Kinetics of mast cell, basophil, and oral food challenge responses in omalizumab-treated adults with peanut allergy.

BACKGROUND: Monoclonal antibodies directed at IgE demonstrate clinical efficacy in subjects with peanut allergy, but previous studies have not addressed the kinetics of the clinical response or the role of mast cells and basophils in the food-induced allergic response. OBJECTIVE: We sought to determine the kinetics of the clinical response to omalizumab and whether clinical improvement is associated with either mast cell or basophil suppression. METHODS: Subjects with peanut allergy were treated with omalizumab for 6 months and assessed for clinical and cellular responses. At baseline, subjects had a double-blind, placebo-controlled oral food challenge (OFC), skin prick test titration (SPTT), and basophil histamine release (BHR) to peanut. BHR was repeated at week 2 and then weekly until it decreased to less than 20% of baseline values. The OFCs and SPTTs were repeated after the BHR reduction (or at week 8 if BHR did not decrease) and again at 6 months. RESULTS: Fourteen subjects enrolled in the study. At the second food challenge, there was a significant increase in the threshold dose of peanut inducing allergic symptoms (80 to 6500 mg, P < .01). Peanut-induced BHR was either completely suppressed (n = 5) or 10-fold more allergen was required to induce maximal BHR (n = 9), and SPTT responses were not significantly changed from baseline. After 6 months of omalizumab, further changes in the OFC threshold dose or BHR were not observed, but a significant suppression in SPTTs was identified. CONCLUSIONS: The clinical response to omalizumab occurs early in treatment when the basophil, but not the mast cell, is suppressed, supporting a role for the basophil in acute food reactions.

Effects of omalizumab on basophil and mast cell responses using an intranasal cat allergen challenge.

BACKGROUND: Omalizumab treatment suppresses FcepsilonRI expression faster on blood basophils than skin mast cells. OBJECTIVE: We used omalizumab to elucidate the relative contributions of basophil versus mast cell FcepsilonRI activation in a nasal allergen challenge (NAC) model. METHODS: Eighteen subjects with cat allergy were enrolled in a 3.5-month, double-blind, randomized (3.5:1), placebo-controlled trial of omalizumab using standard dosing. At baseline, subjects underwent NAC with lavage for prostaglandin D(2) measurement, skin prick test titration (SPTT), and blood sampling for basophil histamine release (BHR) and basophil IgE/FcepsilonRI measurements. Basophil studies were repeated at day 3 and then weekly until cat allergen-induced BHR was <20% of baseline or until day 45. Baseline visit procedures were repeated after the BHR reduction (midstudy NAC) and at the treatment period's completion (final NAC). RESULTS: Subjects treated with omalizumab who completed all NACs (n = 12) demonstrated significant mean reduction in BHR to an optimal dose of cat allergen by midstudy NAC compared with baseline (74% decrease; P = .001). In addition, these subjects demonstrated significant decreases in mean combined nasal symptom scores (50% decrease; P = .007) and total sneeze counts (59% decrease; P = .01) by midstudy NAC relative to baseline NAC. In contrast, measures of mast cell response (SPTT and nasal lavage prostaglandin D(2)) were only significantly reduced by the final NAC. Subjects on placebo (n = 4) did not experience a shift in basophil, NAC symptom, or mast cell measures. CONCLUSION: Reduction in nasal symptom scores occurred when the basophil, but not mast cell, response was reduced on omalizumab, implicating a role for basophils in the acute NAC response.

Cultured peripheral blood mast cells from chronic idiopathic urticaria patients spontaneously degranulate upon IgE sensitization: Relationship to expression of Syk and SHIP-2.

Recently, signaling changes in the FcvarepsilonRI pathway involving inositol lipid phosphatases have been identified in the basophils of chronic idiopathic urticaria (CIU) subjects. Based on the profile of basophil FcvarepsilonRI-mediated histamine degranulation, we have segregated CIU subjects into two groups, CIU Responder (CIU R) or CIU Nonresponder (CIU NR). In the present study, we compared expression of SHIP-1, SHIP-2, and Syk protein to histamine release (HR) from mast cells (MC) cultured from the peripheral blood of CIU R, CIU NR, and normal subjects. The MC of CIU R donors contained significantly increased Syk and decreased SHIP-2 as compared to CIU NR (Syk: p=0.038, SHIP-2: p=0.038) and normals (Syk: p=0.042, SHIP-2: p=0.027). Spontaneous HR from CIU donors was increased two-fold compared to normals (p=0.04). In summary, our results suggest a possible predilection for urticarial MC to spontaneously degranulate upon IgE sensitization contributing to the increased pruritus associated with CIU.

Development of a novel multiplex electrochemiluminescent-based immunoassay for quantification of human serum IgG against 10 Staphylococcus aureus toxins.

An electrochemiluminescent (ECL)-based multiplex immunoassay using Meso-Scale Discovery (MSD) technology was developed for detecting antibody response toward 10 Staphylococcus aureus (S. aureus) exotoxins. These 10 antigens included three different groups of toxins: 1) single component pore-forming toxins such as alpha- and delta-hemolysins, 2) the bicomponent pore-forming toxin Panton-Valentine leukocidin (PVL), comprised of LukS-PV and LukF-PV subunits, and 3) enterotoxin/superantigens - Staphylococcal enterotoxins A (SEA), B (SEB), C1 (SEC1), D (SED), K (SEK) and Toxic shock syndrome toxin-1 (TSST-1). Assay development included optimization steps with a conventional SEB ELISA-based serological assay and then optimized parameters were transferred and re-optimized in a singleplex ECL format. Finally, two pentaplex solid-phase ECL formats were developed. As proof of concept, one set of pentaplex ECL data was compared with conventional ELISA results. During the assay development controls were screened and developed for both the singleplex and multiplex assays. ECL-based multiplex assays were more sensitive with a wide dynamic range and proved more time-efficient than conventional ELISAs. Using the newly developed ECL method we showed, for the first time, that delta-hemolysin toxin can induce an immune response as antibody titers could be detected.

Basophil phenotypes in chronic idiopathic urticaria in relation to disease activity and autoantibodies.

Potentially pathogenic IgG autoantibodies to IgE or its receptor, Fc epsilonRIalpha, have been detected in approximately 40% of chronic idiopathic urticaria (CIU) patients. CIU patients' basophils display distinct altered Fc epsilonRIalpha-mediated degranulation. CIU patients with basophil histamine release in response to polyclonal goat anti-human IgE > or = 10% are classified as CIU responders (CIU-R) and < 10% are CIU non-responders (CIU-NR). We compared the presence of autoantibodies to basophil degranulation phenotypes and to disease status (active or inactive). Sera were collected from non-CIU subjects and CIU subjects who participated in a longitudinal study of disease severity and had defined basophil degranulation phenotypes. Immunoenzymetric assays (IEMA) quantified IgG anti-Fc epsilonRIalpha and anti-IgE. IgG anti-Fc epsilonRIalpha antibody was detected in 57% of CIU-R (n=35), 55% of CIU-NR (n=29), and 57% of non-CIU subjects (n=23), whereas IgG anti-IgE was present in 43% of CIU-R, 45% of CIU-NR, and 30% of non-CIU subjects. Both the autoantibody levels and the functional basophil phenotype remained stable in subjects with active disease (n=16), whereas there was an enhancement in basophil function as subjects evolved into a state of remission (n=6), which appears independent of the presence of autoantibody. IEMAs detected a similar frequency of autoantibodies in CIU-R, CIU-NR, and non-CIU subjects. Basophil function may be independent of IEMA-detected autoantibodies.

Basophil histamine release activity and disease severity in chronic idiopathic urticaria.

BACKGROUND: Altered basophil degranulation phenotypes are found in patients with chronic idiopathic urticaria (CIU). OBJECTIVE: To evaluate CIU disease severity in relation to basophil histamine release (HR) characteristics. METHODS: Patients with CIU were recruited from allergy and dermatology clinics. Patients with recent use of systemic corticosteroids or immunosuppressants were excluded. Patients completed disease severity surveys and had blood basophils isolated and stimulated for HR using polyclonal goat anti-human IgE and N-formyl-met-leu-phe. The HR was measured using automated fluorometry. Multivariate linear regression analyses were used to investigate relationships between HR data and CIU disease measures. RESULTS: Fifty patients completed surveys, of which 34 were further categorized into 2 subgroups based on basophil HR response to anti-IgE stimulation: responders (> or = 10% HR) and nonresponders (< 10% HR). Responders and nonresponders reported similar use of oral corticosteroids, work absences, and quality-of-life impairment but differed in their patterns of medications used for CIU. Basophil responders had a trend of higher use of the emergency department for CIU management. Multivariate regression revealed that patients with the basophil responder phenotype experienced significantly higher current itch scores (P = .02) compared with nonresponders. CONCLUSIONS: Quality-of-life impairment is similar in CIU basophil subsets. Patients with CIU with a basophil responder phenotype report longer disease duration, a higher frequency of emergency department use, and significantly higher itch severity.

Expression of activation markers on basophils in a controlled model of anaphylaxis.

BACKGROUND: Anaphylaxis has variable clinical presentations and lacks reliable biomarkers. Expression of activation markers on basophils has been useful in assessing sensitization in IgE-mediated diseases but has not been examined in vivo in anaphylaxis. OBJECTIVE: The study's goals were to assess the baseline expression of activation markers on basophils in individuals with a sting reaction history, the degree of change in expression of these markers after intentional sting challenge, and the relationship between in vitro and in vivo activation marker expression. METHODS: Patients allergic to insect venom were enrolled and grouped by clinical category defined by a history of a systemic or large local reaction and use of venom immunotherapy. Blood was collected before and after sting challenge. Enriched basophils were analyzed for activation marker expression. In select subjects, basophils were examined after in vitro stimulation with insect venom for activation marker expression and histamine release. RESULTS: Of 35 sting-challenge participants, 21 provided adequate samples for analysis. Pre-sting basophil CD63 expression was significantly higher in systemic reactors on immunotherapy. Following sting challenge, the rise in basophil CD69 expression and CD203c was significantly higher in systemic reactors on immunotherapy. Levels of activation markers on basophils were greater after in vitro venom stimulation than after in vivo challenge. CONCLUSION: Broader shifts in expression of basophil activation markers after in vivo challenge occurred among subjects with a history of in vivo systemic anaphylaxis despite venom immunotherapy. CLINICAL IMPLICATIONS: Basophil activation markers may be potential biomarkers for anaphylaxis.

Basophil FcepsilonRI histamine release parallels expression of Src-homology 2-containing inositol phosphatases in chronic idiopathic urticaria.

BACKGROUND: Basophils are implicated in the pathogenesis of chronic idiopathic urticaria (CIU). Autoantibodies to the IgE receptor (FcepsilonRI) and serum histamine releasing activity have been detected in some subjects with CIU, although their role in vivo is unclear. Basophils of patients with CIU have altered FcepsilonRI-mediated histamine release (HR); however, the mechanism is unknown. In the basophil FcepsilonRI signaling pathway, protein levels of Src-homology 2-containing-5'-inositol phosphatase (SHIP)-1 are inversely correlated with the release of mediators or releasability. A related phosphatase, SHIP-2, is a negative regulator of monocyte IgG receptor (FcgammaR) signaling . We hypothesized that SHIP levels are altered in CIU basophils. METHODS: Blood basophils were isolated from cold urticaria, CIU, or normal donors, and FcepsilonRI-dependent and independent HR were quantified. Protein levels of SHIP-1, SHIP-2, spleen tyrosine kinase, and phosphorylated Akt were determined by Western blotting. Subjects' serum was tested for serum histamine releasing activity and anti-FcepsilonRIalpha antibodies. RESULTS: CIU basophils displayed a bimodal response to anti-IgE activation. One half of CIU subjects' basophils had reductions in anti-IgE-induced HR and were designated nonresponders (CIU NR). CIU NR basophil HR remained diminished at 10-fold to 30-fold higher doses of anti-IgE. CIU anti-IgE responder basophils had HR similar to normal subjects. SHIP-1 and SHIP-2 proteins were increased in CIU NR basophils and were linked to reduced phosphoAkt after anti-IgE stimulation. CIU basophil anti-IgE response was not related to the presence of serologic factors. CONCLUSION: In CIU basophils, the observed changes in FcepsilonRI signaling pathway molecule expression may underlie changes in releasability. CLINICAL IMPLICATIONS: Patients with CIU can be segregated on the basis of basophil functional phenotype.