Validation of a commercial allergen microarray platform for specific immunoglobulin E detection of respiratory and plant food allergens.

BACKGROUND: As the use of multiplex-specific immunoglobulin E (sIgE) detection methods becomes increasingly widespread, proper comparative validation assessments of emerging new platforms are vital. OBJECTIVE: To evaluate the clinical and technical performance of a newly introduced microarray platform, Allergy Explorer (ALEX) (MacroArray Diagnostics), in the diagnosis of pollen (cypress, grass, olive), dust mite (Dermatophagoides pteronyssinus), mold (Alternaria alternata), fruit (apple, peach), and nut (walnut, hazelnut and peanut) allergies and to compare it with those of the ImmunoCAP Immuno Solid-phase Allergen Chip (ISAC) 112 microarray and the ImmunoCAP singleplex method (ThermoFisher Scientific). METHODS: We enrolled 153 patients with allergy and 16 controls without atopy. The sIgE assays were conducted using ISAC112, ALEX version 2 (ALEX2), and ImmunoCAP for whole extracts and major components. Technical validation of ALEX2 was performed by measuring repeatability and interassay, interbatch, and interlaboratory reproducibility. RESULTS: When measured globally (detection by 1 or more allergen components), ALEX2 had adequate sensitivity and specificity for most of the allergens studied, comparable in general with that of ISAC112 (except for olive pollen and walnut) and similar to that of ImmunoCAP whole extract measurements. Component-by-component analysis revealed comparable results for all techniques, except for Ole e 1 and Jug r 3, in both ISAC112 and ImmunoCAP comparisons, and Alt a 1, when compared with ISAC112. Continuous sIgE levels correlate with sIgE by ImmunoCAP. Good reproducibility and repeatability were observed for ALEX2. CONCLUSION: ALEX2 has sound technical performance and adequate diagnostic capacity, comparable in general with that of ISAC112 and ImmunoCAP.

Profilin-mediated food-induced allergic reactions are associated with oral epithelial remodeling.

BACKGROUND: In areas of high exposure to grass pollen, allergic patients are frequently sensitized to profilin, and some experience severe profilin-mediated food-induced reactions. This specific population of patients is ideal to study the relationship between respiratory and food allergies. OBJECTIVE: We sought to determine the role of oral mucosal epithelial barrier integrity in profilin-mediated allergic reactions. METHODS: Thirty-eight patients with profilin allergy stratified into mild or severe according to their clinical history and response to a profilin challenge test and 6 nonallergic subjects were recruited. Oral mucosal biopsies were used for measurement of CD11c, CD3, CD4, tryptase, claudin-1, occludin, E-cadherin, and vascular endothelial growth factor A levels; Masson trichrome staining; and POSTN, IL33, TPSAB, TPSB, and CMA gene expression analysis by using quantitative RT-PCR. Blood samples were used for basophil activation tests. RESULTS: Distinct features of the group with severe allergy included the following: (1) impaired epithelial integrity with reduced expression of claudin-1, occludin, and E-cadherin and decreased numbers of epithelial cells, which is indicative of acanthosis, higher collagen deposition, and angiogenesis; (2) inflammatory immune response in the mucosa, with an increased number of CD11c+ and CD4+ infiltrates and increased expression of the cytokine genes POSTN and IL33; and (3) a 10-fold increased sensitivity of basophils to profilin. CONCLUSIONS: Patients with profilin allergy present with significant damage to the oral mucosal epithelial barrier, which might allow profilin penetration into the oral mucosa and induction of local inflammation. Additionally, severely allergic patients presented with increased sensitivity of effector cells.

Type C feruloyl esterase from Aspergillus ochraceus: a butanol specific biocatalyst for the synthesis of hydroxycinnamates in a ternary solvent system

Background: Aspergillus ochraceus was isolated from coffee pulp and selected as an interesting hydroxycinnamoyl esterase strain producer, using an activity microplate high-throughput screening method. In this work, we purified and characterized a new type C A. ochraceus feruloyl esterase (AocFaeC), which synthesized specifically butyl hydroxycinnamates in a ternary solvent system. Results: AocFaeC was produced by solid state fermentation, reaching its maximal activity (1.1 U/g) after 48 h of culture. After purification, the monomeric protein (34 kDa) showed a specific activity of 57.9 U/mg towards methyl ferulate. AocFaeC biochemical characterization confirmed its identity as a type C feruloyl esterase and suggested the presence of a catalytic serine in the active site. Its maximum hydrolytic activity was achieved at 40°C and pH 6.5 and increased by 109 and 77% with Ca2+ and Mg2+, but decreased by 90 and 45% with Hg2+ and Cu2+, respectively. The initial butyl ferulate synthesis rate increased from 0.8 to 23.7 nmol/min after transesterification condition improvement, using an isooctane:butanol:water ternary solvent system, surprisingly the synthesis activity using other alcohols was negligible. At these conditions, the synthesis specific activities for butyl p-coumarate, sinapinate, ferulate, and caffeate were 87.3, 97.6, 168.2, and 234 U/µmol, respectively. Remarkably, AocFaeC showed 5 folds higher butyl caffeate synthesis rate compared to type B Aspergillus niger feruloyl esterase, a well-known enzyme for its elevated activity towards caffeic acid esters. Conclusions: Type C feruloyl esterase from A. ochraceus is a butanol specific biocatalyst for the synthesis of hydroxycinnamates in a ternary solvent system