Peanut lipids influence the response of bronchial epithelial cells to the peanut allergens Ara h 1 and Ara h 2 by decreasing barrier permeability.

Background: Peanut-allergic individuals react upon their first known ingestion of peanuts, suggesting sensitization occurs through non-oral exposure. Increasing evidence suggests that the respiratory tract is a probable site for sensitization to environmental peanuts. However, the response of the bronchial epithelium to peanut allergens has never been explored. Furthermore, food matrix-derived lipids play an important role in allergic sensitization. Objective: To contribute to a better understanding of the mechanisms of allergic sensitization to peanuts via inhalation, by exploring the direct effect of the major peanut allergens Ara h 1 and Ara h 2 and peanut lipids on bronchial epithelial cells. Methods: Polarized monolayers of the bronchial epithelial cell line 16HBE14o- were stimulated apically with peanut allergens and/or peanut lipids (PNL). Barrier integrity, transport of allergens across the monolayers, and release of mediators were monitored. Results: Ara h 1 and Ara h 2 impacted the barrier integrity of the 16HBE14o- bronchial epithelial cells and crossed the epithelial barrier. Ara h 1 also induced the release of pro-inflammatory mediators. PNL improved the barrier function of the cell monolayers, decreased paracellular permeability and reduced the amount of allergens crossing the epithelial layer. Conclusion: Our study provides evidence of the transport of Ara h 1 and Ara h 2 across the airway epithelium, of the induction of a pro-inflammatory milieu, and identifies an important role for PNL in controlling the amount of allergens that can cross the epithelial barrier. These, all together, contribute to a better understanding of the effects of peanuts exposure on the respiratory tract.

Pru du 1, the Bet v 1-homologue from almond, is a major allergen in patients with birch pollen associated almond allergy.

Background: Almond allergy is common and can manifest in two different forms. Primary almond allergy has been reported to be associated with sensitization to almond legumin Pru du 6. In birchendemic regions, there is a link between birch-pollinosis which is likely based on a cross-reactive Bet v 1 homologue, a yet unidentified allergen in almond. Therefore, we sought to identify and characterize a Bet v 1-homologue in almond. Methods: The expression of a Bet v 1 homologue in almond kernels was confirmed by mass spectrometry. The recombinant protein was produced in Escherichia coli and its cross-reactivity and allergenic potency was analyzed by IgE quantitative and competitive ELISA, immunoblotting and basophil histamine release using sera from 17 almond allergic patients. Results: The identified Bet v 1 homologue received the designation Pru du 1.0101. Pru du 1.0101 bound IgE from 82 % of almond allergic patients. Bet v 1 was able to inhibit IgE-binding to rPru du 1 by 100%, while rPru du 1 inhibited IgE binding to rBet v 1 by 48%. Pru du 1.0101 activated basophils, though 100- to 1000-fold higher concentrations were required for maximum activation in comparison to rBet v 1. Conclusion: Considering the strong inhibition capacity and higher allergenic potency of Bet v 1, the results provide compelling evidence for primary sensitization to Bet v 1 in case of birch pollen associated almond allergy. Combining Pru du 6 and Pru du 1 in diagnostic approaches may help to discriminate between primary and birch-pollen associated almond allergy.

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.

The Major Peanut Allergen Ara h 2 Produced in Nicotiana benthamiana Contains Hydroxyprolines and Is a Viable Alternative to the E. Coli Product in Allergy Diagnosis.

Peanut allergy is a potentially life-threatening disease that is mediated by allergen-specific immunoglobulin E (IgE) antibodies. The major peanut allergen Ara h 2, a 2S albumin seed storage protein, is one of the most dangerous and potent plant allergens. Ara h 2 is posttranslationally modified to harbor four disulfide bridges and three hydroxyprolines. These hydroxyproline residues are required for optimal IgE-binding to the DPYSPOHS motifs representing an immunodominant IgE epitope. So far, recombinant Ara h 2 has been produced in Escherichia coli, Lactococcus lactis, Trichoplusia ni insect cell, and Chlamydomonas reinhardtii chloroplast expression systems, which were all incapable of proline hydroxylation. However, molecular diagnosis of peanut allergy is performed using either natural or E. coli-produced major peanut allergens. As IgE from the majority of patients is directed to Ara h 2, it is of great importance that the recombinant Ara h 2 harbors all of its eukaryotic posttranslational modifications. We produced hydroxyproline-containing and correctly folded Ara h 2 in the endoplasmic reticulum of leaf cells of Nicotiana benthamiana plants, using the plant virus-based magnICON® transient expression system with a yield of 200 mg/kg fresh biomass. To compare prokaryotic with eukaryotic expression methods, Ara h 2 was expressed in E. coli together with the disulfide-bond isomerase DsbC and thus harbored disulfide bridges but no hydroxyprolines. The recombinant allergens from N. benthamiana and E. coli were characterized and compared to the natural Ara h 2 isolated from roasted peanuts. Natural Ara h 2 outperformed both recombinant proteins in IgE-binding and activation of basophils via IgE cross-linking, the latter indicating the potency of the allergen. Interestingly, significantly more efficient IgE cross-linking by the N. benthamiana-produced allergen was observed in comparison to the one induced by the E. coli product. Ara h 2 from N. benthamiana plants displayed a higher similarity to the natural allergen in terms of basophil activation due to the presence of hydroxyproline residues, supporting so far published data on their contribution to the immunodominant IgE epitope. Our study advocates the use of N. benthamiana plants instead of prokaryotic expression hosts for the production of the major peanut allergen Ara h 2.

A chondroitin sulfate proteoglycan 4­specific monoclonal antibody inhibits melanoma cell invasion in a spheroid model.

The overexpression of chondroitin sulfate proteoglycan 4 (CSPG4) is associated with several tumor types, including malignant melanoma, squamous cell carcinoma, triple­negative breast carcinoma, oligodendrocytomas or gliomas. Due to its restricted distribution in normal tissues, CSPG4 has been considered a potential target for several antitumor approaches, including monoclonal antibody (mAb) therapies. The aim of the present study was to characterize the impact of the CSPG4­specific mAb clone 9.2.27 on its own or in combination with the commonly used BRAF­selective inhibitor, PLX4032, on different functions of melanoma cells to assess the potential synergistic effects. The BRAF V600­mutant human melanoma cell lines, M14 (CSPG4­negative) and WM164 (CSPG4­positive), were exposed to the CSPG4­specific 9.2.27 mAb and/or PLX4032. Cell viability and colony formation capacity were evaluated. A 3D­cell culture spheroid model was used to assess the invasive properties of the treated cells. In addition, flow cytometric analysis of apoptosis and cell cycle analyses were performed. Incubation of the WM164 cell line with CSPG4­specific 9.2.27 mAb decreased viability, colony formation ability and the invasive capacity of CSPG4­positive tumor cells, which was not the case for the CSPG4­negative M14 cell line. Combined treatment of the WM164 cells with 9.2.27 mAb plus PLX4032 did not exert any significant additional effect in comparison to treatment with PLX4032 alone in the clonogenic and invasion assays. M14 cell cycle distribution was not influenced by the CSPG4­specific 9.2.27 mAb. By contrast, the exposure of WM164 cells to the mAb resulted in an arrest of the cells in the S phase. Moreover, combined treatment of the WM164 cells led to a significantly increased accumulation of cells in the subG1 phase, combined with a decrease of cells in the G2/M phase. On the whole, findings of the present study indicate that the CSPG4­specific 9.2.27 mAb exerts an anti­invasive effect on CSPG4­positive melanoma spheroids, which is not enhanced by BRAF inhibition. These findings provide the basis for further investigations on the effects of anti­CSPG4­based treatments of CSPG4­positive tumors.

Newly defined allergens in the WHO/IUIS Allergen Nomenclature Database during 01/2019-03/2021.

The WHO/IUIS Allergen Nomenclature Database (http://allergen.org) provides up-to-date expert-reviewed data on newly discovered allergens and their unambiguous nomenclature to allergen researchers worldwide. This review discusses the 106 allergens that were accepted by the Allergen Nomenclature Sub-Committee between 01/2019 and 03/2021. Information about protein family membership, patient cohorts, and assays used for allergen characterization is summarized. A first allergenic fungal triosephosphate isomerase, Asp t 36, was discovered in Aspergillus terreus. Plant allergens contained 1 contact, 38 respiratory, and 16 food allergens. Can s 4 from Indian hemp was identified as the first allergenic oxygen-evolving enhancer protein 2 and Cic a 1 from chickpeas as the first allergenic group 4 late embryogenesis abundant protein. Among the animal allergens were 19 respiratory, 28 food, and 3 venom allergens. Important discoveries include Rap v 2, an allergenic paramyosin in molluscs, and Sal s 4 and Pan h 4, allergenic fish tropomyosins. Paramyosins and tropomyosins were previously known mainly as arthropod allergens. Collagens from barramundi, Lat c 6, and salmon, Sal s 6, were the first members from the collagen superfamily added to the database. In summary, the addition of 106 new allergens to the previously listed 930 allergens reflects the continuous linear growth of the allergen database. In addition, 17 newly described allergen sources were included.

Expression of chondroitin sulfate proteoglycan 4 (CSPG4) in melanoma cells is downregulated upon inhibition of BRAF.

Chondroitin sulfate proteoglycan 4 (CSPG4) is a multifunctional transmembrane proteoglycan involved in spreading, migration and invasion of melanoma. In addition to the activating BRAF V600E mutation, CSPG4 was shown to promote MAPK signaling by mediating the growth­factor induced activation of receptor tyrosine kinases. However, it remains elusive which factors regulate CSPG4 expression. Therefore, the aim of the present study was to examine whether BRAF and MEK inhibitors have an effect on the expression of CSPG4. We exposed a panel of BRAF­mutant CSPG4­positive or ­negative melanoma cell lines to BRAF and MEK inhibitors. Protein levels of CSPG4 were analyzed by flow cytometry (FACS), immunofluorescence microscopy (IF), and western blotting. CSPG4 mRNA levels were determined by quantitative PCR (qPCR). The prolonged exposure of cells to BRAF and MEK inhibitors resulted in markedly reduced levels of the CSPG4 protein in permanent resistant melanoma cells as well as decreased levels of its mRNA. We did not observe increasing levels of CSPG4 shedding into the culture supernatants. In addition, patient­derived matched tumor samples following therapy with kinase inhibitors showed decreased numbers of CSPG4­positive cells as compared to pre­therapy tumor samples. Our results indicate that BRAF and MEK inhibition downregulates CSPG4 expression until the cells have developed permanent resistance. Our findings provide the basis for further investigation of the role of CSPG4 in the development of drug­resistance in melanoma cells.

Fish Allergy Around the World-Precise Diagnosis to Facilitate Patient Management.

The accurate and precise diagnosis of IgE-mediated fish allergy is one of the biggest challenges in allergy diagnostics. A wide range of fish species that belong to evolutionary distant classes are consumed globally. Moreover, each fish species may contain multiple isoforms of a given allergen that often differ in their allergenicity. Recent studies indicated that the cross-reactivity between different fish species is limited in some cases and depends on the evolutionary conservation of the involved allergens. Fish allergens belong to several protein families with different levels of stability to food processing. Additionally, different preparation methods may contribute to specific sensitization patterns to specific fish species and allergens in different geographic regions. Here, we review the challenges and opportunities for improved diagnostic approaches to fish allergy. Current diagnostic shortcomings include the absence of important region-specific fish species in commercial in vitro and in vivo tests as well as the lack of their standardization as has been recently demonstrated for skin prick test solutions. These diagnostic shortcomings may compromise patients' safety by missing some of the relevant species and yielding false negative test results. In contrast, the avoidance of all fish as a common management approach is usually not necessary as many patients may be only sensitized to specific species and allergens. Although food challenges remain the gold standard, other diagnostic approaches are investigated such as the basophil activation test. In the context of molecular allergy diagnosis, we discuss the usefulness of single allergens and raw and heated fish extracts. Recent developments such as allergen microarrays offer the possibility to simultaneously quantify serum IgE specific to multiple allergens and allergen sources. Such multiplex platforms may be used in the future to design diagnostic allergen panels covering evolutionary distant fish species and allergens relevant for particular geographic regions.

Corrigendum: Fish Allergy Around the World-Precise Diagnosis to Facilitate Patient Management.

[This corrects the article DOI: 10.3389/falgy.2021.732178.].

Expanding the allergen repertoire of salmon and catfish.

BACKGROUND: Diagnostic tests for fish allergy are hampered by the large number of under-investigated fish species. Four salmon allergens are well-characterized and registered with the WHO/IUIS while no catfish allergens have been described so far. In 2008, freshwater-cultured catfish production surpassed that of salmon, the globally most-cultured marine species. We aimed to identify, quantify, and compare all IgE-binding proteins in salmon and catfish. METHODS: Seventy-seven pediatric patients with clinically confirmed fish allergy underwent skin prick tests to salmon and catfish. The allergen repertoire of raw and heated protein extracts was evaluated by immunoblotting using five allergen-specific antibodies and patients' serum followed by mass spectrometric analyses. RESULTS: Raw and heated extracts from catfish displayed a higher frequency of IgE-binding compared to those from salmon (77% vs 70% and 64% vs 53%, respectively). The major fish allergen parvalbumin demonstrated the highest IgE-binding capacity (10%-49%), followed by triosephosphate isomerase (TPI; 19%-34%) in raw and tropomyosin (6%-32%) in heated extracts. Six previously unidentified fish allergens, including TPI, were registered with the WHO/IUIS. Creatine kinase from salmon and catfish was detected by IgE from 14% and 10% of patients, respectively. Catfish L-lactate dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase, pyruvate kinase, and glucose-6-phosphate isomerase showed IgE-binding for 6%-13% of patients. In salmon, these proteins could not be separated successfully. CONCLUSIONS: We detail the allergen repertoire of two highly farmed fish species. IgE-binding to fish tropomyosins and TPIs was demonstrated for the first time in a large patient cohort. Tropomyosins, in addition to parvalbumins, should be considered for urgently needed improved fish allergy diagnostics.

Advances and novel developments in molecular allergology.

The continuous search for new allergens and the design of allergen derivatives improves the understanding of their allergenicity and aids the design of novel diagnostic and immunotherapy approaches. This article discusses the recent developments in allergen and epitope discovery, allergy diagnostics and immunotherapy. Structural information is crucial for the elucidation of cross-reactivity of marker allergens such as the walnut Jug r 6 or that of nonhomologous allergens, as shown for the peanut allergens Ara h 1 and 2. High-throughput sequencing, liposomal nanoallergen display, bead-based assays, and protein chimeras have been used in epitope discovery. The binding of natural ligands by the birch pollen allergen Bet v 1 or the mold allergen Alt a 1 increased the stability of these allergens, which is directly linked to their allergenicity. We also report recent findings on the use of component-resolved approaches, basophil activation test, and novel technologies for improvement of diagnostics. New strategies in allergen-specific immunotherapy have also emerged, such as the use of virus-like particles, biologics or novel adjuvants. The identification of dectin-1 as a key player in allergy to tropomyosins and the formyl peptide receptor 3 in allergy to lipocalins are outstanding examples of research into the mechanism of allergic sensitization.

Collagen-An Important Fish Allergen for Improved Diagnosis.

BACKGROUND: Fish collagen is widely used in medicine, cosmetics, and the food industry. However, its clinical relevance as an allergen is not fully appreciated. This is likely due to collagen insolubility in neutral aqueous solutions, leading to low abundance in commercially available in vitro and skin prick tests for fish allergy. OBJECTIVE: To investigate the relevance of fish collagen as an allergen in a large patient population (n = 101). METHODS: Acid-soluble collagen type I was extracted from muscle and skin of Atlantic salmon, barramundi, and yellowfin tuna. IgE binding to collagen was analyzed by ELISA for 101 fish-allergic patients. Collagen-sensitized patients' sera were tested for IgE binding to parvalbumin from the same fish species. IgE cross-linking was analyzed by rat basophil leukemia assay and basophil activation test. Protein identities were confirmed by mass spectrometry. RESULTS: Purified fish collagen contained type I α1 and α2 chains and their multimers. Twenty-one of 101 patients (21%) were sensitized to collagen. Eight collagen-sensitized patients demonstrated absence of parvalbumin-specific IgE to some fish species. Collagen induced functional IgE cross-linking, as shown by rat basophil leukemia assay performed using 6 patients' sera, and basophil activation test using fresh blood from 1 patient. Collagen type I α chains from barramundi and Atlantic salmon were registered at www.allergen.org as Lat c 6 and Sal s 6, respectively. CONCLUSIONS: IgE sensitization and IgE cross-linking capacity of fish collagen were demonstrated in fish-allergic patients. Inclusion of relevant collagen allergens in routine diagnosis is indicated to improve the capacity to accurately diagnose fish allergy.

Development of a novel Ara h 2 hypoallergen with no IgE binding or anaphylactogenic activity.

BACKGROUND: To date, no safe allergen-specific immunotherapy for patients with peanut allergy is available. Previous trials were associated with severe side effects. OBJECTIVE: We sought to determine the relative importance of conformational and linear IgE-binding epitopes of the major peanut allergen Ara h 2 and to produce a hypoallergenic variant with abolished anaphylactogenic activity. METHODS: Wild-type Ara h 2 and a mutant lacking the loops containing linear IgE epitopes were produced in insect cells. Conformational IgE epitopes were removed by unfolding these proteins through reduction and alkylation. IgE binding was tested by means of ELISA with sera from 48 Ara h 2-sensitized patients with peanut allergy. Basophil activation and T-cell proliferation were tested with blood samples from selected patients. Anaphylactogenic potency was tested by using intraperitoneal challenge of mice sensitized intragastrically to peanut extract. RESULTS: Patients' IgE recognized conformational and linear epitopes in a patient-specific manner. The unfolded mutant lacking both types of epitopes displayed significantly lower IgE binding (median ELISA OD, 0.03; interquartile range, 0.01-0.06) than natural Ara h 2 (median ELISA OD, 0.99; interquartile range, 0.90-1.03; P < .01). Basophil activation by unfolded mutant Ara h 2 was low (median area under the curve, 72 vs 138 for native wild-type Ara h 2; P < .05), but its ability to induce T-cell proliferation was retained. Unfolded mutants without conformational epitopes did not induce anaphylaxis in peanut-sensitized mice. CONCLUSIONS: By removing conformational and linear IgE epitopes, a hypoallergenic Ara h 2 mutant with abolished IgE binding and anaphylactogenic potency but retained T-cell activation was generated.

Fish-derived low molecular weight components modify bronchial epithelial barrier properties and release of pro-inflammatory cytokines.

The prevalence of fish allergy among fish-processing workers is higher than in the general population, possibly due to sensitization via inhalation and higher exposure. However, the response of the bronchial epithelium to fish allergens has never been explored. Parvalbumins (PVs) from bony fish are major sensitizers in fish allergy, while cartilaginous fish and their PVs are considered less allergenic. Increasing evidence demonstrates that components other than proteins from the allergen source, such as low molecular weight components smaller than 3 kDa (LMC) from pollen, may act as adjuvants during allergic sensitization. We investigated the response of bronchial epithelial cells to PVs and to LMC from Atlantic cod, a bony fish, and gummy shark, a cartilaginous fish. Polarized monolayers of the bronchial epithelial cell line 16HBE14o- were stimulated apically with fish PVs and/-or the corresponding fish LMC. Barrier integrity, transport of PVs across the monolayers and release of mediators were monitored. Intact PVs from both the bony and the cartilaginous fish were rapidly internalized by the cells and transported to the basolateral side of the monolayers. The PVs did not disrupt the epithelial barrier integrity nor did they modify the release of proinflammatory cytokines. In contrast, LMC from both fish species modified the physical and immunological properties of the epithelial barrier and the responses differed between bony and cartilaginous fish. While the barrier integrity was lowered by cod LMC 24 h after cell stimulation, it was increased by up to 2.3-fold by shark LMC. Furthermore, LMC from both fish species increased basolateral and apical release of IL-6 and IL-8, while CCL2 release was increased by cod but not by shark LMC. In summary, our study demonstrated the rapid transport of PVs across the epithelium which may result in their availability to antigen presenting cells required for allergic sensitization. Moreover, different cell responses to LMC derived from bony versus cartilaginous fish were observed, which may play a role in different allergenic potentials of these two fish classes.

Patients Allergic to Fish Tolerate Ray Based on the Low Allergenicity of Its Parvalbumin.

BACKGROUND: Clinical reactions to bony fish species are common in patients with allergy to fish and are caused by parvalbumins of the ß-lineage. Cartilaginous fish such as rays and sharks contain mainly α-parvalbumins and their allergenicity is not well understood. OBJECTIVE: To investigate the allergenicity of cartilaginous fish and their α-parvalbumins in individuals allergic to bony fish. METHODS: Sensitization to cod, salmon, and ray among patients allergic to cod, salmon, or both (n = 18) was explored by prick-to-prick testing. Clinical reactivity to ray was assessed in 11 patients by food challenges or clinical workup. IgE-binding to ß-parvalbumins (cod, carp, salmon, barramundi, tilapia) and α-parvalbumins (ray, shark) was determined by IgE-ELISA. Basophil activation tests and skin prick tests were performed with ß-parvalbumins from cod, carp, and salmon and α-parvalbumins from ray and shark. RESULTS: Tolerance of ray was observed in 10 of 11 patients. Prick-to-prick test reactions to ray were markedly lower than to bony fish (median wheal diameter 2 mm with ray vs 11 mm with cod and salmon). IgE to α-parvalbumins was lower (median, 0.1 kU/L for ray and shark) than to ß-parvalbumins (median, ≥1.65 kU/L). Furthermore, α-parvalbumins demonstrated a significantly reduced basophil activation capacity compared with ß-parvalbumins (eg, ray vs cod, P < .001; n = 18). Skin prick test further demonstrated lower reactivity to α-parvalbumins compared with ß-parvalbumins. CONCLUSIONS: Most patients allergic to bony fish tolerated ray, a cartilaginous fish, because of low allergenicity of its α-parvalbumin. A careful clinical workup and in vitro IgE-testing for cartilaginous fish will improve patient management and may introduce an alternative to bony fish into patients' diet.

Seafood allergy: A comprehensive review of fish and shellfish allergens.

Seafood refers to several distinct groups of edible aquatic animals including fish, crustacean, and mollusc. The two invertebrate groups of crustacean and mollusc are, for culinary reasons, often combined as shellfish but belong to two very different phyla. The evolutionary and taxonomic diversity of the various consumed seafood species poses a challenge in the identification and characterisation of the major and minor allergens critical for reliable diagnostics and therapeutic treatments. Many allergenic proteins are very different between these groups; however, some pan-allergens, including parvalbumin, tropomyosin and arginine kinase, seem to induce immunological and clinical cross-reactivity. This extensive review details the advances in the bio-molecular characterisation of 20 allergenic proteins within the three distinct seafood groups; fish, crustacean and molluscs. Furthermore, the structural and biochemical properties of the major allergens are described to highlight the immunological and subsequent clinical cross-reactivities. A comprehensive list of purified and recombinant allergens is provided, and the applications of component-resolved diagnostics and current therapeutic developments are discussed.