rebound at week 36 (47 copies/ml) and immediately reverted to the conventional dose schedule of FTC/TDF/EFV; plasma HIV-RNA was undetectable after four weeks. No major changes on liver and kidney function tests, lipid levels and us-CRP were observed. Although no profound modifications of T CD4 count were observed during follow-up, the CD4/CD8 ratio increased significantly at week 48 compared to the baseline (p<0.05). In conclusion, 48-week DODO administration of the fixed dose FTC/TDF/EFV STR combination was safe and effective in maintaining HIV viral replication below the detection limit in a selected group of HIV-1-infected individuals.

Validation of a self-reported HIV symptoms list: the ISS-HIV symptoms scale.

BACKGROUND: To describe the development and the psychometric properties of the Istituto Superiore di Sanità-HIV symptoms scale (lSS-HIV symptoms scale). METHODS: The ISS-HIV symptom scale was developed by an Italian working team including researchers, physicians and people living with HIV. The development process went through the following steps: (1) review of HIV/AIDS literature; (2) focus group; (3) pre-test analysis; (4) scale validation. RESULTS: The 22 symptoms of HIV-ISS symptoms scale were clustered in five factors: pain/general discomfort (7 items); depression/anxiety (4 items); emotional reaction/psychological distress (5 items); gastrointestinal discomfort (4 items); sexual discomfort (2 items). The internal consistence reliability was for all factors within the minimum accepted standard of 0.70. CONCLUSIONS: The results of this study provide a preliminary evidence of the reliability and validity of the ISS-HIV symptoms scale. In the new era where HIV infection has been transformed into a chronic diseases and patients are experiencing a complex range of symptoms, the ISS-HIV symptoms scale may represent an useful tool for a comprehensive symptom assessment with the advantage of being easy to fill out by patients and potentially attractive to physicians mainly because it is easy to understand and requires short time to interpret the results.