LAD2 mast cell activation test associates with the reaction severity and diagnoses BAT nonresponders in Hymenoptera venom allergy.

BACKGROUND AND OBJECTIVE: The usefulness of the mast cell activation test (MAT) in diagnosing patients with uninterpretable basophil activation test (BAT) caused by nonresponding basophils has not yet been addressed. It should be further evaluated if the results of MAT are associated with the severity of the allergic reaction. METHODS: We recruited 39 Hymenoptera venom allergic (HVA) patients, 22 non-sensitized controls, and 37 BAT nonresponding HVA patients. Specific IgE levels for honey bee venom (HBV), yellow jacket venom (YJV) and total IgEs were quantified using the Immulite system. BAT and MAT with LAD2 cells in response to HBV and YJV were performed. RESULTS: We first optimized the susceptibility of LAD2 cells to IgE-mediated degranulation in HVA and showed that prestimulation with IL-33 and IL-6 significantly increased the LAD2 cells´ responsiveness to allergen stimulation (P<0.01). LAD2 MAT results correlated with BAT results, and patients with severe sting reactions (Mueller grades IV or III) had a median 2-fold higher LAD2 MAT than the patients with nonsevere sting reactions (Mueller grades II, I or LLR) (P<0.05). Further, LAD2 MAT provided conclusive results in 54.1% (20 of 37) of HVA patients with nonresponding basophils in the BAT. CONCLUSION: The LAD2 MAT represents a new diagnostic tool for HVA patients with nonresponding basophils. Further, LAD2 MAT can identify patients at risk of severe sting reactions and thus can help guide recommendations for venom immunotherapy and improve the management of patients with HVA.

Quantitative, Absolute Count-Based T-Cell Analysis of CD69 Upregulation as a New Methodology for In Vitro Diagnosis of Delayed-Type Hypersensitivity Reaction to Nickel.

BACKGROUND: T cells play a major role in delayed-type hypersensitivity reactions. Their reactivity can be assessed by measuring the upregulation of the activation marker CD69, followed by assessment of proliferation and cytokine production. The aim of our study was to develop a novel, whole blood-based, quantitative, absolute count activation index (AI) for analysis of CD69 upregulation in various subsets of T cells in nickel-hypersensitive patients and compare it with previously reported approaches. METHODS: The study population comprised 10 patients with nickel allergy and 9 healthy controls. CD69 expression of CD3+, CD3+CD4+, and CD3+CD8+ T cells in heparinized blood was determined with flow cytometry after incubation with nickel sulfate for 48 hours. The absolute count of CD69+ cells was determined using microbeads. Production of the cytokines IL-2, IL-5, IL-13, and IFN-γ was determined after stimulation of peripheral blood mononuclear cells with nickel sulfate for 48 hours. RESULTS: We showed absolute AI to be the most sensitive approach. The index was calculated as the ratio of the absolute count of nickel-stimulated CD69-positive T cells to the absolute count of CD69-positive T cells in nonstimulated blood. This novel quantitative approach was more discriminative than previously reported approaches in which the T-cell CD69 percentage AI and cytokine production are measured. CONCLUSIONS: Our results demonstrated that measuring the absolute CD69 AI is a novel and accurate approach for quantification of antigen-specific T cells in the blood of patients with hypersensitivity reactions to nickel. This approach may be useful for better in vitro assessment of patients with delayed-type hypersensitivity reactions.

Quantitative, absolute count-based T-Cell analysis of CD69 upregulation as a new methodology for in vitro diagnosis of delayed-type hypersensitivity reaction to nickel

BACKGROUND: T cells play a major role in delayed-type hypersensitivity reactions. Their reactivity can be assessed by measuring the upregulation of the activation marker CD69, followed by assessment of proliferation and cytokine production. The aim of our study was to develop a novel, whole blood-based, quantitative, absolute count activation index (AI) for analysis of CD69 upregulation in various subsets of T cells in nickel-hypersensitive patients and compare it with previously reported approaches. METHODS: The study population comprised 10 patients with nickel allergy and 9 healthy controls. CD69 expression of CD3+, CD3+CD4+, and CD3+CD8+ T cells in heparinized blood was determined with flow cytometry after incubation with nickel sulfate for 48 hours. The absolute count of CD69+ cells was determined using microbeads. Production of the cytokines IL-2, IL-5, IL-13, and IFN-γ was determined after stimulation of peripheral blood mononuclear cells with nickel sulfate for 48 hours. RESULTS: We showed absolute AI to be the most sensitive approach. The index was calculated as the ratio of the absolute count of nickel-stimulated CD69-positive T cells to the absolute count of CD69-positive T cells in nonstimulated blood. This novel quantitative approach was more discriminative than previously reported approaches in which the T-cell CD69 percentage AI and cytokine production are measured. CONCLUSIONS: Our results demonstrated that measuring the absolute CD69 AI is a novel and accurate approach for quantification of antigen-specific T cells in the blood of patients with hypersensitivity reactions to nickel. This approach may be useful for better in vitro assessment of patients with delayed-type hypersensitivity reactions

ANTECEDENTES: Los linfocitos T juegan un papel importante en las reacciones de hipersensibilidad de tipo retardado. Su actividad puede evaluarse midiendo la expresión del marcador de activación CD69, seguido de la proliferación y la producción de citocinas. El objetivo de nuestro estudio ha sido el desarrollar un novedoso análisis cuantitativo del índice de activación absoluto (AI) en sangre completa de la expresión de CD69, en diferentes subconjuntos de linfocitos T, en pacientes con hipersensibilidad al níquel, y compararlo con los métodos existentes. MÉTODOS: Se estudiaron diez pacientes con alergia al níquel y nueve controles sanos. La expresión de CD69 de los linfocitos T CD3+, CD3+CD4+ y CD3+ CD8+ en sangre heparinizada se determinó con citometría de flujo, después de una incubación con sulfato de níquel durante 48 h. El recuento absoluto de células CD69+ se determinó con microesferas. La producción de las citocinas IL-2, IL-5, IL-13 e IFN-γ se cuantificó después de la estimulación de células mononucleares periféricas, durante 48 h, con sulfato de níquel. RESULTADOS: Se demuestra que la determinación del índice AI absoluto es la metodología más sensible. Se calculó como la relación entre el recuento absoluto de linfocitos T CD69-positivos estimulados con níquel y el recuento absoluto de linfocitos T CD69-positivos en sangre no estimulada. Este nuevo enfoque cuantitativo fue más discriminativo que los enfoques publicados previamente en los que se midió el porcentaje de CD69 de linfocitos T y la producción de citocinas. CONCLUSIONES: Nuestros resultados demostraron que la medición del AI absoluto de CD69 es un enfoque nuevo y preciso para cuantificar los linfocitos T específicos de antígeno en la sangre de pacientes con reacciones de hipersensibilidad al níquel. Este enfoque puede ser útil para una mejor evaluación in vitro de los pacientes con reacciones de hipersensibilidad de tipo retardado

Allergic Rhinitis and its Impact on Asthma (ARIA) guidelines: 2016 revision

BACKGROUND: Allergic rhinitis (AR) affects 10% to 40% of the population. It reduces quality of life and school and work performance and is a frequent reason for office visits in general practice. Medical costs are large, but avoidable costs associated with lost work productivity are even larger than those incurred by asthma. New evidence has accumulated since the last revision of the Allergic Rhinitis and its Impact on Asthma (ARIA) guidelines in 2010, prompting its update. OBJECTIVE: We sought to provide a targeted update of the ARIA guidelines. METHODS: The ARIA guideline panel identified new clinical questions and selected questions requiring an update. We performed systematic reviews of health effects and the evidence about patients' values and preferences and resource requirements (up to June 2016). We followed the Grading of Recommendations Assessment, Development, and Evaluation (GRADE) evidence-to-decision frameworks to develop recommendations. RESULTS: The 2016 revision of the ARIA guidelines provides both updated and new recommendations about the pharmacologic treatment of AR. Specifically, it addresses the relative merits of using oral H1-antihistamines, intranasal H1-antihistamines, intranasal corticosteroids, and leukotriene receptor antagonists either alone or in combination. The ARIA guideline panel provides specific recommendations for the choice of treatment and the rationale for the choice and discusses specific considerations that clinicians and patients might want to review to choose the management most appropriate for an individual patient. CONCLUSIONS: Appropriate treatment of AR might improve patients' quality of life and school and work productivity. ARIA recommendations support patients, their caregivers, and health care providers in choosing the optimal treatment.

Drug allergy passport and other documentation for patients with drug hypersensitivity - An ENDA/EAACI Drug Allergy Interest Group Position Paper.

The strongest and best-documented risk factor for drug hypersensitivity (DH) is the history of a previous reaction. Accidental exposures to drugs may lead to severe or even fatal reactions in sensitized patients. Preventable prescription errors are common. They are often due to inadequate medical history or poor risk assessment of recurrence of drug reaction. Proper documentation is essential information for the doctor to make sound therapeutic decision. The European Network on Drug Allergy and Drug Allergy Interest Group of the European Academy of Allergy and Clinical Immunology have formed a task force and developed a drug allergy passport as well as general guidelines of drug allergy documentation. A drug allergy passport, a drug allergy alert card, a certificate, and a discharge letter after medical evaluation are adequate means to document DH in a patient. They are to be handed to the patient who is advised to carry the documentation at all times especially when away from home. A drug allergy passport should at least contain information on the culprit drug(s) including international nonproprietary name, clinical manifestations including severity, diagnostic measures, potential cross-reactivity, alternative drugs to prescribe, and where more detailed information can be obtained from the issuer. It should be given to patients only after full allergy workup. In the future, electronic prescription systems with alert functions will become more common and should include the same information as in paper-based documentation.

Immunological and clinical factors associated with adverse systemic reactions during the build-up phase of honeybee venom immunotherapy.

BACKGROUND: Adverse systemic reactions (SRs) are more common in honeybee venom immunotherapy (VIT) than in wasp VIT. Factors that might be associated with SRs during the honeybee VIT are poorly understood. OBJECTIVE: Our aim was to evaluate risk factors for SRs during the build-up phase of honeybee venom immunotherapy. METHODS: We included 93 patients who underwent ultra-rush honeybee VIT. The adverse SRs and their severity was compared to various immunological (sIgE, tIgE, basophil CD63 response, baseline tryptase, and skin tests), patient-specific (age, sex, cardiovascular conditions and medications, and other allergic diseases), and sting-specific factors (anaphylaxis severity, time interval to onset of symptoms, and absence of cutaneous symptoms). RESULTS: Twenty-three patients (24.7%) experienced mild SRs and 13 patients (14%) severe SRs. In five patients with severe SRs, the build-up was stopped. High basophil allergen sensitivity, evaluated as dose-response curve metrics of EC15, EC50, CD-sens, AUC, or the response to submaximal 0.01 µg/mL of venom concentration, was the most significant risk factor and only independent predictor of severe SRs and/or build-up stop. Time interval of <5 min after sting to onset of symptoms and lower specific IgEs to rApi m1 was also associated with severe SRs. There was no difference in other immunological, patient-specific, or sting-specific factors, including the baseline tryptase. None of the studied factors was associated with mild SRs. CONCLUSION AND CLINICAL RELEVANCE: High basophil allergen CD63 sensitivity phenotype was a major indicator of severe adverse SRs during the build-up phase of honeybee VIT. Possibly role was also showed for short latency to filed sting reaction and low sIgE to rApi m1. Before honeybee VIT, measurement of basophil allergen sensitivity should be used to identify patients with a high risk for severe side-effects.

Sustained effect of grass pollen subcutaneous immunotherapy on suppression of allergen-specific basophil response; a real-life, nonrandomized controlled study.

BACKGROUND: An important advantage of allergen immunotherapy as compared to pharmacotherapy for allergic rhinitis is the long-term effect that persists after completing immunotherapy. The mechanism of the sustained effect of allergen immunotherapy is not completely understood. METHODS: We conducted a 7-year study of monitoring allergen-specific basophil response and serological markers in 20 subjects with moderate-to-severe grass pollen-allergic rhinitis just before beginning and after up-dosing of subcutaneous grass pollen immunotherapy, before the first pollen season, and 1-2 years after completion of 3-5 years of treatment. Comparable untreated rhinitis subjects were followed at the same time points. Clinical outcomes included assessment of symptoms, use of rescue medication, and quality of life. The basophil response was also monitored after removal of IgG antibodies. RESULTS: Basophil response assessed as area under the curve (AUC) halved during initiation of SCIT and was 55% lower 1-2 years after completing SCIT. In the untreated group, the basophil response remained comparable. Although immunotherapy-induced grass pollen-specific IgG4 levels decreased to near pre-immunotherapy levels after completing SCIT, the removal of IgG antibodies resulted in an increase in basophil response almost to the pre-immunotherapy levels. In untreated subjects, removal of IgG did not have any effect on basophil response. CONCLUSIONS: Grass pollen immunotherapy induces sustained suppression of the allergen-specific basophil response that persists after completion of treatment and could account for long-term clinical tolerance. It also seems to be associated with persistent blocking activity of IgG antibodies.

Rhinitis symptoms caused by grass pollen are associated with elevated basophile allergen sensitivity and a larger grass-specific immunoglobulin E fraction.

BACKGROUND: The mechanisms responsible for the difference between clinically irrelevant IgE-sensitization and allergic rhinitis are not fully understood. OBJECTIVE: We evaluated the humoral and cellular mechanisms that may be associated with the presence of allergic rhinitis symptoms. METHODS: We selected 26 subjects with positive grass pollen skin tests and IgE antibodies to Timothy (g6) and the major grass allergens rPhl p 1, 5b. Fourteen of those patients reported a history of allergic rhinitis. During winter, we performed a grass pollen CD63 basophile activation test using four log allergen concentrations, followed by a grass nasal provocation test (NPT). We obtained symptom scores in the subsequent pollination season. RESULTS: We showed that subjects with a positive NPT have significantly higher CD63 basophile grass pollen responsiveness than NPT-negative subjects, preferably at submaximal allergen concentrations, which represent cellular sensitivity. Moreover, basophile sensitivity positively correlated with the size of the grass-specific IgE fraction in relation to total IgE, and it was highly predictive of allergic rhinitis symptoms in the following pollination season. CONCLUSION AND CLINICAL RELEVANCE: Allergic rhinitis symptoms are significantly associated with allergen-specific basophile sensitivity. In vitro evaluation of basophile sensitivity should prove useful for distinguishing clinical phenotype of allergic sensitization.