Oral food challenge (OFC) is the criterion standard to assess peanut allergy (PA), but it involves a risk of allergic reactions of unpredictable severity.
Our aim was to identify biomarkers for risk ...of severe reactions or low dose threshold during OFC to peanut.
We assessed Learning Early about Peanut Allergy study, Persistance of Oral Tolerance to Peanut study, and Peanut Allergy Sensitization study participants by administering the basophil activation test (BAT) and the skin prick test (SPT) and measuring the levels of peanut-specific IgE, Arachis hypogaea 2–specific IgE, and peanut-specific IgG4, and we analyzed the utility of the different biomarkers in relation to PA status, severity, and threshold dose of allergic reactions to peanut during OFC.
When a previously defined optimal cutoff was used, the BAT diagnosed PA with 98% specificity and 75% sensitivity. The BAT identified severe reactions with 97% specificity and 100% sensitivity. The SPT, level of Arachis hypogaea 2–specific IgE, level of peanut-specific IgE, and IgG4/IgE ratio also had 100% sensitivity but slightly lower specificity (92%, 93%, 90%, and 88%, respectively) to predict severity. Participants with lower thresholds of reactivity had higher basophil activation to peanut in vitro. The SPT and the BAT were the best individual predictors of threshold. Multivariate models were superior to individual biomarkers and were used to generate nomograms to calculate the probability of serious adverse events during OFC for individual patients.
The BAT diagnosed PA with high specificity and identified severe reactors and low threshold with high specificity and high sensitivity. The BAT was the best biomarker for severity, surpassed only by the SPT in predicting threshold. Nomograms can help estimate the likelihood of severe reactions and reactions to a low dose of allergen in individual patients with PA.
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The monoclonal anti‐immunoglobulin E (IgE) antibody, omalizumab, was the first drug approved for use in patients with chronic idiopathic/spontaneous urticaria (CIU/CSU) who remain symptomatic despite ...H1‐antihistamine treatment. Omalizumab binds to free IgE, which lowers free IgE levels and causes FcεRI receptors on basophils and mast cells to be downregulated. It has been shown to improve symptoms of CIU/CSU, but its mechanism of action is not currently understood. Potential mechanisms in CIU/CSU include reducing mast cell releasability, reversing basopenia and improving basophil IgE receptor function, reducing activity of IgG autoantibodies against FcεRI and IgE, reducing activity of IgE autoantibodies against an antigen or autoantigen that has yet to be definitively identified, reducing the activity of intrinsically ‘abnormal’ IgE, and decreasing in vitro coagulation abnormalities associated with disease activity. However, none of these theories alone or in combination fully account for the pattern of symptom improvement seen with omalizumab therapy, and therefore, no one mechanism is likely to be the definitive mechanism of action. Additional research is needed to further clarify the involvement of omalizumab in relieving symptoms associated with the complex, multifactorial pathogenesis of CIU/CSU.
Basophils are primarily associated with immunomodulatory functions in allergic diseases and parasitic infections. Recently, it has been demonstrated that both activated human and mouse basophils can ...form extracellular DNA traps (BETs) containing mitochondrial DNA and granule proteins. In this report, we provide evidence that, in spite of an apparent lack of phagocytic activity, basophils can kill bacteria through BET formation.
Summary
Background
Basophils are important effector cells involved in the pathogenesis of inflammatory skin diseases including chronic urticaria which is associated by increased IL‐31 serum levels. ...So far the effects of IL‐31 on human basophils are unknown.
Objective
To analyse the functional role of IL‐31 in basophil biology.
Methods
IL‐31 expression was evaluated in skin samples derived from chronic spontaneous urticaria patients. Oncostatin M receptor (OSMR), IL‐31 receptor A (RA) and IL‐31 protein expressions were analysed on human basophils from healthy donors. Basophil responses to IL‐31 were assessed for chemotaxis, externalization of CD63 and CD203c as well as the release of histamine, IL‐4 and IL‐13.
Results
IL‐31RA and OSMR were expressed on human basophils. IL‐31 was strongly expressed in the skin of patients with chronic spontaneous urticaria and was released from isolated basophils following either anti‐IgE, IL‐3 or fMLP stimulation. IL‐31 induced chemotaxis and the release of IL‐4 and IL‐13 which was specifically inhibited by anti‐IL‐31RA and anti‐OSMR. Conversely, IL‐31 had no effect on CD63 and CD203c externalization or histamine release.
Conclusions and Clinical Relevance
Human basophils are a source of –and are activated by – IL‐31 with the release of pro‐inflammatory cytokines and the induction of chemotaxis indicating an important novel function of IL‐31 in basophil biology.
Stimulated human basophils exhibit different degranulation patterns with release of mediators and appearance of activation markers such as CD63 and CD203c. Traditionally, released mediators are ...quantified in the supernatant of activated cells, whereas the expression of activation markers by individual cells is analyzed by flow cytometry. Alternatively, intracellular histamine and its release by basophils and mast cells have been repeatedly studied applying an enzyme-affinity-gold method based on the affinity of the histaminase diamine oxidase for its substrate histamine.
To develop a flow cytometric technique enabling to study histamine release by individual basophils in combination with the expression of activation markers. To elucidate the principles of basophil degranulation on a single cell level.
Intracellular histamine and its release is analyzed flow cytometrically by an enzyme-affinity method using diamine oxidase conjugated to laser-excitable fluorochromes. Phenotyping of cells implied flow cytometric quantification of CD63 and CD203c. Stimuli such as allergen, anti-IgE, N-formyl-met-leu-phe (fMLP), phorbol 12-myristate 13-acetate (PMA), ionomycin and interleukin (IL-)3 are applied to obtain different degranulation profiles.
Stimulation with anti-IgE, allergen, fMLP and PMA±ionomycin induces a rapid release of histamine that can be analyzed flow cytometrically. Analyses on a single cell level reveal that histamine release is not restricted to cells showing significant up-regulation of CD63. Alternatively, up-regulation of CD203c does not per se indicate histamine release. In some patients, priming of cells with IL-3 not only facilitates basophil responsiveness but also implies an increased ability of DAO to label the cells.
This study provides the proof-of-concept that histamine and its release can be studied by multicolor flow cytometry on a single cell level (HistaFlow). Coupling the data to simultaneous phenotyping of activated basophils confirms that histamine release principally results from anaphylactic degranulation and in a lesser extent from piecemeal degranulation.
► Histamine release by basophils can be analyzed flowcytometrically (HistaFlow). ► HistaFlow allows simultaneous analyses of activation markers and histamine release. ► HistaFlow confirms the existence of different activation and degranulation states.
IgE, mast cells, basophils, and eosinophils Stone, Kelly D., MD, PhD; Prussin, Calman, MD; Metcalfe, Dean D., MD
Journal of allergy and clinical immunology,
02/2010, Letnik:
125, Številka:
2
Journal Article
Recenzirano
Odprti dostop
IgE, mast cells, basophils, and eosinophils are essential components of allergic inflammation. Antigen-specific IgE production, with subsequent fixation of IgE to FcϵRI receptors on mast cells and ...basophils, is central to the initiation and propagation of immediate hypersensitivity reactions. Mast cells, basophils, and eosinophils are central effector cells in allergic inflammation, as well as in innate and adaptive immunity. This review highlights what is known about these components and their roles in disease pathogenesis.
The molecular characterization of immune subsets is important for designing effective strategies to understand and treat diseases. We characterized 29 immune cell types within the peripheral blood ...mononuclear cell (PBMC) fraction of healthy donors using RNA-seq (RNA sequencing) and flow cytometry. Our dataset was used, first, to identify sets of genes that are specific, are co-expressed, and have housekeeping roles across the 29 cell types. Then, we examined differences in mRNA heterogeneity and mRNA abundance revealing cell type specificity. Last, we performed absolute deconvolution on a suitable set of immune cell types using transcriptomics signatures normalized by mRNA abundance. Absolute deconvolution is ready to use for PBMC transcriptomic data using our Shiny app (https://github.com/giannimonaco/ABIS). We benchmarked different deconvolution and normalization methods and validated the resources in independent cohorts. Our work has research, clinical, and diagnostic value by making it possible to effectively associate observations in bulk transcriptomics data to specific immune subsets.
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•Characterization of 29 human immune cell type by RNA-seq and flow cytometry•Modules of specific, co-expressed, and housekeeping genes are defined•The mRNA heterogeneity and abundance are cell type specific•The proposed normalization approach enables absolute deconvolution
Monaco et al. generate an RNA-seq dataset on 29 immune cell types and identify modules of cell type-specific, co-expressed, and housekeeping genes. The mRNA heterogeneity and abundance of the different cell types were examined. Absolute deconvolution of PBMCs was obtained by taking into account mRNA abundance when normalizing the signature matrix.
Background Threshold levels for peanut allergy determined by using oral challenges are important for the food industry with regard to allergen labeling. Moreover, the utility of biological markers in ...predicting threshold levels is uncertain. Objective We sought to use a modified oral food challenge regimen that might determine threshold levels for peanut allergy mimicking a more real-life exposure and to correlate the eliciting dose (ED) and severity of clinical reaction in children with peanut allergy with B-cell, T-cell, and effector cell markers. Methods A modified food challenge procedure with doses scheduled 2 hours apart was used in 63 children with peanut allergy. All children received a maximum of 8 semi-log increasing titration steps of roasted peanuts ranging from 3 to 4500 mg of peanut protein until objective allergic reactions occurred. Severity of symptoms was graded from I to V. Biological markers were measured before challenge. Results Forty-five of 63 patients showed objective symptoms after greater than 30 minutes, with a median latency of clinical reaction of 55 minutes. By using a log-normal dose-distribution model, the ED5 was calculated to be 1.95 mg of peanut protein. The ED was significantly and inversely correlated with peanut- and Ara h 2–specific IgE levels, skin prick test responses, basophil activation, and TH 2 cytokine production by PBMCs. Symptom severity did not correlate with any of the markers or the ED. Conclusion This modified food challenge procedure might better reflect threshold levels for peanut allergy than the standard procedure because most of the patients reacted at a time interval of greater than 30 minutes. By using this model, threshold levels, but not severity, could be correlated with biological markers.
The inflammatory response after myocardial infarction (MI) is a precisely regulated process that greatly affects subsequent remodeling. Here, we show that basophil granulocytes infiltrated infarcted ...murine hearts, with a peak occurring between days 3 and 7. Antibody-mediated and genetic depletion of basophils deteriorated cardiac function and resulted in enhanced scar thinning after MI. Mechanistically, we found that basophil depletion was associated with a shift from reparative Ly6Clo macrophages toward increased numbers of inflammatory Ly6Chi monocytes in the infarcted myocardium. Restoration of basophils in basophil-deficient mice by adoptive transfer reversed this proinflammatory phenotype. Cellular alterations in the absence of basophils were accompanied by lower cardiac levels of IL-4 and IL-13, two major cytokines secreted by basophils. Mice with basophil-specific IL-4/IL-13 deficiency exhibited a similarly altered myeloid response with an increased fraction of Ly6Chi monocytes and aggravated cardiac function after MI. In contrast, IL-4 induction in basophils via administration of the glycoprotein IPSE/α-1 led to improved post-MI healing. These results in mice were corroborated by the finding that initially low counts of blood basophils in patients with acute MI were associated with a worse cardiac outcome after 1 year, characterized by a larger scar size. In conclusion, we show that basophils promoted tissue repair after MI by increasing cardiac IL-4 and IL-13 levels.
Physical trauma disrupts skin barrier function. How the skin barrier recovers is not fully understood. We evaluated in mice the mechanism of skin barrier recovery after mechanical injury inflicted by ...tape stripping. Tape stripping disrupted skin barrier function as evidenced by increased transepidermal water loss. We show that tape stripping induces IL-1–, IL-23–, and TCRγδ+-dependent upregulation of cutaneous Il17a and Il22 expression. We demonstrate that IL-17A and IL-22 induce epidermal hyperplasia, promote neutrophil recruitment, and delay skin barrier function recovery. Neutrophil depletion improved the recovery of skin barrier function and decreased epidermal hyperplasia. Single-cell RNA sequencing and flow cytometry analysis of skin cells revealed basophil infiltration into tape-stripped skin. Basophil depletion upregulated Il17a expression, increased neutrophil infiltration, and delayed skin barrier recovery. Comparative analysis of genes differentially expressed in tape-stripped skin of basophil-depleted mice and Il17a−/− mice indicated that basophils counteract the effects of IL-17A on the expression of epidermal and lipid metabolism genes important for skin barrier integrity. Our results demonstrate that basophils play a protective role by downregulating Il17a expression after mechanical skin injury, thereby counteracting the adverse effect of IL-17A on skin barrier function recovery, and suggest interventions to accelerate this recovery.
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