Molecular allergy diagnosis is sensitive and avoids misdiagnosis in patients sensitized to seasonal allergens.

BACKGROUND: The specificity of extract-based pollen allergy diagnosis is decreased due to cross-reactivity via cross-reactive carbohydrate determinants (CCDs) or panallergens such as profilins or polcalcins. This study aimed to explore the prevalence of sensitization to seasonal extracts, CCDs, profilin and polcalcin and investigate the sensitivity and specificity of seasonal molecular allergy diagnosis (MAD) using commercially available test methods. METHODS: 2948 patients were screened for specific immunoglobulin E to ash, birch, mugwort, ragweed and timothy grass pollen extracts and grouped according to the number of positive tests (1-5). 100 patients from each group and a control group were randomly selected to calculate the prevalence of CCD and panallergen sensitization. With 742 patients, sensitivity and specificity of MAD (Alt a 1, Fra/Ole e 1, Bet v 1, Phl p 1, Art v 1, and Amb a 1) was determined. RESULTS: 1627 patients (55.2%) were positive to at least one, and 1002 patients (34.0%) were positive to multiple of the five pollen allergens investigated; 18.5% of the pollen-sensitized patients had sensitization to CCDs or panallergens. Specifically, sensitization to CCDs, profilins, and polcalcins was observed in 8.7%, 10.9%, and 2.9% of these patients, respectively. The sensitivity of MAD was high, with sensitivities between 96.2% and 100% using ImmunoCAP and 91.5% and 100% using ALEX2 . Specificity was 100% for both assays. CONCLUSIONS: Due to cross-reactivity, about one-fifth of pollen-sensitized patients is at risk of misdiagnosis. However, MAD is sensitive, specific and helps to avoid misdiagnosis and select primary allergen sources for immunotherapy.

Identification of Potentially Tolerated Fish Species by Multiplex IgE Testing of a Multinational Fish-Allergic Patient Cohort.

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.

One Year in the Extreme Isolation of Antarctica-Is This Enough to Modulate an "Allergic" Sensitization?

(1) Background: After spending a year wintering in Antarctica, individual expedition members have reported increased or even new allergic reactions to environmental allergens after their return. (2) Methods: Blood samples from five overwintering crews were analyzed using the chip based multiplex ALEX Allergy Explorer (MacroArray Diagnostics GmbH, Austria). (3) Results: About one third of the 39 participants displayed specific IgEs against pollen. In most individuals, kinetics showed a reduction in the specific IgE at the time about nine months after deployment to Antarctica. Five participants had the highest specific IgE levels after returning to the "normal" world. The examination of the specific IgE relative to house dust mites and storage mites showed different kinetics. Six out of 10 had the highest specific IgE concentrations at the inner Antarctic measurement time point. These data corresponded well to the general situation in the stations. At the stations themselves, there were almost no pollen particle load, especially at Concordia. (4) Conclusions: Antarctic long-term confinement can induce an altered immune function, which is in some individuals pronounced after return to the familiar allergen environment. Future prospective studies in larger cohorts are needed to further specify these first results.

IgE reactivity patterns in Asian and central European cockroach-sensitized patients reveal differences in primary sensitizing allergen sources.

Background: The prevalence of cockroach (CR) sensitization and its relevance as a trigger of allergy symptoms differs greatly in different geographic areas. Objective: This study aimed to compare molecular IgE reactivity profiles in CR-sensitized patients with perennial allergy symptoms from Hong Kong (HK) and Austria and identify the main primary sensitizers. Methods: IgE sensitization was assessed by skin prick test and/or IgE reactivity with CR extract. Molecular IgE reactivity profiles were analyzed via multiplex assay for sensitization to allergens and extracts from CR, house dust mite (HDM), shellfish, and 3 additional insect species. Results: HDM was the main primary sensitizer in both cohorts. In the HK group, genuine sensitization to CR was found in 45%, but none of the patients in the Austrian cohort was truly sensitized to that allergen source. Most patients from HK were cross-sensitized to other insects and/or shellfish, presumably by broad reactivity to tropomyosin and arginine kinase. About half of Austrian subjects lacked IgE to these pan-allergens, indicating co- but not cross-sensitization to insects and/or shellfish. Regarding IgE recognition frequencies, arginine kinases (64% HK, 10% Austria) and tropomyosins (42% HK, 15% Austria) were most frequently recognized; Bla g 4 (lipocalin) was detected in HK patients only (42%). Tropomyosin (Per a 7) was significantly more frequently recognized in patients with asthma. Sera from HDM-sensitized subjects from HK showed a higher proportion of sensitization to minor mite allergens. Conclusion: Molecular profiling identified differences between CR-sensitized allergic patients from HK and Austria in terms of primary sensitizers and molecular IgE reactivity patterns. Tropomyosin from American cockroach (Per a 7) was shown to be significantly associated with asthma symptoms and might be suitable as biomarker for more severe respiratory allergy symptoms.

Mite Molecular Profile in the Th2-Polarized Moderate-to-Severe Persistent Asthma Endotype Subjected to High Allergen Exposure.

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.

Irreversible inactivation of ISG15 by a viral leader protease enables alternative infection detection strategies.

In response to viral infection, cells mount a potent inflammatory response that relies on ISG15 and ubiquitin posttranslational modifications. Many viruses use deubiquitinases and deISGylases that reverse these modifications and antagonize host signaling processes. We here reveal that the leader protease, Lbpro, from foot-and-mouth disease virus (FMDV) targets ISG15 and to a lesser extent, ubiquitin in an unprecedented manner. Unlike canonical deISGylases that hydrolyze the isopeptide linkage after the C-terminal GlyGly motif, Lbpro cleaves the peptide bond preceding the GlyGly motif. Consequently, the GlyGly dipeptide remains attached to the substrate Lys, and cleaved ISG15 is rendered incompetent for reconjugation. A crystal structure of Lbpro bound to an engineered ISG15 suicide probe revealed the molecular basis for ISG15 proteolysis. Importantly, anti-GlyGly antibodies, developed for ubiquitin proteomics, are able to detect Lbpro cleavage products during viral infection. This opens avenues for infection detection of FMDV based on an immutable, host-derived epitope.

Interaction of 2A proteinase of human rhinovirus genetic group A with eIF4E is required for eIF4G cleavage during infection.

In enteroviruses, the inhibition of protein synthesis from capped host cell mRNA is catalyzed by the virally encoded 2A proteinase (2Apro), which cleaves eukaryotic initiation factors (eIF) 4GI and 4GII. Despite much investigation, the exact mechanism of 2Apro cleavage remains however unclear. Here, we identify the domains responsible for the eIF4E/HRV2 2Apro interaction using molecular modelling and describe mutations that impair this interaction and delay in vitro cleavage of eIF4G isoforms. Furthermore, we produced HRV1A viruses bearing the mutation L17R, Y32A or Y86A in the 2Apro sequence. All three viruses showed reduced yield and were appreciably delayed during infection in eIF4GI cleavage. Thus, we propose for genetic group A HRVs that the eIF4E/2Apro interaction is essential for successful viral replication. In contrast, HRV4 2Apro and coxsackievirus B4 2Apro failed to form complexes with eIF4E, suggesting that the mechanism of eIF4G isoform cleavage in these and related viruses is different.

NMR analysis of the interaction of picornaviral proteinases Lb and 2A with their substrate eukaryotic initiation factor 4GII.

Messenger RNA is recruited to the eukaryotic ribosome by a complex including the eukaryotic initiation factor (eIF) 4E (the cap-binding protein), the scaffold protein eIF4G and the RNA helicase eIF4A. To shut-off host-cell protein synthesis, eIF4G is cleaved during picornaviral infection by a virally encoded proteinase; the structural basis of this reaction and its stimulation by eIF4E is unclear. We have structurally and biochemically investigated the interaction of purified foot-and-mouth disease virus (FMDV) leader proteinase (Lb(pro)), human rhinovirus 2 (HRV2) 2A proteinase (2A(pro)) and coxsackievirus B4 (CVB4) 2A(pro) with purified eIF4GII, eIF4E and the eIF4GII/eIF4E complex. Using nuclear magnetic resonance (NMR), we completed (13)C/(15) N sequential backbone assignment of human eIF4GII residues 551-745 and examined their binding to murine eIF4E. eIF4GII551-745 is intrinsically unstructured and remains so when bound to eIF4E. NMR and biophysical techniques for determining stoichiometry and binding constants revealed that the papain-like Lb(pro) only forms a stable complex with eIF4GII(551-745) in the presence of eIF4E, with KD values in the low nanomolar range; Lb(pro) contacts both eIF4GII and eIF4E. Furthermore, the unrelated chymotrypsin-like 2A(pro) from HRV2 and CVB4 also build a stable complex with eIF4GII/eIF4E, but with K(D) values in the low micromolar range. The HRV2 enzyme also forms a stable complex with eIF4E; however, none of the proteinases tested complex stably with eIF4GII alone. Thus, these three picornaviral proteinases have independently evolved to establish distinct triangular heterotrimeric protein complexes that may actively target ribosomes involved in mRNA recruitment to ensure efficient host cell shut-off.

Specificity of human rhinovirus 2A(pro) is determined by combined spatial properties of four cleavage site residues.

The 2A proteinase (2A(pro)) of human rhinoviruses cleaves the virally encoded polyprotein between the C terminus of VP1 and its own N terminus. Poor understanding of the 2A(pro) substrate specificity of this enzyme has hampered progress in developing inhibitors that may serve as antiviral agents. We show here that the 2A(pro) of human rhinovirus (HRV) 1A and 2 (rhinoviruses from genetic group A) cannot self-process at the HRV14 (a genetic group B rhinovirus) cleavage site. When the amino acids in the cleavage site of HRV2 2A(pro) (Ile-Ile-Thr-Thr-Ala*Gly-Pro-Ser-Asp) were singly or doubly replaced with the corresponding HRV14 residues (Asp-Ile-Lys-Ser-Tyr*Gly-Leu-Gly-Pro) at positions from P3 to P2', HRV1A and HRV2 2A(pro) cleavage took place at WT levels. However, when three or more positions of the HRV1A or 2 2A(pro) were substituted (e.g. at P2, P1 and P2'), cleavage in vitro was essentially eliminated. Introduction of the full HRV14 cleavage site into a full-length clone of the HRV1A and transfection of HeLa cells with a transcribed RNA did not give rise to viable virus. In contrast, revertant viruses bearing cysteine at the P1 position or proline at P2' were obtained when an RNA bearing the three inhibitory amino acids was transfected. Reversions in the enzyme affecting substrate specificity were not found in any of the in vivo experiments. Modelling of oligopeptide substrates onto the structure of HRV2 2A(pro) revealed no appreciable differences in residues of HRV2 and HRV14 in the respective substrate binding sites, suggesting that the overall shape of the substrate is important in determining binding efficiency.