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

Improved recombinant Api m 1- and Ves v 5-based IgE testing to dissect bee and yellow jacket allergy and their correlation with the severity of the sting reaction.

BACKGROUND: No study has assessed the diagnostic sensitivity of rApi m 1 and rVes v 5 on Immulite testing system. OBJECTIVE: To compare the diagnostic sensitivity of commercially available venom recombinant allergens between the currently available immunoassays [ImmunoCAP (CAP) and Immulite (LITE)] and establish their correlation with the severity of the sting reaction. METHODS: This study evaluated 95 bee venom and 110 yellow jacket venom-allergic subjects. We measured the levels of sIgE to rApi m 1, rVes v 5 (LITE and CAP), rApi m 2 (LITE), rVes v 1 (CAP) and total IgE (CAP). Forty-nine healthy subjects served as controls. RESULTS: The diagnostic sensitivity of rApi m 1 and rVes v 5 was significantly higher with the LITE than with the CAP system (71% vs. 88% and 82% vs. 93%). The specificity of both assays for both allergens was between 94% and 98%. Twenty-nine patients that tested negative for rApi m 1 or rVes v 5 with CAP were positive with LITE, but none of the patients that tested negative with LITE were positive with CAP. The positive values of rApi m 1 and rVes v 5 were on average 2.7 and 2.3 times higher, with the LITE than with the CAP system. The combination of rApi m 1 and rApi m 2 (LITE) and the combination of rVes v 5 (LITE) and rVes v 1 (CAP) almost matched the sensitivity of native venoms (95% and 97%, respectively), whereas the diagnostic sensitivity of the combination of rVes v 5 and rVes v 1 (CAP) did not reach the sensitivity of rVes v 5 (LITE) alone (90% vs. 93%). IgE levels to venom recombinants and total IgE did not correlate with the severity of sting reaction. CONCLUSIONS & CLINICAL RELEVANCE: The use of rApi m 1 and rVes v 5 with the LITE system significantly enhanced diagnostic utility of venom recombinants and should improve the dissection of bee and yellow jacket venom allergy.

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.

Short-term venom immunotherapy induces desensitization of FcεRI-mediated basophil response.

BACKGROUND: The precise immunological mechanisms for the early clinical protection of venom immunotherapy (VIT) have not yet been explained. Our aim was to evaluate whether high-affinity IgE receptor (FcεRI) and the related basophil function have a role in the induction of short-term VIT protection. METHODS: We included 60 adults and 48 children. Basophil threshold sensitivity (CD-sens) to anti-FcεRI stimulation, and FcεRI gene and cell-surface expression were assessed at the beginning and just before the first maintenance dose (MD) of 100 µg of ultra-rush VIT (day 5) and at the beginning, during buildup, and just before the first MD of 70 µg and of 100 µg of semi-rush VIT (weeks 1-2 and 5). RESULTS: We demonstrated a significant reduction in CD-sens to anti-FcεRI stimulation before the first MD in both ultra-rush and semi-rush VIT in all included subjects. FcεRI gene and/or cell-surface expression was decreased in 34-100% of subjects, with different dynamics between VIT protocols. CONCLUSION: We found a marked desensitization of FcεRI-activated basophils after short-term VIT. This suppression, which could be highly relevant for the development of early protective mechanisms, might be also related to the changes at the level of FcεRI expression.

High non-specific T lymphocyte response to the adjuvanted H1N1 vaccine in comparison with the H1N1/H3N2/B-Brisbane vaccine without adjuvant.

Shortly after the report of pandemic 2009 influenza A (H1N1), vaccine manufacturers, in conjunction with public agencies, started developing a H1N1 vaccine. In 2009, various approaches were implemented around the globe. The United States and Australia finally approved only non-adjuvanted H1N1 influenza vaccines, whereas Canada and the EU also approved adjuvanted vaccines. In 2010, seasonal influenza vaccine without adjuvant was again widely accepted in both hemispheres. The addition of adjuvant to the vaccine enhances the immunogenity of the vaccine in the presence of a relatively low amount of antigen. However, it might also induce undesirable non-specific immune response. For this reason, we conducted a prospective observational study to monitor T cell absolute count and H1N1-specific immunogenicity after 2009 and 2010 immunization. Fourteen healthy volunteers received the monovalent H1N1 AS03 adjuvanted influenza vaccine (3.5 µg of H1N1 and squalene-based adjuvant) in October 2009. The immunization was associated with a significant increase in T lymphocyte absolute count (P < 0.0001), reaching abnormal values in 57% of subjects. During this period, none of the subject showed any manifestation of severe viral infection or inflammation. Acute infection by CMV or EBV viruses was also excluded. In October 2010, the same subjects received a seasonal non-adjuvanted influenza vaccine (15 µg of each: H1N1, H3N2, and B-Brisbane). However, after 2010 immunization, no change in T lymphocyte absolute count was observed. H1N1-induced immunogenicity was good for both vaccines. Our results suggest a pronounced non-specific T cell response after AS03-adjuvanted 2009 H1N1 vaccination.

Basophil response and the induction of a tolerance in venom immunotherapy: a long-term sting challenge study.

BACKGROUND: There is no in vitro test to predict the induction of long-term tolerance in patients treated with venom immunotherapy (VIT). The aim of this study was to investigate whether immunotherapy-induced changes in basophil responsiveness reflect a state of protection and the induction of a tolerance. METHODS: Twenty-three patients with allergic reaction after Hymenoptera sting (11 wasp and 12 honeybee) were treated with VIT. In all patients, a CD63 basophil activation test was performed before the beginning of immunotherapy, after 1 year and after completing 4-6.5 years of immunotherapy (approximately 1 year after stopping). The tolerance was then evaluated by a sting challenge test. The basophil activation test was repeated 3-6 months after the challenge. RESULTS: Twenty-two subjects showed a negative sting challenge, and one subject, a positive sting challenge. Allergen-specific basophil response remained unchanged after 1 year of immunotherapy. However, after immunotherapy, a significant and approximately fourfold decrease was demonstrated in all tolerant subjects mainly in response to submaximal 0.1 µg/ml allergen concentration. This depression was sustained and did not change with the sting challenge test. In a nontolerant patient with a positive sting challenge, basophil response did not change. CONCLUSIONS: Our results suggest that the depression of allergen-specific basophil response seems to be associated with the induction of a tolerance after completing a course of VIT.

Association of ORMDL3, STAT6 and TBXA2R gene polymorphisms with asthma.

Asthma is one of the most common chronic diseases in childhood. It is well known that genetic variability contributes to asthma risk. One of the most replicated asthma candidate genes is ORM1-like 3 (Saccharomyces cerevisiae) (ORMDL3), which has been associated with childhood asthma susceptibility. Another asthma candidate gene is signal transducer and activator of transcription 6 (STAT6), a regulator of IgE class switching. Gene coding thromboxane A2 receptor (TBXA2R), involved in chronic airway inflammation, has been associated with asthma in several genetic studies. We have studied the association of polymorphism rs4795405 in ORMDL3, rs324011 in STAT6 as well as rs8113232 and rs3786989 in TBXA2R with asthma risk, various asthma phenotypes and asthma-related symptoms. The study group consisted of 154 children with asthma, in whom clinical parameters were measured and whose asthma control and atopic status were determined. A control group comprised 71 healthy children. Genotyping was performed using an allelic discrimination assay. The ORMDL3 polymorphism rs4795405 was suggestively associated with asthma risk. Furthermore, it was significantly associated with nonatopic asthma and asthma without rhinitis. No association was detected between the STAT6 polymorphism rs324011 or the TBXA2R polymorphisms rs8113232 and rs3786989 and asthma susceptibility. However, an association between rs324011 in STAT6 with recurrent wheezing in early childhood and a suggestive association between rs8113232 in TBXA2R with rhinitis in children with asthma were observed. Our results confirmed ORMDL3 as a candidate gene for childhood asthma susceptibility. STAT6 and TBXA2R polymorphisms were not associated with asthma risk, but they were associated with asthma-related symptoms.

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.

Immunoglobulin G-dependent changes in basophil allergen threshold sensitivity during birch pollen immunotherapy.

BACKGROUND: The effects of pollen immunotherapy on effector cells of allergic inflammation, such as mast cells and basophils, are poorly understood. OBJECTIVE: For this reason, we conducted an open study on basophil allergen threshold sensitivity during birch pollen immunotherapy. METHODS: Basophil sensitivity was measured by CD63 flow cytometry in 14 patients with moderate-severe intermittent birch pollen rhinitis using four log allergen concentrations. In nine patients, we analysed the basophil sensitivity before and during treatment with subcutaneous birch immunotherapy (perennial scheme with allergoid). We also included eight birch-allergic donor subjects for IgG inhibition experiments and eight control subjects. RESULTS: There was a decrease in basophil allergen threshold sensitivity after 2, 3, and 5 months of immunotherapy. This decrease was correlated with an improvement in patients' symptoms measured on a visual analogue scale. The serum obtained after immunotherapy induced a significant decrease in allergen threshold sensitivity in donor birch-allergic basophils. This inhibition was not observed after IgG depletion from the serum. CONCLUSIONS: In this study, we showed that birch immunotherapy-induced IgG antibodies are associated with a reduction in basophil allergen threshold sensitivity. Further studies are needed to show whether the changes in basophil sensitivity are of clinical relevance in pollen immunotherapy.

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.