Comparision of two-flow cytometry methods for basophil degranulation in patients sensitized to grass pollen

BACKGROUND: Two-flow cytometry methods for quantification of degranulated basophil after allergen-specific activation were discussed. The methods are discerned by used membrane receptors--FcepsilonRI or IL-3Ralpha Our goal was to evaluate the diagnostic potential of the methods and to correlate them to allergen-specific IgE detection and skin prick test (SPT). METHODS: Patient's and control's groups were studied with Bulgarian grass pollen allergen B1 simultaneously by flow cytometry kits: Basotest and BD FastImmune test. Allergy diagnosis was based on clinical history and SPT. The determination of specific IgE was performed by ELISA--RIDASCREEN. RESULTS: There were no significant differences between the patient's results from Basotest and FastImmune (P>0.05). A significant correlation between values, analyzed by Basotest and by FastImmune was found (r=0.88). The sensitivity and specificity of the Basotest, FastImmune, specific IgE and SPT were 85%, 72%, 92% and 92% sensitivity and 100%, 92%, 100% and 85% specificity respectively. The efficiency was between 82% and 97%. There were a significant correlation between the specific IgE and flow cytometry tests: tau=0.92 (Basotest) and tau=0.71 (FastImmune) and a moderate significant correlation between the SPT and the in vitro tests: tau=0.26 (Basotest) and tau=0.31 (FastImmune). CONCLUSION: The successful use of the Bulgarian grass pollen allergen B1 and both flow cytometry tests was presented. These methods could be as specific tools for IgE-mediated diagnosis especially FastImmune in the case of low IgE receptor expression.     

Hymenoptera-venom-induced upregulation of the basophil activation marker ecto-nucleotide pyrophosphatase/phosphodiesterase 3 in sensitized individuals

BACKGROUND: Bee and wasp venoms are potent allergens capable of inducing severe clinical reactions. To detect immediate-type hypersensitivity to these allergens, a rapid in vitro test was developed that relies on the upregulation of ecto-nucleotide pyrophosphatase/phosphodiesterase 3 (E-NPP3) on activated basophils. METHODS: Blood basophils of 13 healthy donors and 22 patients allergic to bee or wasp venom were analyzed for E-NPP3 (CD203c) expression using monoclonal antibody 97A6. Basophils were analyzed by flow cytometry after activation with anti-IgE antibody or allergen. Venom-induced E-NPP3 upregulation on basophils was compared with diagnostic parameters, including skin tests and assessment of specific IgE. In selected samples, the increase in E-NPP3 expression on activated basophils was compared with histamine release and CD63 upregulation. RESULTS: In 20/22 patients sensitized to wasp or bee venom, E-NPP3 expression on basophils was upregulated in response to activation by allergen or anti-IgE. The maximum increase in E-NPP3 expression (above ten times of baseline) was achieved after 15 min of stimulation with 1 microg/ml of allergen or anti-IgE antibody. Sensitized individuals who failed to upregulate E-NPP3 in response to IgE receptor cross-linking also failed to induce histamine release and CD63 upregulation. CONCLUSIONS: Flow cytometric determination of hymenoptera-venom-induced upregulation of E-NPP3 is a novel in vitro test to identify sensitized individuals.     

Molecular cloning, expression and immunological characterisation of Pas n 1, the major allergen of Bahia grass Paspalum notatum pollen

Bahia grass, Paspalum notatum, is a clinically important subtropical grass with a prolonged pollination season from spring to autumn. We aimed to clone and characterise the major Bahia grass pollen allergen, Pas n 1. Grass pollen-allergic patients presenting to a tertiary hospital allergy clinic were tested for IgE reactivity with Bahia grass pollen extract by skin prick testing, ImmunoCAP, ELISA and immunoblotting. Using primers deduced from the N-terminal peptide sequence of a group 1 allergen of Bahia grass pollen extract separated by two-dimensional gel electrophoresis, the complete Pas n 1 cDNA was obtained by rapid amplification of cDNA ends and cloned. Biological relevance of recombinant Pas n 1 expressed in Escherichia coli was assessed by serum IgE reactivity and basophil activation. Twenty-nine of 34 (85%) consecutive patients presenting with grass pollen allergy were skin prick test positive to Bahia grass pollen. The Pas n 1 cDNA has sequence homology with the beta-expansin 1 glycoprotein family and is more closely related to the maize pollen group 1 allergen (85% identity) than to ryegrass Lol p 1 or Timothy grass Phl p 1 (64 and 66% identity, respectively). rPas n 1 reacted with serum IgE in 47 of 55 (85%) Bahia grass pollen-allergic patients, activated basophils and inhibited serum IgE reactivity with the 29 kDa band of Bahia grass pollen extract. In conclusion the cDNA for the major group 1 allergen of the subtropical Bahia grass pollen, Pas n 1, was identified and cloned. rPas n 1 is immunologically active and is a valuable reagent for diagnosis and specific immunotherapy of grass pollen allergy.     

Increased expression of surface activation markers on neutrophils following migration into the nasal lumen

BACKGROUND : The sequence of events following the recruitment of a free-flowing neutrophil in the peripheral circulation, via adhesion, migration and release of mediators, to a neutrophil on the surface of the nasal epithelium is a co-ordinated process. Little is known about the state of neutrophil activation following this course of events. OBJECTIVES : To investigate the expression of surface activation markers on neutrophils, reflecting activation during their recruitment to the nose, and to see whether the inflammatory process during allergic rhinitis influences this process. METHOD : Nine healthy controls and 12 patients with grass pollen-induced intermittent allergic rhinitis were investigated during the peak of the pollen season. The expression of CD11b, CD66b and CD63 on the neutrophil cell surface, as a reflection of activation, was analysed using flow cytometry. Neutrophils were derived from peripheral blood and nasal lavage fluid. In addition, eosinophil cationic protein (ECP) and myeloperoxidase (MPO) as well as L-, P- and E-selectins in the nasal lavage fluid were analysed using RIA and ELISA, respectively. RESULTS : A marked increase in the expression of all three CD markers on the neutrophil cell surface was noticed following migration from the bloodstream to the surface of the nasal mucosa. At the peak of the grass pollen season, the MPO levels increased, reflecting an increase in the total number of nasal fluid neutrophils. In parallel, the expression of CD11b was further augmented. The expression of the CDb11b was reduced on neutrophils remaining in the circulation. In addition, the level of L-selectin was reduced on neutrophils derived from the blood during allergic inflammation. CONCLUSION : Neutrophils might become activated during their transfer from the blood to the surface of the nasal mucosa, but these changes may also be due to depletion of activated neutrophils in the blood via activated endothelial/epithelial adhesion and chemoattractant measures. The increased expression of surface activation markers during allergic rhinitis suggests roles for neutrophils in the inflammatory process.     

Allergen-induced basophil activation: CD63 cell expression detected by flow cytometry in patients allergic to Dermatophagoides pteronyssinus and Lolium perenne

BACKGROUND: In this study, we determined by flow cytometry the percentage of basophils activated after in vitro stimulation by allergens and expressing the CD63 marker. The diagnostic reliability of the technique was assessed as well as its correlation with other in vitro diagnostic parameters. METHODS: Fifty-three patients suffering from asthma and/or allergic rhinitis following sensitization to Dermatophagoides pteronyssinus and 51 patients sensitized to Lolium perenne were investigated. Twenty-four atopic patients not sensitive to these allergens and 38 healthy subjects were also selected as controls. The basophil activation test determines the percentage of basophils which express CD63 as an activation marker, by means of flow cytometry, after in vitro stimulation with allergen, using double labelling with monoclonal antibody anti-CD63-PE and anti-IgE-FITC. RESULTS: No differences in basal values (non-activated control) were found between sensitized patients, atopic controls and healthy controls. On the other hand, sensitized patients showed a significantly higher percentage of activated basophils after stimulation by allergens in vitro than both control groups (P < 0.001). We found a significant correlation between skin tests and basophil activation tests (r = 0.72, P < 0.001). We also found a positive and significant correlation between basophil activation tests and histamine release tests (r = 0.80, P < 0.001), allergen-specific sulphidoleukotriene production (r = 0.7, P < 0.001) and the occurrence of serum allergen-specific IgE (r = 0.71, P < 0.001). CONCLUSION: The basophil activation test is a highly reliable technique in the diagnosis of allergy to inhalant allergens. The sensitivity of the basophil activation test was 93.3%, and its specificity 98.4%, when using a cut-off point of 15% activated basophils as positive result.     

Confirmation of the diagnosis of natural rubber latex allergy by the Basotest method

BACKGROUND: The flow cytometry CD63-based basophil activation test (Basotest has already been validated for the diagnosis of immediate-type allergy such as venom, house dust mite or cypress pollen allergies. The aim of this study was to evaluate the performance (specificity and sensitivity) of Basotest in the diagnosis of natural rubber latex allergy. METHODS: We included 46 latex allergic patients (clinical symptoms of latex allergy, positive latex skin prick tests and/or latex specific IgE) and 33 control subjects and performed Basotest on all subjects. RESULTS: The sensitivity and specificity of Basotest were 84.8 and 87.9%, respectively, when we considered the theoretic cut-off at 15% of CD63-positive cells. Using ROC curves, the optimal cut-off was evaluated at 22%, for which sensitivity and specificity were 79.3 and 96.7%, respectively. CONCLUSION: The Basotest is a reliable test in addition to clinical history and tests already validated (such as skin prick tests and specific IgE) to confirm the diagnosis of natural rubber latex allergy.     

Recruitment of CD1a+ Langerhans cells to the nasal mucosa in seasonal allergic rhinitis and effects of topical corticosteroid therapy

BACKGROUND: Local antigen presentation may be necessary for both primary and recall T-cell responses to grass pollen in hay fever patients. We examined the effect of seasonal allergen exposure on nasal mucosal antigen-presenting cell (APC) populations and the effects of topical corticosteroid therapy. METHODS: Nasal biopsies were collected from 46 grass pollen-sensitive seasonal rhinitis patients before the grass-pollen season. A second biopsy was collected during the pollen season, when patients had received 6 weeks' treatment with either fluticasone propionate (200 microg, twice daily) or placebo. Cell populations in biopsy sections were quantified by immunocytochemistry. RESULTS: Significant increases in submucosal and epithelial CD1a+ Langerhans cells, but not CD68 + macrophages or CD20 + B cells, were observed during the pollen season. Seasonal increases in CD1a+ Langerhans cells were inhibited by corticosteroid therapy. CONCLUSIONS: Recruitment of CD1a+ Langerhans cells to the nasal mucosa during natural seasonal allergen exposure may contribute to local T cell responses. Topical corticosteroids may act, at least in part, by inhibiting effective allergen presentation to T cells through inhibition of recruitment of Langerhans cells to the nasal mucosa.     

Measurement of basophil-activating capacity of grass pollen allergens, allergoids and hypoallergenic recombinant derivatives by flow cytometry using anti-CD203c

BACKGROUND: The assessment of the basophil-activating potential is an important aspect in the development of improved preparations for specific immunotherapy. The aim of the study was to evaluate the suitability of CD203c expression as a measure of basophil activation to compare allergoids with original allergen extracts, and recombinant hypoallergenic allergen derivatives with recombinant wild-type and natural allergens. METHODS: Heparinized whole blood samples from grass pollen allergic subjects were stimulated with grass pollen allergens and allergen derivatives followed by labelling of the basophils with PE-conjugated anti-CD203c. After lysis of the erythrocytes and fixation, the basophils were detected by flow cytometry. In some experiments, histamine release was determined simultaneously. RESULTS: Grass pollen allergoids revealed a 10-10 000-fold reduction of basophil-activating capacity measured by CD203c expression. The deletion mutant DM4 of rPhl p 5b showed stronger hypoallergenic characteristics in a range of 50-10 000-fold reduction, whereas a combination mutant of rPhl p 5b and Phl p 6 revealed less hypoallergenic features. Histamine release experiments led to a similar outcome as CD203c measurement. CONCLUSIONS: The measurement of CD203c expression on basophils by flow cytometry provides a rapid and sensitive method for the estimation of the allergic or hypoallergenic features of allergen preparations. The results demonstrated the hypoallergenicity of grass pollen allergoids and of the rPhl p 5b variant DM4, which may be a candidate in future preparations for specific immunotherapy.     

The degree of natural allergen exposure modifies eosinophil activity markers in the circulation of patients with mild asthma

We have previously found that CD9, CD11b, and intracellular ECP (EG2) may be used as activity markers for eosinophils in vitro . The main object of the present study was to determine whether these markers can reflect eosinophil activation in vivo in relation to allergen exposure. For this purpose, six patients with a history of allergic rhinitis and occasional asthma symptoms during the pollen season participated. Blood donors served as controls. Peripheral blood eosinophils were analyzed according to the FOG method and flow cytometry, before and during one birch pollen season with high pollen load (HPL) and one with low pollen load (LPL). The CD9 expression on peripheral eosinophils from the patients was significantly increased both before (P<0.05) and during (P<0.01) HPL season, and CD11b expression solely during HPL season (P = 0.01) as compared to controls. The intracellular expression of the EG2 epitope was increased before (P<0.01) and during (P<0.05) HPL season, and increased significantly (P<0.05) during season as compared to before. No changes were observed before and during LPL season. The proportion of eosinophils was increased both before (P<0.05) and during (P<0.001) the HPL season as compared to controls. The markers CD9, EG2, and, to a lesser extent, CD11b seem to detect activated eosinophils in the circulation, whereas EG2 may also reflect increased antigen exposure during season.     

A novel tool for the detection of allergic sensitization combining protein microarrays with human basophils

Background Protein microarray (PM) is a powerful alternative to costly or labour‐intensive diagnostic for the large‐scale detection of allergen‐specific IgE. In this study, we established a proof‐of‐concept that coupling the diversity of protein array with the biological output of basophilic cells is a feasible proposition. Method Human basophils purified from the peripheral blood of healthy donors were stripped, re‐sensitized with the serum or IgE preparation to be tested, and incubated with manually spotted protein array chips (FAST slides). The basophilic cell lines KU‐812 and RBL‐703/21 likewise sensitized were compared with peripheral blood basophils by the same approach. Purified basophils or other basophilic cells were incubated with FAST slides for various periods of time, washed, and cell binding was visualized by light microscopy. Basophil activation, indicating the effective cross‐linking of IgE by allergens, was monitored via up‐regulation of basophil activation surface marker (CD 63). Results Purified stripped peripheral basophils, re‐sensitized with the serum of a grass pollen‐allergic patient, displayed strong binding to anti‐IgE antibody and grass pollen extract with relatively low unspecific binding. Similar results were obtained with RBL‐703/21, which may be a good replacement for peripheral basophils to avoid the costly, cumbersome and time‐consuming basophil purification. Conclusion Our data suggest that coupling the diversity of a PM approach with the potential functionality and biological activity of a cell‐based test is feasible and may result in a new system to detect allergic sensitization.     

Flow cytometric basophil activation test by detection of CD63 expression in patients with immediate‐type reactions to betalactam antibiotics

BACKGROUND: In this study, we used flow cytometry to determine the percentage of activated basophils that expressed the CD63 marker after in vitro stimulation by different betalactam antibiotics. The diagnostic reliability of the technique was assessed, as well as its correlation with specific IgE. METHODS: Fifty-eight patients with clinical allergy to betalactam antibiotics and presenting positive skin tests to at least one of the allergens (minor determinant mixture (MDM), benzylpenicilloyl-polylysine (PPL), penicillin, ampicillin, amoxicillin, cephalosporins) were tested. Thirty subjects non-allergic to betalactams were also studied as controls. The flow assay stimulation test (FAST) uses flow cytometry to determine the percentage of basophils that express CD63 as an activation marker after in vitro stimulation with allergen. Double labelling with monoclonal antibodies anti-CD63-PE and anti-IgE FITC was used. RESULTS: The allergic patients show a statistically greater number of activated basophils than the control subjects, after the incubation of cells with all the betalactams at various concentrations. The sensitivity of the technique is 50%, the specificity 93.3%, the likelihood ratio for a positive value 7.46 and the likelihood ratio for a negative value 0.54. In spite of having a greater sensitivity (37.9%) and specificity (86.7%) than CAP, differences between sensitivity and specificities of both techniques (CAP and FAST) do not reach statistical significance. CONCLUSION: The basophil activation test is a particularly useful technique in the diagnosis of patients with IgE-mediated allergy to betalactams and allows the identification of 50% of patients. Used in conjunction with CAP, it allows the identification of 65.5% of such patients.     

Recombinant allergens promote expression of CD203c on basophils in sensitized individuals

BACKGROUND: Traditionally, the diagnosis of type I allergies is based on clinical data, skin test results, and laboratory test results with allergen extracts. During the past few years, several attempts have been made to refine diagnostic assays in clinical allergy by introducing recombinant allergens and novel markers of IgE-dependent cell activation. OBJECTIVES: We have identified the ectoenzyme CD203c as a novel basophil antigen that is upregulated on IgE receptor cross-linkage. In this study we applied CD203c and a panel of recombinant allergens to establish a novel basophil test that allows for a reliable quantification of IgE-dependent responses at the effector cell level. METHODS: Patients allergic to birch (Bet v 1, n = 15; Bet v 2, n = 8) and grass (Phl p 1, n = 15; Phl p 2, n = 10; Phl p 5, n = 14) pollen allergens, as well as 10 nonallergic donors, were examined. Basophils were exposed to various concentrations of recombinant allergens for 15 minutes and then examined for expression of CD203c by means of flow cytometry. CD203c upregulation was correlated with the increase in CD63. RESULTS: Exposure to recombinant allergens resulted in a dose-dependent increase in expression of CD203c on peripheral blood basophils in sensitized individuals, whereas no increase was seen in healthy control subjects. The effects of the recombinant allergens on CD203c expression were also time dependent. There was a good correlation between allergen-induced upregulation of CD203c and upregulation of CD63 (R = 0.76). CONCLUSION: Flow cytometric quantitation of CD203c on blood basophils exposed to recombinant allergens is a useful approach to determine the allergic state in sensitized individuals and represents a basis for a sensitive novel allergy test.     

Marked improvement of the basophil activation test by detecting CD203c instead of CD63

BACKGROUND: The flow cytometric basophil activation test by detection of CD63 expression has been developed as an alternative method for in vitro diagnosis of IgE-mediated reactions to various allergens. Despite promising initial studies, the test remains disappointing in terms of sensitivity. CD203c has recently been demonstrated as a specific activation marker of basophils that is rapidly up-regulated after allergen challenge in sensitized patients. OBJECTIVE: The goal of the present study was to compare basophil activation tests by using either CD203c or CD63 in the diagnosis of immediate-type allergy to latex. METHODS: Twenty-seven patients (health care workers of our institution) who developed clinical features evocative of allergy after contact with latex were included and classified into two groups. Group 1 (n = 16) comprised true allergic patients who presented with typical signs of immediate allergic reaction associated with a positive skin test (prick test). Group 2 (n = 11) consisted of patients whose clinical history was not typical and had negative skin test. Twelve healthy subjects were also studied as controls. We compared the sensitivity of two triple-staining flow cytometric protocols measuring basophil activation after latex stimulation: CD45-IgE-CD63 and CD45-IgE-CD203c. RESULTS: The CD203c protocol showed a higher sensitivity than the CD63 protocol (75% vs. 50%). In comparison, latex-specific IgE sensitivity was found to be 69%. Furthermore, the magnitude of the basophil response was significantly higher with CD203c in comparison with CD63. Specificity was 100% for both protocols. CONCLUSION: Due to superior gating of basophils and a higher range of activation in response to allergen, the basophil activation test is markedly improved by use of CD203c instead of CD63.     

Cytokine production in peripheral blood cells during and outside the pollen season in birch-allergic patients and non-allergic controls

BACKGROUND: The naturally occurring pollen season permits observation of the kinetic changes in the process of allergic inflammation. We examined cytokine production in peripheral blood (PB) T cells and monocytes obtained from birch-allergic patients both during and outside the pollen season. METHODS: PB from 16 patients and six healthy controls was obtained during the alder pollen season, at the beginning and the peak of the birch pollen season and outside the pollen season. Mononuclear cells (MNC) were stimulated with allergen and polyclonal activators. For flow cytometric analysis, MNC were stained with monoclonal antibodies (MoAbs) against the cell surface markers CD3, CD8, CD14 and the intracellular cytokines IL-4, IL-5, IL-10, IL-12, IL-13, granulocyte macrophage-colony stimulating factor (GM-CSF) and IFN-gamma. RESULTS: In allergic patients, significant increases in clinical symptoms, use of medication, eosinophil numbers and birch-specific IgE were found during the pollen season. In vitro allergen stimulation increased the number of GM-CSF+ monocytes (P<0.01) and this increase was dependent on allergen exposure. The IL-4/IFN-gamma ratio rose (P<0.001) at the peak of birch pollen season and the ratio correlated with symptom scores during the birch season. In the CD4+ cell population, the numbers of GM-CSF+ cells were higher throughout the alder and birch seasons compared with outside the pollen season (P<0.05). No such changes were seen in the healthy controls. CONCLUSIONS: The main finding of our study was the increased percentage of GM-CSF+ monocytes in atopic subjects compared with healthy controls. In allergic patients, natural seasonal pollen exposure resulted in increased numbers of GM-CSF+ cells among both monocytes and CD4+ T cells. We have also shown that a seasonal change in Th2/Th1 cytokine ratio requires an adequate and prolonged allergen stimulation that is seen late in the pollen season.     

Hemin,a heme oxygenase substrate analog,inhibits the cell surface expression of CD11b and CD66b on human neutrophils

BACKGROUND: Neutrophils are signaled to sites of infection and inflammation by different chemotactic stimuli. In order to reach the airways they have to adhere to, and then migrate through, the endothelium of pulmonary vessels. Carbon monoxide (CO) is a gaseous mediator, endogenously produced in the human airways. Increased CO production has been demonstrated during airway inflammation and CO as well as hemin, a substrate for CO producing enzymes, has been shown to affect neutrophil migration. Our objective was to investigate if the neutrophil cell surface expression of CD11b, CD66b and CD63 was changed during intermittent allergic rhinitis and to establish whether CO could affect the expression of these markers of cellular activation. METHODS: Blood from 10 healthy volunteers was drawn and incubated with different concentrations of hemin. Blood from 12 other healthy volunteers and from 12 patients with intermittent allergic rhinitis was also drawn during grass pollen season. Neutrophils were then isolated from all these three sets, and their expression of CD antigens measured using flow cytometry. RESULTS: Patients with symptomatic intermittent allergic rhinitis exhibited lower levels of CD11b and CD66b on the neutrophil cell surface. Incubation with hemin decreased the expression of CD11b and CD66b. CD63 was generally weakly expressed and not significantly affected by hemin incubation. CONCLUSION: Our results demonstrate that expressions of neutrophil cell surface glycoproteins are changed during the season in patents with intermittent allergic rhinitis and that hemin, a substrate for CO production, may act as an inhibitor of neutrophil activation. This indicates a possible role for CO in the immune defense system.     

Nasal challenge testing in grass pollen hay fever.

Nasal sensitivity to rye grass pollen allergens was evaluated by provocation testing in patients with hay fever due to grass pollen using measurements of nasal airways resistance (NAR), a reproducible system for delivery of allergen, and stringent criteria for allergen storage. Reproducibility was assessed in 24 subjects with hay fever by nasal provocation with serial dilutions of Lolium perenne allergens on 3 occasions: during the grass pollen season, immediately after the season, and in early winter. Threshold doses of allergen required to double the saline control NAR or to provoke persistent sneezing and rhinorrhea were slightly higher 1 mo after the pollen season, but there was no significant differences between threshold doses during the pollen season and 8 mo later. When the threshold doses during challenges were exceeded, there were late reactions in 4 of 24 patients. Normal subjects and patients with perennial rhinitis and with negative skin tests to L. perenne extract were unresponsive in nasal challenge tests.     

Individual hymenoptera venom compounds induce upregulation of the basophil activation marker ectonucleotide pyrophosphatase/phosphodiesterase 3 (CD203c) in sensitized patients

BACKGROUND: Bee and wasp venom extracts contain potent allergens capable of inducing severe clinical reactions. To analyze immediate-type hypersensitivity to defined hymenoptera venom components, a recently developed in vitro test was applied that is based on the upregulation of CD203c expression on basophils. METHODS: CD203c expression on blood basophils of 9 healthy donors and 39 patients allergic to bee and/or wasp venom was analyzed by flow cytometry before and after activation with the purified bee venom allergens phospholipase A2 (Api m 1), hyaluronidase (Api m 2) and melittin (Api m 4), or the purified wasp venom allergens phospholipase A1 (Ves v 1), hyaluronidase (Ves v 2) and the recombinant antigen 5 (Ves v 5). Venom-induced CD203c upregulation on basophils was compared with skin tests and assessment of specific IgE. Basophils of nonresponders were preincubated with 10 ng/ml interleukin-3 (IL-3) prior to allergen stimulation. RESULTS: CD203c upregulation on basophils was induced by defined hymenoptera venom components in 35/39 patients with a diagnosed allergy to wasp and/or bee venom. Twenty-seven of the 34 tested patients with wasp allergy showed CD203c upregulation in response to Ves v 5, 26 of these patients also reacted with Ves v 2 and 17 with Ves v 1. Nine of 13 patients with bee allergy reacted with Api m 1, 13 individuals with Api m 2 and none of these patients with the minor allergen Api m 4. A diagnosed wasp allergy could also be confirmed in the prestimulated basophils (IL-3) of 2 nonresponder individuals who failed to upregulate CD203c in response to IgE receptor cross-linking prior to culture with IL-3. CONCLUSIONS: Flow-cytometric determination of CD203c upregulation on basophils activated by molecularly defined allergens is a powerful method to identify the precise allergen reactivity in sensitized individuals.     

The effect of anti-IgE treatment on in vitro leukotriene release in children with seasonal allergic rhinitis

BACKGROUND: Binding of allergens with IgE to the IgE receptors on mast cells and basophils results in the release of inflammatory mediators as sulfidoleukotrienes (SLTs), triggering allergic cascades that result in allergic symptoms, such as asthma and rhinitis. OBJECTIVE: We sought to investigate whether anti-IgE (Oma-lizumab), a humanized monoclonal anti-IgE antibody, in addition to specific immunotherapy (SIT) affects the leukotriene pathway. METHODS: Ninety-two children (age range, 6-17 years) with sensitization to birch and grass pollens and with seasonal allergic rhinitis were included in a phase III, placebo- controlled, multicenter clinical study. All subjects were randomized to one of 4 treatment groups. Two groups subcutaneously received birch SIT and 2 groups received grass SIT for at least 14 weeks before the start of the birch pollen season. After 12 weeks of SIT titration, placebo or anti-IgE was added for 24 weeks. The primary clinical efficacy variable was symptom load (ie, the sum of daily symptom severity score and rescue medication score during pollen season). Blood samples taken at baseline and at the end of study treatment after the grass pollen season were used for separation of leukocytes in this substudy. After in vitro stimulation of the blood cells with grass and birch pollen allergens, SLT release (LTC4, LTD4, and LTE4) was quantified by using the ELISA technique. RESULTS: Before the study treatment, SLT release to birch and grass pollen exposure did not differ significantly among the 4 groups. Under treatment with anti-IgE + SIT-grass (n = 23), a lower symptom load occurred during the pollen season compared to placebo + SIT-grass (n = 24, P =.012). The same applied to both groups receiving birch SIT (n = 23 and n = 22, respectively; P =.03). At the end of treatment, the combination of anti-IgE plus grass SIT, as well as anti-IgE plus birch SIT, resulted in significantly lower SLT release after stimulation with the corresponding allergen (416 ng/L [5th-95th percentile, 1-1168] and 207 ng/L [1-860 ng/L], respectively) compared with placebo plus SIT (2490 ng/L [384-6587 ng/L], P =.001; 2489 ng/L [1-5670 ng/L], P =.001). In addition, treatment with anti-IgE was also followed by significantly lower SLT releases to the allergens unrelated to SIT (grass SIT: 300 ng/L [1-2432 ng/L] in response to birch allergen; birch SIT: 1478 ng/L [1-4593 ng/L] in response to grass pollen) in comparison with placebo (grass SIT: 1850 ng/L [1-5499 ng/L], P =.001; birch SIT: 2792 ng/L [154-5839 ng/L], P =.04]. CONCLUSION: Anti-IgE therapy reduces leukotriene release of peripheral leukocytes stimulated with allergen in children with allergic rhinitis undergoing allergen immunotherapy independent of the type of SIT allergen used.     

Seasonal idiopathic rhinitis with local inflammatory response and specific IgE in absence of systemic response

Background: Patients with idiopathic rhinitis (IR) are considered to be nonallergic because they have a negative skin prick test (SPT) and allergen specific‐IgE in serum. The concept of localized mucosal allergy in the absence of atopy has recently been proposed. The immunological mechanisms involved in seasonal IR have not been sufficiently studied. We examined nasal mucosa inflammation, the presence of nasal specific‐IgE and the response to nasal allergen provocation test (NAPT) in patients with seasonal IR who presented symptoms only in spring. Methods: We evaluated 32 patients with seasonal IR and 35 with persistent allergic rhinitis to pollen (PAR‐P) and compared these with healthy controls and persons with PAR to house dust mite during the pollen season, as well as by NAPT out‐of‐season with grass and Olea europea. We measured the nasal leukocyte–lymphocyte phenotype (CD45, CD33, CD16, CD3, CD4 and CD8), eosinophil‐cationic‐protein, and total and specific‐IgE to grass and olive pollen in serum and nasal lavage and performed NAPT. Results: In the IR group, 62.5% had a positive NAPT (IR‐PosNAPT), 20/32 to grass, with four of these having a positive NAPT to olive pollen as well. IR‐PosNAPT patients showed a similar nasal leukocyte–lymphocyte profile to the PAR‐P patients and different to controls. We detected nasal specific‐IgE in 35% of IR‐PosNAPT patients. Conclusions: These results support the hypothesis that a subgroup of patients with IR have seasonal symptoms with evidence of a nasal allergic immune reaction in the absence of a positive SPT or serum specific IgE.