Background: Many fish-allergic patients only react to certain fish species and may tolerate others, mostly because of IgE-mediated recognition of specific epitopes on the major allergen parvalbumin. However, the considerable number of fish species consumed makes it challenging to identify which species are allergenic and which are tolerated by individual patients. Objective: In order to improve the diagnosis of fish allergy, we investigated IgE-mediated reactivity to parvalbumins from 12 freshwater fish species that are largely underrepresented in diagnostic tests. Methods: Parvalbumins were purified from 12 freshwater fish species belonging to 8 families, and their isoform composition was analyzed using mass spectrometry. IgE specific for each parvalbumin was quantified in serum samples from 66 fish-allergic individuals, and basophil activation tests were performed for 5 patients. Crosswise inhibition assays were carried out for all parvalbumins for 7 patients to investigate cross-reactivity between the parvalbumins from the different species. Results: IgE binding and cross-linking potency of the parvalbumins differed, with the strongest reactivities observed for 4 parvalbumins from the salmonid family (results positive for 89%-95% of patients) and the weakest for parvalbumins from Wels catfish, European eel, and tench (results negative for ≥50% of patients). Ninety percent of the patients with negative results for Wels catfish parvalbumin also had negative results for additional parvalbumins from multiple species. Inhibition assays revealed variable recognition of epitopes by several patients, with the primary sensitizers most frequently being parvalbumins from salmonids and percids. Conclusion: Including freshwater salmonids in the diagnostic work-up for fish allergy may help to identify most fish-allergic patients. IgE to Wels catfish could help distinguish between polysensitized and oligosensitized patients.

The ALEX2-test (MacroArray Diagnostics, Vienna, Austria) is a diagnostic multiplex IgE-test for the simultaneous detection of IgE to 178 allergens and 117 extracts, in addition to total IgE. Test results from more than 90 countries are stored on a GDPR-compliant cloud server for backup, customer support, and continuous postmarket surveillance. To improve the coverage of exposomes on a global scale and to further increase the sensitivity of the test, the allergen panel was updated from ALEX2 to ALEX3. By mid-2023, when ALEX3 was designed, almost 400,000 real-world ALEX2 test results were available. Analysing prevalences and average sIgE-levels of individual allergen preparations, coverage of extracts by components, and co-reactivity of members of the same allergen family provided a rationale for updating the array. In parallel, based on the scientific literature and clinical studies, new allergens were selected. On ALEX3, 218 allergens and 82 extracts will be represented, including 52 new allergens. Allergen preparations with low prevalence and clinical relevance, as well as redundant allergens and extracts, were discontinued. New allergens encompass, e.g., cyclophilins, alpha-gal, and additional markers from respiratory and food allergen sources. Using a large dataset of ALEX2 test results exemplifies the targeted, data-driven improvement of a diagnostic IgE-macroarray.

Background: Recent studies indicated that fish-allergic patients may safely consume certain fish species. Multiplex IgE testing facilitates the identification of species tolerated by individual patients. Methods: Sera were collected from 263 fish-allergic patients from Austria, China, Denmark, Luxembourg, Norway and Spain. Specific (s) IgE to parvalbumins (PVs) from 10 fish species along with IgE to 7 raw and 6 heated fish extracts was quantified using a research version of the ALEX 2 assay. IgE-signatures of individual patients and patient groups were analyzed using SPSS and R. Results: sIgE to alpha-PV from ray, a cartilaginous fish, was not detected in 78% of the patients while up to 41% of the patients, depending on their country of origin, tested negative for at least one beta-PV. sIgE values were highest for mackerel and tuna PVs (>10 kUA/L) and significantly lower for cod (4.9 kUA/L) and sole PVs (2.55 kUA/L). 17% of the patients, although negative for PVs, tested positive for the respective fish extracts. Based on the absence of IgE to PVs and extracts, up to 21% of the patients were identified as potentially tolerating one or more bony fish. Up to 90% of the patients tested negative for ray. The probability of negativity to one fish based on negativity to others was calculated. Negativity to tuna and mackerel emerged as a good marker of negativity to additional bony fish. Conclusion: Measuring sIgE to PVs and extracts from evolutionary distant fish species indicates bony and cartilaginous fish species for tolerance-confirming food challenges.

With great interest, we read the controversy between González-Pérez et al., 2022 González-Pérez R. Poza-Guedes P. Pineda F. Castillo M. Matheu V. Mederos-Luis E. et al. Atopic dermatitis and frequency of Der p 11 binding as a major allergen revisited. J Invest Dermatol. 2022; 142: 721-723 Abstract Full Text Full Text PDF PubMed Scopus (2) Google Scholar , challenging the supposedly elevated prevalence of IgE sensitization to Der p 11 from the house dust mite species Dermatophagoides pteronyssinus in patients with atopic dermatitis (AD), and Gattinger et al., 2022 Gattinger P. Huang H.J. Valenta R. Vrtala S. Response to González-Pérez et al. J Invest Dermatol. 2022; 142: 723-726 Abstract Full Text Full Text PDF PubMed Scopus (1) Google Scholar , scrutinizing the data published by González-Pérez. However, in our opinion, Gattinger's response conveys several disputable claims and possibly misleading designations that might cause a biased view on the actual relevance of Der p 11 as a surrogate marker for severe AD and on technical aspects of the two multiplex IgE tests that were used by the authors, which we believe deserve to be commented. Timely Wound Healing Is Dependent on Endothelial but Not on Hair Follicle Stem Cell Toll-Like Receptor 2 SignalingJournal of Investigative DermatologyVol. 142Issue 11PreviewAs a part of innate immunity, toll-like receptor 2 (TLR2) plays an important function in most defensive responses of the organism, including but not limited to infections. Cutaneous injury, one of the most common challenges for mammals, mobilizes a number of cell types, including epithelial, immune, and vascular cells, for timely tissue repair. However, in contrast to immune cells, little is known about TLR2 function on nonimmune cells during skin regeneration. In this study, we used two tissue-specific conditional Tlr2-knockout mouse lines to address the effects of TLR2 in endothelial and hair follicle stem cells (HFSCs) on cutaneous wound healing. Full-Text PDF Open ArchiveAtopic Dermatitis and Frequency of Der p 11 Binding as a Major Allergen RevisitedJournal of Investigative DermatologyVol. 142Issue 3PreviewType 2 inflammation plays a central role in the pathogenesis of atopic dermatitis (AD) encompassing a specific CD4+ T helper type (Th) 2 cell response and the activation of other T-cell lineages, such as Th1, Th17/IL-23, and Th22 (Bakker et al., 2021). House dust mites (HDMs) allergens are likely the most relevant airborne allergen for AD in sensitized and genetically predisposed individuals worldwide (Calderón et al., 2015; Thomas, 2010). Accurate knowledge of the HDM allergens in the patient's environment turns out mandatory in the diagnosis and prescription of effective allergen immunotherapy (Bumbacea et al., 2020; González-Pérez and Poza-Guedes, 2021; González-Pérez et al., 2021). Full-Text PDF Open Archive

We aimed to identify the molecular profile and distribution patterns of allergens from House Dust Mites (HDM) and Storage Mites (SM) in a selected population of children with persistent moderate-to-severe asthma. We selected non-consecutive pediatric patients sensitized to any of the following mites Dermatophagoides pteronyssinus, Acarus siro, Blomia tropicalis, Lepidoglyphus destructor, Glyciphagus domesticus, and Tyrophagus putrescentiae with moderate to severe persistent asthma according to the GINA guidelines. Skin testing (SPT) with mite extracts, specific IgE (sIgE) evaluation using Precision allergy molecular diagnostic applications (PAMD@) -ALEX MacroArray Diagnostics, Austria- including 15 allergens and a Western Blot immunoassay were performed. All 22 (mean age 16 y.o.) patients (15 with moderate asthma and 7 with severe asthma) showed a positive response to the selected panel of mites. The mean of total IgE and absolute eosinophils were above 100 IU/mL and 300 Eos/mm3. Group 1, 2 allergens were identified in all subjects regardless of their asthma severity. Der p 23 and group 5 allergens were also present in all patients. The cluster analysis of mite allergens included >15 distinct individual profiles. Detected mean sIgE levels were significantly (p<.05) higher for HDM in respect to SM. A highly pleomorphic aggregation of mite molecules recognized by IgE was identified in a selected cohort of paediatric moderate-to-severe asthmatics. PAMD@ promotes insight in the complexity of interactions among mite species -including both HDM and SM- in childhood T2 asthma.

BACKGROUND:Although recent studies indicated that many fish-allergic patients may safely consume certain fish species, no clinical guidelines are available for identification of the exact species tolerated by specific patients.OBJECTIVE:To investigate whether multiplex immunoglobulin E (IgE) testing reveals potentially tolerated fish through absence of IgE to parvalbumin (PV) and extracts from specific species.METHODS:Sera from 263 clinically well-defined fish-allergic patients from Austria, China, Denmark, Luxembourg, Norway, and Spain were used in a research version of the ALEX2 multiplex IgE quantification assay. Specific IgE to PVs from 10 fish species (9 bony and 1 cartilaginous), and to extracts from 7 species was quantified. The IgE signatures of individual patients and patient groups were analyzed using SPSS and R.RESULTS:Up to 38% of the patients were negative to cod PV, the most commonly used molecule in fish allergy diagnosis. Forty-five patients (17%) tested negative to PVs but positive to the respective fish extracts, underlining the requirement for extracts for accurate diagnosis. Between 60% (Spain) and 90% (Luxembourg) of the patients were negative to PV and extracts from ray, a cartilaginous fish, indicating its potential tolerance. Up to 21% of the patients were negative to at least 1 bony fish species. Of the species analyzed, negativity to mackerel emerged as the best predictive marker of negativity to additional bony fish, such as herring and swordfish.CONCLUSIONS:Parvalbumins and extracts from multiple fish species relevant for consumption should be used in fish-allergy diagnosis, which may help identify potentially tolerated species for individual patients.

Sensitization profile to house dust mites (HDM) in respiratory allergy may differ depending on specific geographical areas. The role of mites is controversial concerning type 2 inflammation in asthma. The present study aims to characterize the immunological pattern in a selected asthma population focusing on their HDM sensitization. We selected 45 non-consecutive patients sensitized to Dermatophagoides pteronyssinus (D. pteronyss.) with moderate-severe persistent asthma according to the GINA Guidelines. Skin prick test (SPT) with standardized extracts of D. pteronyss. Serum blood samples were obtained from all participating subjects. Total IgE and sIgE including a custom-made panel of 9 HDM allergens (Der p 1, Der p 2, Der p 5, Der p 7, Der p 10, Der p 11, Der p 20 Der p 21, and Der p 23) were quantified. All 45 asthmatic subjects showed a positive SPT to D. pteronyss. with different molecular sensitization patterns. Median total IgE and absolute eosinophils were 898 UI/mL and 400 Eos/mm3 respectively. Regarding major allergens (Der p 1, Der p 2 and Der p 23), the median titers of sIgE to both Der p 2 (26.19 kU/L) and Der p 23 (21.81 kU/L) were higher to Der p 1 (18.96 kU/L), while mid-tier allergens (Der p 5, Der p 7 and Der p21) reached serodominance (>50%) throughout all samples. Both major and mid-tier HDM allergens endorsed serodominance in the current type-2 inflammation asthma population. The putative role of these serominant allergens in the pathobiology of this moderate-severe asthma phenotype is yet to be determined.

The relevance of storage mites (SM) in the pathogenesis of respiratory allergy remains uncertain. We sought to investigate the serological molecular profile of a selected asthma population with a high environmental exposure focusing on SM sensitization. We selected non-consecutive patients sensitized to SM (i.e. Blomia tropicalis, Lepidoglyphus destructor, Glycyphagus domesticus and Tyrophagus putrescentiae) with moderate-severe persistent asthma according to the GINA Guidelines. Skin prick test (SPT) to SM and serum samples were obtained from all subjects. Total IgE and sIgE including a comprehensive panel of 6 allergens (Blo t 5, Blo t 10, Blo t 21, Lep d 2, Gly d 2 and Tyr p 2) were quantified. Forty-five asthmatic subjects showed at least a positive SPT to any of the above mentioned SM with different molecular sensitization patterns. Median serum total IgE was 898 UI/mL. Regarding major allergens, median titers of group 2 allergens sIgE Lep d 2 (11.74 kU/L), Gly d 2 (9.67 kU/L) were higher than Blo t 5 (6.96 kU/L) and Blo t 21 (7.95 kU/L). Lep d 2, Gly d 2 and Tyr p 2) and Blo t 5 were serodominant throughout all samples Group 2 along with Blo t 5 and Blo t 21 allergens from Lepidoglyphus destructor, Glyciphagus domesticus, Tyrophagus putrescentiae and Blomia tropicalis, showed serodominance in the selected asthma population. Clinical and molecular cross-reactivitity among mite species needs to be considered in terms of a genuine diagnosis and a customized therapy.

Background: The association among the IgE responses to prevailing groups of house dust mite (HDM) allergens in the concurrent asthma phenotypes has not been determined. Objective: The aim of the present study lays on a component-resolved diagnosis (CRD) model to investigate the mite molecular signature in subjects with type-2 inflammation asthma. Methods: We selected patients showing a clinically relevant sensitization to HDMs with moderate-to-severe persistent asthma. Skin prick test (SPT) with standardized mite extracts, a broad customized CRD serum sIgE panel including 9 Dermatophagoides pteronyssinus allergens and the related protein allergenic characterization, was investigated in all serum samples. Results: Ninety out of 93 (96.77%) patients with a positive SPT to HDM showed a concordant sIgE (≥0.35 kUA/L) to the crude extract of D. pteronyssinus. Major allergens (Der p 2, Der p 23, and Der p 1) were present in >70% of all subjects, with mid-tier allergens (Der p 5, Der p 7, and Der p 21) reaching up to 51% in the present cohort. A complex pleomorphic repertoire of HDM molecules recognized by IgE was depicted, including 38 distinct profiles. Conclusions and Clinical Relevance: The proposed CRD panel approach, containing the most prevalent HDM allergens, appeared to be sufficient to obtain a precise D. pteronyssinus molecular diagnosis in asthmatics with a climate-dependent high-mite allergen exposure and coexisting sensitization. A dominant role of both major and mid-tier allergens has been confirmed in moderate and severe persistent asthmatics with the preponderant Th2-high endotype.

The objective of this analysis was to compare the multiplex ImmunoCAP ISAC (Thermo Fisher Scientific, Sweden) and the multiplex Alex Allergy Explorer (Macro Array Diagnostics GmbH, Austria) platform on specific IgE to grass pollen (Phl p 1, Phl p 5), tree pollen (Bet v 1), house dust mites (Der p 1, Der p 2) and cat (Fel d 1) allergens in allergic patients. Our findings demonstrate a good correlation of presently used methods to detect serum sIgE. Multiplex testing of allergen-specific IgE can be the method of choice for a prospective component-resolved diagnosis of type I allergy, and the basis for the design and monitoring of a patient-tailored specific therapy.

Die Verfügbarkeit von Einzelallergenen und deren Einsatz in der „Mikroarray“-Technologie gestattet die simultane Bestimmung von sIgE im Multiplex-Verfahren gegen eine Vielzahl unterschiedlicher Allergene (> 100) aus kleinsten Serummengen. So lassen sich in einer Bestimmung umfangreiche individuelle Sensibilisierungsprofile erstellen. Sie ermöglichen in Zusammenschau mit der Anamnese, Kreuzreaktionen leichter zu erkennen, das Risiko für schwere Reaktionen besser einzuschätzen und die Indikation zur spezifischen Immuntherapie besonders bei Polysensibilisierten gezielter zu stellen.

The availability of single allergens and their use in microarray technology enables the simultaneous determination of specific IgE (sIgE) to a multitude of different allergens (> 100) in a multiplex procedure requiring only minute amounts of serum. This allows extensive individual sensitization profiles to be determined from a single analysis. Combined with a patient’s medical history, these profiles simplify identification of cross-reactivity; permit a more accurate estimation of the risk of severe reactions; and enable the indication for specific immunotherapy to be more precisely established, particularly in cases of polysensitization. Strictly speaking, a multiplex assay is not a single test, but instead more than 100 simultaneous tests. This places considerable demands on the production, quality assurance, and interpretation of data. The following chapter describes the multiplex test systems currently available and discusses their characteristics. Performance data are presented and the sIgE values obtained from multiplex and singleplex assays are compared. Finally, the advantages and limitations of molecular allergy diagnostics using multiplex assays in clinical routine are discussed, and innovative possibilities for clinical research are described. The multiplex diagnostic tests available for clinical routine have now become well established. The interpretation of test results is demanding, particularly since all individual results need to be checked for their plausibility and clinical relevance on the basis of previous history (patient history, clinical symptoms, challenge test results). There is still room for improvement in certain areas, for example with respect to the overall test sensitivity of the method, as well as the availability and quality of particular allergens. The current test systems are just the beginning of a continuous development that will influence and most likely change clinical allergology in the coming years.

The availability of single allergens and their use in microarray technology enables the simultaneous determination of specific IgE (sIgE) to a multitude of different allergens (> 100) in a multiplex procedure requiring only minute amounts of serum. This allows extensive individual sensitization profiles to be determined from a single analysis. Combined with a patient's medical history, these profiles simplify identification of cross-reactivity; permit a more accurate estimation of the risk of severe reactions; and enable the indication for specific immunotherapy to be more precisely established, particularly in cases of polysensitization. Strictly speaking, a multiplex assay is not a single test, but instead more than 100 simultaneous tests. This places considerable demands on the production, quality assurance, and interpretation of data. The following chapter describes the multiplex test systems currently available and discusses their characteristics. Performance data are presented and the sIgE values obtained from multiplex and singleplex assays are compared. Finally, the advantages and limitations of molecular allergy diagnostics using multiplex assays in clinical routine are discussed, and innovative possibilities for clinical research are described. The multiplex diagnostic tests available for clinical routine have now become well established. The interpretation of test results is demanding, particularly since all individual results need to be checked for their plausibility and clinical relevance on the basis of previous history (patient history, clinical symptoms, challenge test results). There is still room for improvement in certain areas, for example with respect to the overall test sensitivity of the method, as well as the availability and quality of particular allergens. The current test systems are just the beginning of a continuous development that will influence and most likely change clinical allergology in the coming years.

Profilin from wheat (Triticum aestivum) has been identified as an allergen (Tri a 12). The recombinant 14 kDa protein was produced in Escherichia coli, purified and crystallized using the hanging-drop vapour-diffusion method. A diffraction-data set was collected in-house from a single crystal to a resolution of 3.3 angstrom. The crystals belonged to space group P3(2)21, with unit-cell parameters a = b = 58.9 angstrom, c = 82.5 angstrom, alpha= beta = 90 degrees and gamma = 120 degrees. (doi: 10.5562/cca1790)