Basophil histamine release in the diagnosis of house dust mite and dander allergy of asthmatic children

The aim of the study is to compare the glass fibre-based basophil histamine release test with skin test (Phazet), RAST (Phadebas) and bronchial provocation test in children with allergic asthma. The study comprised 68 selected children with a case history of extrinsic allergic asthma to danders (cat and dog) and house-dust mite. Skin prick test, RAST, and histamine release were performed in all children and the bronchial provocation test was used as a reference of "true allergic asthma". A total of 81 allergen bronchial challenges were performed and 44 children experienced 49 positive provocations. In 2.9% (2/68) of the children histamine release could not be performed due to technical difficulties (low histamine release with anti-IgE). Concordances in the range 76-87% were observed with no significant difference between the tests. The highest concordance (87%) was found between histamine release and bronchial provocation test followed by skin prick test vs bronchial provocation (84%) and RAST vs bronchial provocation (80%). The sensitivity and specificity were calculated for each test. All tests showed sensitivities in the range 90-94% and no significant difference between them was observed. The specificity of histamine release, skin prick test, and RAST was 0.78, 0.69, and 0.63, respectively. The specificity of histamine release was better than RAST demonstrated by 95% confidence intervals. In conclusion, it was found that the histamine release test is a convenient diagnostic method and the study indicates a diagnostic value comparable to the common diagnostic methods in clinical allergy.     

Comparison of fiberglass-based histamine assay with a conventional automated fluorometric histamine assay, case history, skin prick test, and specific serum IgE in patients with milk and egg allergic reactions

A microfiberglass-based histamine assay (HR_M) was compared with an automated flourometric histamine assay (HR_A). Twenty-four with and 24 without a case history (CH) of milk and/or egg allergy were tested by HR_M and HR_A, skin prick test (SPT), and specific serum IgE (RAST). Six different concentrations of milk, egg, and anti-IgE to stimulate washed leukocytes (250 μ for HR_A and whole blood samples (25 μ for HR_M) in parallel. When we compared scores representing basophil senditivity, correlation coefficients (r_s) were positive (r(_anti-IgE))=0.88, r(_egg) = 0.95, r(_milk) = 0.88, P< 0.001), but no significant correlation were found after found after exclusion of the negatives in both tests. In some individual dose-response curves, the scores obtained by HR_M were shifted to higher allergen and anti-IgE concentrations. A high degree of concordance was found in positive and negative responses between the two: anti-IgE 91%, egg 92%, milk 86%. Finally, we found a good concordance between, on one other, CH, SPT, and RAST (HR_M vs. CH/SPT/RAST) 92/82/82%; milk 89/74/67%. We conclude that HR_M is in good qualitative, but poor quantitative, agreement with the autoanalyzer-based fluorometric histamine assay.     

Passive sensitization of basophil leukocytes from a non-atopic adult by plasma from allergic children

Basophil leukocytes from a non-atopic donor, who responded well to anti-IgE, were depleted of their native membrane-bound IgE by acid treatment and passively sensitized with plasma containing either Phleum pratense-, Dermatophagoides pteronyssinus- or dog dander- specific IgE obtained from 18 allergic children. Histamine release was then performed on the passively sensitized cells and the results were compared with those of bronchial provocation test (BPT), allergen-specific serum IgE (RAST), skin prick test (SPT), and conventional histamine release test (HR). A high coincidence rate was found between BPT, RAST and histamine release after passive sensitization (HR-PS), but compared to HR, it was lower. This could be because several of the patients had non-responding basophils (i.e. no release after challenge with anti-IgE) in the conventional histamine release assay. The lower rate was not related to a lack of antigen-specific IgE, since after passive sensitization of basophils, anti-IgE and allergen provocation could induce release. It is concluded that plasma from allergic children with non-responding basophil leukocytes contain antigen-specific IgE capable of binding to Fc-receptors on the basophils of a non-atopic donor. In addition it was found that the plasma could change the cell reactivity (maximal histamine release) of the donor cells, since the amount of histamine released varied according to the plasma used for passive sensitization. The lack of histamine release response in some patients could be because their own membrane-bound IgE is unable to induce mediator release or, more likely, activation of one or more of the subcellular steps involved in the release is impaired.     

Basophil activation during pollen season in patients monosensitized to grass pollens

BACKGROUND: Basotest is a new basophil-activation test based upon the expression of CD63 (gp53) in the presence of allergens. It is an effective diagnostic test for pollen-allergic patients. However, it is not known whether Basotest results differ during the pollen season. METHODS: We examined the activation of basophils by Basotest in 13 patients sensitized only to grass pollen, before and during the pollen season, in order to assess whether Basotest could be used as a diagnostic test during the pollen season. Dose-response curves with 10-fold increasing concentrations of timothy grass pollen (10-4 to 100 AU) were carried out. RESULTS: Basophils were not activated spontaneously during the pollen season since the CD63 expression was below detectable levels before in vitro cell activation. A decreased percentage of activated basophils at the peak of activation was found in comparing the pre- and in-season tests, but all patients had a positive test. When basophil activation was at its peak, the allergen concentration was similar during the two periods. Moreover, the median area under the curve was significantly (P < 0.02) reduced during the season as compared to before the season. CONCLUSION: Basotest can therefore be used as a diagnostic test during the pollen season, but the allergen exposure needs to be characterized if quantitative studies are performed.     

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??Objective To explore theeffect ofinhaled budesonide suspension on the activation of peripheral blood basophils in children with asthma. Methods Forty-eight children with asthma admitted between January 2013 and March 2014 in Shanghai Children’s Medical Center Affiliated to Shanghai Jiao Tong University School of Medicine were adopted in asthma group. Fifty-three children with pneumonia in the same period and 49 children performed physical
examination in Department of Child Care were adopted in pneumonia group and normal control group??respectively. Flow cytometry combined with dustmitestimulation was used to measure the expression of CD63 in blood basophils of the three groups?? and the results were compared. The asthmatic children were detected the expression of CD63 in blood basophil again after 6 months of therapy with budesonide suspension?? and the results were compared before and after treatment. Results Compared with the pneumonia group and normal control group?? the basophilsfluorescence intensity of CD63 wasincreased significantly in asthmagroup before and after dustmitestimulation??P??0.01??. The asthmatic children receieved 6 months of treatment with inhaled budesonide suspension. The basophilsfluorescence intensity of CD63 didnot change before dustmitestimulation??P??0.05????butdecreased significantly after dustmitestimulation??P??0.05??. Conclusion The detectionofbasophil activation can be used to monitor thestatus of
asthmacontrol.The basophilis one of the importanttargets forinhaled corticosteroidwhich affects children asthma.     

Poor association between allergen-specific serum immunoglobulin E levels, skin sensitivity and basophil degranulation: a study with recombinant birch pollen allergen Bet v 1 and an immunoglobulin E detection system measuring immunoglobulin E capable of binding to FcɛRI

BACKGROUND: Results from several studies indicate that the magnitude of immediate symptoms of type I allergy caused by allergen-induced cross-linking of high-affinity Fc epsilon receptors on effector cells (mast cells and basophils) is not always associated with allergen-specific IgE levels. OBJECTIVE: To investigate the association of results from intradermal skin testing, basophil histamine release and allergen-specific IgE, IgG1-4, IgA and IgM antibody levels in a clinical study performed in birch pollen-allergic patients (n = 18). METHODS: rBet v 1-specific IgEs were measured by quantitative CAP measurements and by using purified Fc epsilon RI-derived alpha-chain to quantify IgE capable of binding to effector cells. Bet v 1-specific IgG subclasses, IgA and IgM levels were measured by ELISA, and basophil histamine release was determined in whole blood samples. Intradermal skin testing was performed with the end-point titration method. RESULTS: Our study demonstrates on the molecular level that the concentrations of allergen-specific IgE antibodies capable of binding to Fc epsilon RI and biological sensitivities are not necessarily associated. A moderate association was found between cutaneous and basophil sensitivity. CONCLUSION: Our results highlight the quantitative discrepancies and limitations of the present diagnostic tools in allergy, even when using a single allergenic molecule. The quantity of allergen-specific serum IgE is only one component of far more complex cellular systems (i.e. basophil-based tests, skin tests) used as indirect diagnostic tests for IgE-mediated allergic sensitivity.     

Biological characterization of glutaraldehyde-modified Parietaria judaica pollen extracts

BACKGROUND: Allergoids are widely used in specific immunotherapy (SIT) for the treatment of IgE-mediated allergic diseases, but all techniques for standardization of conventional allergic extracts may not be appropriate for standardization of a glutaraldehyde (GA)-modified extract because of the unique characteristics of these extracts. OBJECTIVE: To assess an accurate methodology for standardization of chemically modified extracts. METHODS: GA-modified extracts from Parietaria judaica pollen were purified by diafiltration. Biochemical properties were investigated by determination of amino groups, chromatography, and SDS-PAGE. The IgE-binding activity was determined by skin prick test, enzyme allergosorbent test inhibition, basophil activation, and histamine release tests. Peripheral blood mononuclear cells (PBMCs) from P. judaica pollen-allergic subjects were stimulated with either native or allergoid extracts, and proliferation was measured. RESULTS: Biochemical data indicated a high degree of allergen polymerization resulting in extract components higher than 100 kDa. IgE-binding activity, both in vivo and in vitro, was reduced by more than 99.8%. Both allergen and allergoid induced PBMC proliferation and synthesis of blocking IgG antibodies at similar rates. Moreover, no evidence of introduction of new determinants by chemical modification was found. CONCLUSIONS: The preparation of GA-modified extracts by diafiltration is faster and more reliable than previous chromatographic methods. These modified extracts have drastically reduced their allergenicity while maintaining their immunogenicity, and therefore they can be used in safer and shortened schedules of SIT.     

Basophil activation in idiopathic mixed cryoglobulinemia

We studied basophil activation in patients affected by idiopathic mixed cryoglobulinemia in order to investigate the role of the basophil cell in the pathogenesis of the vasculitis typical of this disease. We selected 13 patients and we performed thein vitro basophil degranulation test with cryoprecipitate; in 5 cases the test was positive when it was performed in whole blood, while it was negative when it was performed with cells washed and resuspended in Tyrode's buffer. In 6 subjects it was not possible to perform the test because of the very low number of circulating basophil cells. Finally, in 2 subjects thein vitro basophil degranulation test with cryoprecipitate was negative; this result may be related to the very low levels of complement factors in the sera of these subjects. These findings sugges that the pathogenetic mechanism of vasculitis in idiopathic mixed cryoglobulinemia could be started by basophil activation.     

免疫诊断在哮喘免疫治疗中的意义

128例哮喘患者按皮试,ELISA测定血清中特异性IgE值和嗜碱粒细胞脱颗粒试验(HBDT)对粉尘螨检出情况随机分为二组,治疗组用粉尘螨浸液作免疫治疗,每周一次,共六个月。对照组以对症药物治疗。结果表明HBDT判定为阳性者有效率最高达92.59%,其次为ELISA,有效率为83.33%,皮试的有效率最低,为79.41%,但均明显高于对照组。治疗组内HBDT判定为阳性者的有效率明显高于阴性者,而ELISA和皮试判定为阳性者的有效率虽高于阴性者,但无显著差异。     

Basophil Histamine Release and Humoral Changes during Immunotherapy

An in vitro pilot study was performed to determine whether basophil cell-bound specific IgE was correlated to serum-specific IgE and to basophil cell sensitivity during immunotherapy with a well-documented treatment. The findings showed a clear dissociation between these three parameters, which is in contrast to the situation before hyposensitization and to our previous study in a comparable group of non-hyposensitized patients, where highly significant correlations were observed. Further investigations are in progress to clarify whether the dissociation is involved in the clinical improvement.