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.

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.

Basophil responsiveness in patients with insect sting allergies and negative venom-specific immunoglobulin E and skin prick test results.

BACKGROUND: Current guidelines do not adequately address the question of how best to manage patients with a convincing history of insect allergy, but negative venom-specific IgE and skin test results. METHODS: Forty-seven patients out of a total of 1219 (4%), with a positive history of sting allergy, were recruited over a period of 4.5 years. All recruited patients had a convincing history of a severe or a life-threatening anaphylactic reaction of Mueller grade II-IV (median grade III) after Hymenoptera sting, but negative venom-specific IgE and skin prick test results. Diagnostic work-up was prospectively followed by the CD63 basophil activation test and by intradermal skin testing. A control group of 25 subjects was also assessed. RESULTS: Thirty-five out of 47 (75%) patients demonstrated a positive basophil CD63 response after stimulation with bee and/or wasp venom. Intradermal venom skin tests were performed for 37 patients, 17 (46%) of whom showed positive results. Out of 20 patients who demonstrated negative intradermal test results, 12 patients showed a positive CD63 response (60%). In contrast, out of 9 patients who showed a negative CD63 response, only one was detected by intradermal testing (11%). In the control group, only two out of 25 (4%) subjects displayed a positive basophil response and/or intradermal test. CONCLUSION: Here we show that, in complex cases with inconclusive diagnostic results, the CD63 activation test could be particularly useful and more sensitive than intradermal skin testing.

High sensitivity of basophils predicts side-effects in venom immunotherapy.

BACKGROUND: Systemic side-effects of venom immunotherapy (VIT) represent a considerable problem in the treatment of patients allergic to Hymenoptera venom. We examined the hypothesis whether basophil responsiveness might be connected with the adverse reactions to VIT. METHODS: Basophil surface expression of activation marker CD63 induced by different concentrations of honeybee and wasp venom (0.1 and 1 mug/ml) was measured by flow cytometry in 34 patients with history of systemic anaphylactic reactions to Hymenoptera sting just before rush honeybee or wasp VIT. RESULTS: Eleven of 34 patients had systemic anaphylactic reaction (Mueller grades I-III) and one patient a large local reaction to VIT. In those 12 patients, median percentage of activated basophils after stimulation with VIT-specific venom in concentration of 0.1 microg/ml was 99% (range: 17-195) of value reached with stimulation with 1 microg/ml. Side-effects occurred in all patients with 0.1/1 ratios over 92% (eight of 12). In contrast, in 22 patients with no side-effects, the median 0.1/1 ratio was 25% (range: 2-92). These concentration-dependent activation ratios were significantly different between the groups with and without side reactions (P < 0.0001). We also show significant positive correlation of the occurrence/clinical grade of the side-effects with individual ratios of CD63 basophil response (r = 0.73, P < 0.0001). CONCLUSION: The results suggest that increased basophil sensitivity to allergen-specific in vitro stimulation is significantly associated with major side-effects of VIT.