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.

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.

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.

Airway angiogenesis in patients with rhinitis and controlled asthma.

BACKGROUND: Airway angiogenesis may be an important part of structural remodelling in the pathogenesis of asthma. The development of asthma is frequently preceded by rhinitis. OBJECTIVE: We sought to determine whether the levels of angiogenesis-related factors are elevated in airways of patients with rhinitis or controlled asthma. METHODS: We analysed the induced sputum of 18 rhinitis patients, 16 asthmatic patients, and 15 healthy controls. The concentrations of angiogenin, vascular endothelial growth factor (VEGF), IL-8, fibroblast growth factor (bFGF), and TNF-alpha were measured by cytometric bead arrays. RESULTS: We found significantly increased angiogenin and VEGF concentrations in the induced sputum supernatant of both rhinitis and asthma patients compared with that of the healthy control group (P< or =0.0005). With the exception of TNF-alpha, there was no difference in the other angiogenic factors; TNF-alpha levels were higher in the rhinitis group than in the control group (P=0.02). CONCLUSION: These in vivo results suggest increased airway angiogenesis in patients with rhinitis without asthma as well as in corticosteroid-treated and well-controlled asthma patients.

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.