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Allergy Diagnosis (allergy + diagnosis)
Selected AbstractsCD203c-based basophil activation test in allergy diagnosis: Characteristics and differences to CD63 upregulation,CYTOMETRY, Issue 5 2010Eva M. Sturm Abstract Background: The basophil activation test (BAT) based on CD203c upregulation has been validated as a reliable tool for the diagnosis of IgE-mediated allergies. Nevertheless, CD203c-based BAT is hardly comparable with that of CD63-based tests, as the mechanisms of CD203c versus CD63 induction differ considerably. The aim of the present study was to identify potent influencing factors of the CD203c-based BAT and to emphasize differences between CD63 and CD203c detection. Methods: CD203c-based BAT was determined in 82 healthy controls and in 79 allergic patients. The effects of interleukin (IL)-3 and degranulation enhancing substances were investigated and compared with CD63 upregulation. Furthermore, the influence of different storage conditions and incubation times was evaluated and the impact of antiallergic drugs on the test results was assessed. Results: CD203c and CD63 expression was rapidly upregulated reaching a maximum after 20,30 min. Basophil CD203c upregulation assayed after storage times up to 48 h declined already after 4 h. IL-3 treatment increased CD203c and CD63 baseline levels and decreased basophil CD203c responses in a dose-dependent manner. In contrast, cytochalasin B and latrunculin B did not affect CD203c responses but decreased CD63-based BAT. Finally, therapeutic concentrations of dimetindene and desloratadine did not affect CD203c upregulation. Conclusion: CD203c-based basophil activation test should be performed preferentially within 4 h after taking the blood samples. Priming and degranulation-enhancing factors are not required for CD203c-based BAT. In contrast to skin testing, CD203c-based BAT can be performed in patients undergoing antiallergic treatment. © 2010 International Clinical Cytometry Society [source] Simultaneous flow cytometric detection of basophil activation marker CD63 and intracellular phosphorylated p38 mitogen-activated protein kinase in birch pollen allergy,CYTOMETRY, Issue 1 2009Nicolaas E. Aerts Abstract Background: Phosphorylation of p38 MAPK is a crucial step in IgE-receptor signaling in basophils. The relation of p38 MAPK to the well-validated diagnostic cell surface marker CD63 has not been evaluated in a clinical allergy model. Methods: Expression of CD63 and phosphorylation of p38 MAPK were analyzed flow cytometrically in anti-IgE-gated basophils from 18 birch pollen allergic patients, five grass pollen allergic patients, and five healthy individuals, after 3 and 20 min of stimulation with recombinant major birch pollen allergen (rBet v 1). Additional time points and the influence of p38 MAPK inhibitor SB203580 were studied in birch pollen allergic patients. Results: Phospho-p38 MAPK and CD63 were expressed dose-dependently in birch pollen allergic patient basophils within 1 minute of rBet v 1 stimulation. P38 MAPK phosphorylation was fastest and subsided gradually while CD63 expression remained elevated for at least 20 min. Inhibition of p38 MAPK significantly inhibited CD63 upregulation. With optimal stimulation of the cells (1 ,g/mL), sensitivity and specificity for the discrimination between patients and a group of control individuals (grass pollen allergic patients and healthy controls) were 94% and 100% for CD63 at 3 and 20 min and for phospho-p38 MAPK at 3 min. Conclusion: Antigen-induced p38 MAPK phosphorylation in human basophils essentially contributes to CD63 upregulation. It is a sensitive and specific intracellular marker for allergy diagnosis and offers new insight into the mechanisms of basophil activation. © 2008 Clinical Cytometry Society [source] Evaluation and standardisation of different matrices used for double-blind placebo-controlled food challenges to fishJOURNAL OF HUMAN NUTRITION & DIETETICS, Issue 5 2010E. Vassilopoulou Abstract Background:, Fish allergens represent one of the most common causes of adverse reactions to food worldwide. Double-blind placebo-controlled food challenges (DBPCFC) are the gold standard for food allergy diagnosis. However, no standardised recipes are available for common food allergens such as fish, and a well trained dietitian is essential for creating and standardising them. The present study aimed to create and standardise recipes for use in DBPCFCs to fish. Methods:, Three recipes were prepared. Employing a standardised procedure, a total of 35 panelists evaluated the different matrices using an evaluation form. A paired comparison test was used to estimate total evaluation's outcome. Fish allergic patients were challenged with different fish species blinded with the selected matrix and evaluated the recipe using the same form. Results:, From a base recipe and step-by-step modifications, a low fat recipe was selected among other recipes tested, which proved to be appropriate for fish blinding, in terms of taste, odour, appearance and blinding. Patients challenged with the final matrix found it acceptable, no matter which fish type was used. Conclusions:, In this pilot study, a recipe with satisfactory organoleptic characteristics was developed and validated for DBPCFC to fish. [source] IgE cross-reactivity between Ascaris and domestic mite allergens: the role of tropomyosin and the nematode polyprotein ABA-1ALLERGY, Issue 11 2009N. Acevedo Background:, Analysis of cross-reactivity between the nematode Ascaris ssp. and dust mites, two important allergen sources in the tropics, will contribute in understanding their influence on asthma and atopy. The objective of this study was to investigate immunoglobulin E (IgE) cross-reactivity between Ascaris and two domestic mites in the tropics. Methods:, Sera from 24 asthmatic patients were used in ELISA and immunoblotting IgE-binding inhibition assays using Ascaris, Blomia tropicalis and Dermatophagoides pteronyssinus extracts and the recombinants Blo t 10, ABA-1 and Blo t 13 as competitors. Identification of Ascaris allergens was confirmed by mass spectrometry (LC-MS/MS). Results:, We detected at least 12 human IgE-binding components in Ascaris extract. Blomia tropicalis and D. pteronyssinus inhibited 83.3% and 79% of IgE-binding to Ascaris, while Ascaris inhibited 58.3% and 79.3% to B. tropicalis and D. pteronyssinus respectively. Mite tropomyosin inhibited 85% of IgE-binding to Ascaris. Affinity-purified human IgE to rBlo t 10 identified an allergen of 40 kDa in Ascaris extract, further confirmed as tropomyosin by LC-MS/MS. We found no evidence of IgE cross-reactivity between rABA-1 and any allergen component in mite extracts, including rBlo t 13. Conclusions:, There is cross-reactivity between Ascaris and mites, determined by several allergens including tropomyosin and glutathione- S -transferase. In addition to its potential impact on asthma pathogenesis, Ascaris infection and mite allergy diagnosis relying on the determination of specific IgE could be affected by this cross-reactivity. ABA-1 has no cross-reactive counterpart in mite extracts, suggesting its usefulness as a more specific marker of Ascaris infection. [source] GA2LEN skin test study I: GA²LEN harmonization of skin prick testing: novel sensitization patterns for inhalant allergens in EuropeALLERGY, Issue 10 2009L. M. Heinzerling Background:, Skin prick testing is the standard for diagnosing IgE-mediated allergies. However, different allergen extracts and different testing procedures have been applied by European allergy centres. Thus, it has been difficult to compare results from different centres or studies across Europe. It was, therefore, crucial to standardize and harmonize procedures in allergy diagnosis and treatment within Europe. Aims:, The Global Asthma and Allergy European Network (GA²LEN), with partners and collaborating centres across Europe, was in a unique position to take on this task. The current study is the first approach to implement a standardized procedure for skin prick testing in allergies against inhalant allergens with a standardized pan-European allergen panel. Methods:, The study population consisted of patients who were referred to one of the 17 participating centres in 14 European countries (n = 3034, median age = 33 years). Skin prick testing and evaluation was performed with the same 18 allergens in a standardized procedure across all centres. Results:, The study clearly shows that many allergens previously regarded as untypical for some regions in Europe have been underestimated. This could partly be related to changes in mobility of patients, vegetation or climate in Europe. Conclusion:, The results of this large pan-European study demonstrate for the first time sensitization patterns for different inhalant allergens in patients across Europe. The standardized skin prick test with the standardized allergen battery should be recommended for clinical use and research. Further EU-wide monitoring of sensitization patterns is urgently needed. [source] Micro-arrayed wheat seed and grass pollen allergens for component-resolved diagnosisALLERGY, Issue 7 2009C. Constantin Background:, Wheat is a potent allergen source and can cause baker's asthma, food and pollen allergy. The aim of the study was to develop an allergen micro-array for differential diagnosis of baker's asthma, wheat-induced food allergy and grass pollen allergy. Methods:, We analysed the immunoglobulin-E reactivity profiles of patients suffering from baker's asthma, wheat-induced food allergy and grass pollen allergy to micro-arrayed recombinant wheat flour allergens and grass pollen allergens and compared these results with clinical results and diagnostic tests based on crude wheat flour, wheat pollen and grass pollen allergen extracts. Results:, We identified recombinant wheat flour allergens, which are specifically recognized by patients suffering from baker's asthma, but not from patients with food allergy to wheat or pollen allergy. rPhl p 1 and rPhl p 5 were identified as marker allergens specific for grass pollen allergy. They can be used to replace grass pollen extracts for allergy diagnosis and to identify grass pollen allergic patients among patients suffering from baker's asthma and wheat-induced food allergy. Profilin was identified as a cross-reactive allergen recognized by patients suffering from baker's asthma, food and pollen allergy. Conclusions:, Our results indicate that it will be possible to design serological tests based on micro-arrayed recombinant wheat seed and grass pollen allergens for the discrimination of baker's asthma, wheat-induced food allergy and grass pollen allergy. [source] Breaking frontiers for better early allergy diagnosisALLERGY, Issue 9 2004P. A. Eigenmann No abstract is available for this article. [source] Production and characterization of an allergen panel for component-resolved diagnosis of celery allergyMOLECULAR NUTRITION & FOOD RESEARCH (FORMERLY NAHRUNG/FOOD), Issue S2 2008Merima Bublin Abstract In celery a relevant food allergen source, three allergens have been identified so far: Api g 1 and Api g 4, and one glycosylated protein, Api g 5. For component-resolved food allergy diagnosis high amounts of well-defined allergens are needed. Depending on the individual celery allergen, protocols for heterologous production and purification from natural source, respectively, were established to obtain homogenous protein batches. Afterwards the purified recombinant allergens, Api g 1, Api g 4 and natural Api g 5 were characterized regarding their structural integrity and immunological activity. Therefore, several methods were applied. Proteins were identified by partial N-terminal sequencing, protein mass was verified by MS and sequence integrity by MALDI-TOF and N-terminal sequencing after tryptic digestion. Presence of isoforms in natural allergen preparations was identified by 2-DE. Secondary and tertiary structures were evaluated by circular dichroism (CD) spectroscopy and NMR analysis. Finally, IgE binding capacity was verified using selected sera from celery allergic patients in IgE immunoblots and IgE ELISA. These well-defined celery allergens will be used to prove the concept of component-resolved diagnosis and will contribute to improve food allergy diagnosis in the future. [source] Analysis of basophil activation by flow cytometry in pediatric house dust mite allergyPEDIATRIC ALLERGY AND IMMUNOLOGY, Issue 4 2008Miguel González-Muñoz Detection of allergen-induced basophil activation by flow cytometry has been shown to be a useful tool for allergy diagnosis. The aim of this study was to assess the potential of this technique for the diagnosis of pediatric house dust mite allergy. Quantification of total and specific IgE and basophil activation test were performed to evaluate mite allergic (n = 24), atopic (n = 23), and non-allergic children (n = 9). Allergen-induced basophil activation was detected as a CD63-upregulation. Receiver operating characteristics (ROC) curve analysis was performed to calculate the optimal cut-off value of activated basophils discriminating mite allergic and non-allergic children. ROC curve analysis yielded a threshold value of 18% activated basophils when mite-sensitized and atopic children were studied [area under the curve (AUC) = 0.99, 95% confidence interval (CI) = 0.97,1.01, p < 0.001] with a sensitivity and specificity of 96% for 16 ,g/ml mite extract. Analysis of the data obtained with 1.6 ,g/ml mite extract defined a cut-off value of 8% activated basophils (AUC = 0.96, 95% CI = 0.91,1.01; p < 0.001) with a sensitivity of 82% and specificity of 100%. Comparison between mite allergic and non-allergic children produced a cut-off of 8% activated basophils (AUC = 1.0) with 16 ,g/ml allergen extract and a sensitivity and specificity of 100%. The same threshold and specificity values were obtained with 1.6 ,g/ml extract (AUC = 97%, 95% CI = 0.92,1.02; p < 0.001) but sensitivity decreased to 83%. Two atopic children showed negative skin prick and basophil activation tests and high specific IgE (>43 kU/l) values for Dermatophagoides pteronyssinus allergen. They also showed positive prick (wheal diameter >1.0 cm) and basophil activation (>87%) tests and high specific IgE (>100 kU/l) with shrimp allergen. Shrimp sensitization was demonstrated by high levels of Pen a 1-specific IgE (>100 kU/l). Cross-reactivity between mite and shrimp was confirmed by fluorescence enzyme immunoassay (FEIA-CAP) inhibition study in these two cases. This study demonstrated that the analysis of allergen-induced CD63 upregulation by flow cytometry is a reliable tool for diagnosis of mite allergy in pediatric patients, with sensitivity similar to routine diagnostic tests and a higher specificity. Furthermore, this method can provide additional information in case of disagreement between in vivo and in vitro test results. [source] Suspension-cultured BY-2 tobacco cells produce and mature immunologically active house dust mite allergensPLANT BIOTECHNOLOGY JOURNAL, Issue 1 2007David Lienard Summary The replacement of crude allergen extracts by selected allergens currently represents a major goal for the improvement of allergy diagnosis and immunotherapy. Indeed, the development of molecularly defined vaccines would facilitate both standardization and enhance batch-to-batch reproducibility as well as treatment specificity. In this study, we have investigated the potential of tobacco plant cells to produce biologically active forms of the two major allergens from the house dust mite. A detailed characterization of these plant-made allergens has shown similar proteolytic maturation and folding as well as comparable immunoreactivity to their natural counterparts. Altogether, our results exemplify that suspension-cultured BY-2 tobacco cells represent a low cost and environmentally safe expression system suitable to produce recombinant allergens from Dermatophagoides pteronyssinus under a form appropriate for diagnostic and therapeutic purposes. [source] Secretome analysis of novel IgE-binding proteins from Penicillium citrinumPROTEOMICS - CLINICAL APPLICATIONS, Issue 1 2008Li-Li Chiu Abstract The Penicillium genus of fungi is a frequently reported cause of allergic reactions. However, only a limited number of allergens have been reported. In Penicillium spp., many allergens show higher IgE-binding activity in culture filtrate extracts than in cellular extracts. In order to investigate the IgE-reactive profile of mold-sensitized patients, secreted IgE-reactive proteins from Penicillium citrinum were identified by 2-DE, serum immunoblotting, and nanoLC-MS/MS. Among the IgE-reactive spots, one known allergen, Pen c 13, and four novel allergens were identified. The cDNAs coding for Pen c 32 and Pen c 30 were cloned using designed primers based on nanoLC-MS/MS analysis. The amino acid sequences of Pen c 32 and Pen c 30 were, respectively, found to have extensive similarity with those of pectate lyases and catalases from various fungi. Native Pen c 30 was shown to have catalase activity and to bind to serum IgE from 48% of mold-allergic patients and induced immediate type skin reactions in a sensitized patient. Here, we present a proteome approach which resulted in the identification of four novel secreted allergens. These novel allergens might be useful in allergy diagnosis and in the treatment of mold-allergic disorders. [source] | |||||||||||||