Sensitisation to peach allergen Pru p 7 is associated with severe clinical symptoms in a Spanish population.

BACKGROUND: Pru p 7 has been reported as a major allergen in peach allergy, associated with severe clinical symptoms and related to IgE sensitisation to cypress pollen. The main objective of this study was to prospectively evaluate the frequency of sensitisation to Pru p 7 and its clinical relevance amongst pediatric patients with peach allergy in Madrid (Spain). METHODS: Patients with a history of IgE-mediated symptoms (oral allergy syndrome, urticaria/angioedema, rhinoconjunctivitis/asthma, gastrointestinal symptoms, or anaphylaxis) occurring within 2 h after peach intake or contact were prospectively recruited from February 2020 to September 2021. Skin tests, sIgE by ImmunoCAP® (Pru p 1, Pru p 3, Pru p 4, Pru p 7, and Cupressus arizonica) and oral food challenge (OFC) were performed. The study was approved by the local Ethics Committee (PI-4513). RESULTS: Ninety-two patients were included (53.3% male); median age, 10 (IQR 6.0-14.75) years. Seventy-four (80.4%) patients had a reaction after ingestion of fresh peach (25.0% from peel, 23.9% from pulp, and 44.6% from both). Fifteen (16.3%) patients were sensitised to Pru p 7. Upper airway symptoms, anaphylaxis, and grade 2 reactions were statistically more frequent in patients sensitised to Pru p 7. Seven (7.9%) patients presented with exercise as a cofactor, four of whom were sensitised to Pru p 7 (p = .001). Patients sensitised to Pru p 7 were significantly more likely to have a positive OFC result than patients who were not (p = .008). Four patients who reacted to peach at OFC were sensitised to Pru p 7. Specific IgE against Cupressus arizonica pollen was positive in 25 (62.5%) patients. CONCLUSIONS: Pru p 7 sensitisation was observed in 16.3% of our population and was related to severe reactions, upper airway symptoms, anaphylaxis, and the presence of an eliciting cofactor.

Tools for Etiologic Diagnosis of Drug-Induced Allergic Conditions.

Drug hypersensitivity reactions are a serious concern in clinical practice because they can be severe and result in lifelong sequelae. An accurate diagnosis and identification of the culprit drug is essential to prevent future reactions as well as for the identification of safe treatment alternatives. Nonetheless, the diagnosis can be challenging. In vivo and in vitro tests can be helpful, although none are conclusive; therefore, the tests are not usually performed in isolation but as part of a diagnostic algorithm. In addition, some in vitro tests are only available in research laboratories, and standardization has not been fully accomplished. Collaborating research is needed to improve drug hypersensitivity reaction diagnosis. In this review, we update the current available in vivo and in vitro tools with their pros and cons and propose an algorithm to integrate them into clinical practice.

Fish muscle processing into seafood products reduces ß-parvalbumin allergenicity.

Fish is one of the eight major foods causing type-I food allergy, and the prevalence of its allergy is increasing in part due to changes in consumption habits. One of the main drivers for these changes has been the processing developments transforming the fish muscle into seafood products. Most fish allergic patients react to the Ca2+-binding protein ß-parvalbumin (ß-PV) abundant in muscle. Here we have analyzed the effect of processing in the content and allergenic properties of the ß-PV. We found that the transformation process decreases the ß-PV content (4.7 ± 0.3 mg/g muscle, 0.24 ± 0.03 mg/g surimi, ≤0.003 ± 0.001 mg/g in seafood products), reduces the specific-IgE binding and prevents allergy relevant properties such the protease resistance and amyloid aggregation. These results suggest seafood products as potentially tolerable foods for fish allergic patients, but milk and egg allergic patients should be aware of the presence relevant additives.

Quercus ilex pollen allergen, Que i 1, responsible for pollen food allergy syndrome caused by fruits in Spanish allergic patients.

BACKGROUND: Pollen food allergy syndrome (PFAS) related to PR10 from vegetables is common in northern Europe, whereas in Mediterranean countries PFAS has been preferentially associated with profilins. However, there are pollen-allergic patients reactive to Bet v 1 in birch-free regions. Since it cannot be the primary sensitizer, there has to be another culprit. Quercus ilex is a good candidate as it belongs to the order Fagales. This order includes trees with highly sensitizing pollen such as alder, hazel, hornbeam, oak and chestnut because of the presence of PR10 allergens. PR10 allergens have indeed been described in other Quercus species. OBJECTIVE: Our goals were to determine the rate of sensitization to Q. ilex in central Spain and the associated frequency of PFAS; secondly to identify and clone the Q. ilex allergen PR10. METHODS: We included 224 allergic patients with respiratory symptoms to estimate the rate of sensitization. A skin prick test (SPT) and ImmunoCAP were performed. A total of 38 Q. ilex-sensitized patients were tested using Western blotting to determine the rate of Que i 1. Peptides from Que i 1 were analysed by MALDI-TOF/TOF and Orbitrap LC-MSMS. The Que i 1 sequence was first obtained from the Holm oak transcriptome then cloned and expressed in bacteria. RESULTS: 59.8% of pollen-allergic children were sensitized to Q. ilex. We described and cloned the Q. ilex PR10, Que i 1, which has a sensitization rate of 60.5% and was recognized by 65.4% patients reporting PFAS. CONCLUSION AND CLINICAL RELEVANCE: Sensitization to Q. ilex pollen has increased significantly since 1995. This sensitization could be important, as the presence of PFAS in this population is higher than in patients not sensitized to Q. ilex. The first Q. ilex allergen has been described and is related to PFAS in Spanish patients sensitized to PR10 but not exposed to birch pollen.

Influence of Instant Controlled Pressure Drop (DIC) on Allergenic Potential of Tree Nuts.

Pistachio and cashew contain allergenic proteins, which causes them to be removed from the diet of allergic people. Previous studies have demonstrated that food processing (thermal and non-thermal) can produce structural and/or conformational changes in proteins by altering their allergenic capacity. In this study, the influence of instant controlled pressure drop (DIC) on pistachio and cashew allergenic capacity has been studied. Western blot was carried out using IgG anti-11S and anti-2S and IgE antibodies from sera of patients sensitized to pistachio and cashew. DIC processing causes changes in the electrophoretic pattern, reducing the number and intensity of protein bands, as the pressure and temperature treatment increment, which results in a remarkable decrease in detection of potentially allergenic proteins. The harshest conditions of DIC (7 bar, 120 s) markedly reduce the immunodetection of allergenic proteins, not only by using IgG (anti 11S and anti 2S) but also when IgE sera from sensitized patients were used for Western blots. Such immunodetection is more affected in pistachio than in cashew nuts, but is not completely removed. Therefore, cashew proteins are possibly more resistant than pistachio proteins. According these findings, instant controlled pressure drop (DIC) can be considered a suitable technique in order to obtain hypoallergenic tree nut flour to be used in the food industry.

Reconstruction of fish allergenicity from the content and structural traits of the component ß-parvalbumin isoforms.

Most fish-allergic patients have anti-ß-parvalbumin (ß-PV) immunoglobulin E (IgE), which cross-reacts among fish species with variable clinical effects. Although the ß-PV load is considered a determinant for allergenicity, fish species express distinct ß-PV isoforms with unknown pathogenic contributions. To identify the role various parameters play in allergenicity, we have taken Gadus morhua and Scomber japonicus models, determined their ß-PV isoform composition and analyzed the interaction of the IgE from fish-allergic patient sera with these different conformations. We found that each fish species contains a major and a minor isoform, with the total PV content four times higher in Gadus morhua than in Scomber japonicus. The isoforms showing the best IgE recognition displayed protease-sensitive globular folds, and if forming amyloids, they were not immunoreactive. Of the isoforms displaying stable globular folds, one was not recognized by IgE under any of the conditions, and the other formed highly immunoreactive amyloids. The results showed that Gadus morhua muscles are equipped with an isoform combination and content that ensures the IgE recognition of all PV folds, whereas the allergenic load of Scomber japonicus is under the control of proteolysis. We conclude that the consideration of isoform properties and content may improve the explanation of fish species allergenicity differences.

Exploring tumourigenic potential of the parasite Anisakis: a pilot study.

Anisakiasis is a global disease caused by consumption of raw or lightly cooked fish parasitised with Anisakis spp. third-stage larvae. Cases in the literature show colocalised anisakiasis and colorectal cancer, and the incidental finding of Anisakis larvae at the tumour site was reported. Data from our group suggested an epidemiological link between previous infection and gastrointestinal cancer. Furthermore, it has recently been reported that Anisakis products lead to inflammation and DNA damage. Based on these facts, the aim was to investigate whether Anisakis antigens are able to induce changes in the proliferation of epithelial cells in vitro or in the expression of serum microRNA (miRNA) in Sprague-Dawley rats. Anisakis complete extract (CE) induced increases in cell proliferation and decreases in apoptosis compared with nontreated cells, which resulted in a significant increase in the absolute number of viable cells at 48 h of exposure (P < .05). Furthermore, the miRNAs mmu-miR-1b-5p and mmu-miR-10b-5p (a cancer-related miRNA) were significantly decreased (P < .05) in sera from the rats inoculated with Anisakis CE, compared with control rats inoculated with saline. Additionally, based on their relative quantification values, four other cancer-related miRNAs were considered to be differently expressed, rno-miR-218a-5p and mmu-miR-224-5p (decreased) and rno-miR-125a-3p and rno-miR-200c-3p (increased). Anisakis CE was able to induce changes both in epithelial cells in vitro and in an animal model. The results obtained with Anisakis CE, in terms of increasing cell proliferation, decreasing apoptosis and inducing changes in the expression of serum cancer-related miRNAs in rats, suggest that Anisakis could have tumourigenic potential.

Amyloid Assembly Endows Gad m 1 with Biomineralization Properties.

Acid proteins capable of nucleating Ca2+ and displaying aggregation capacity play key roles in the formation of calcium carbonate biominerals. The helix-loop helix EF-hands are the most common Ca2+-binding motifs in proteins. Calcium is bound by the loop region. These motifs are found in many proteins that are regulated by calcium. Gad m 1, an Atlantic cod ß-parvalbumin isoform, is a monomeric EF-hand protein that acts as a Ca2+ buffer in fish muscle; the neutral and acid apo-forms of this protein can form amyloids. Since Ca2+-nucleating proteins have a propensity to form extended ß-strand structures, we wondered whether amyloid assemblies of an EF-hand protein were able to influence calcium carbonate crystallization in vitro. Here, we used the Gad m 1 chain as a model to generate monomeric and amyloid assemblies and to analyze their effect on calcite formation in vitro. We found that only amyloid assemblies alter calcite morphology.

The amyloid fold of Gad m 1 epitopes governs IgE binding.

Amyloids are polymeric structural states formed from locally or totally unfolded protein chains that permit surface reorganizations, stability enhancements and interaction properties that are absent in the precursor monomers. ß-Parvalbumin, the major allergen in fish allergy, forms amyloids that are recognized by IgE in the patient sera, suggesting a yet unknown pathological role for these assemblies. We used Gad m 1 as the fish ß-parvalbumin model and a combination of approaches, including peptide arrays, recombinant wt and mutant chains, biophysical characterizations, protease digestions, mass spectrometry, dot-blot and ELISA assays to gain insights into the role of amyloids in the IgE interaction. We found that Gad m 1 immunoreactive regions behave as sequence-dependent conformational epitopes that provide a 1000-fold increase in affinity and the structural repetitiveness required for optimal IgE binding and cross-linking upon folding into amyloids. These findings support the amyloid state as a key entity in type I food allergy.

Biomarkers in Occupational Asthma.

PURPOSE OF REVIEW: Work-related asthma is a common disorder among adult asthma patients, and in the case of occupational asthma, it is induced by workplace exposures. RECENT FINDINGS: Occupational asthma provides an excellent model and benchmark for identifying and testing different allergy or inflammatory biomarkers associated with its inception or progression. Moreover, specific inhalation challenge with the incriminated agent represents an experimental setting to identify and validate potential systemic or local biomarkers. Some biomarkers are mainly blood-borne, while local airway biomarkers are derived from inflammatory or resident cells. Genetic and gene-environment interaction studies also provide an excellent framework to identify relevant profiles associated with the risk of developing these work-related conditions. Despite significant efforts to identify clinically relevant inflammatory and genomic markers for occupational asthma, apart from the documented utility of airway inflammatory biomarkers, it remains elusive to define specific markers or signatures clearly associated with different endpoints or outcomes in occupational asthma.

Previous Exposure to the Fish Parasite Anisakis as a Potential Risk Factor for Gastric or Colon Adenocarcinoma.

Anisakiasis is a global disease caused by consumption of raw or lightly cooked fish contaminated with L3 Anisakis spp. larvae. High rates of parasitization of fish worldwide make Anisakis a serious health hazard. In fact, anisakiasis is a growing disease in countries such as Spain, Italy, and Japan, where consumption of raw/marinated fish is high. Some parasitic infections have been recognized as a causative factor for human cancer. Suggested mechanisms include chronic inflammation elicited by the parasite, and a possible tumorigenic effect from certain parasitic secretions. Anisakis can produce persistent local inflammation and granuloma, and larvae have been incidentally found in gastrointestinal (GI) tumors. Our aim was to discover possible differences in the prevalence of unnoticed or asymptomatic previous Anisakis infection in GI cancer patients compared with healthy individuals. Serum levels of specific antibodies against Anisakis antigens were used as a reliable marker of previous contact with their larvae. Ninety-four participants without a previous history of Anisakis infection were prospectively allocated into 1 of 2 groups: 47 patients with GI cancer and 47 controls. Specific IgE, IgA1, and IgG1 against the Anisakis recombinant antigens Ani s 1, Ani s 5, Ani s 9, and Ani s 10 were determined by an ELISA assay. The ratio of positivity to sIgA1, rAni s 1, or rAni s 5 was significantly higher in the cancer patients than in the controls (38.30% vs 6.38%, P < 0.001) and (42.55% vs 10.64%, P < 0.001, respectively). When disaggregated by type of tumor, the patients with gastric cancer showed a higher proportion of positive results for sIgA1 to rAni s 1 (P < 0.001), whereas a higher proportion of colon cancer patients were shown to be positive for sIgA1 to both rAni s 1 (P < 0.05) and rAni s 5 (P < 0.01). Earlier Anisakis infection might be a risk factor for the development of stomach or colon cancer.

Allergenicity of two Anisakis simplex allergens evaluated in vivo using an experimental mouse model.

Anisakis (Anisakidae) is one of the most important causes of helminth-induced allergic reactions and elicits clinical responses that include urticaria, rhinitis, bronco-constriction, cough, and/or gastrointestinal symptoms. More than 13 reactive allergens have been identified in the serum of Anisakis allergy patients, but the allergenicity of only a few of these have been evaluated in vivo using a mouse model. To evaluate the allergenicity of two important allergens, Ani s 1 and Ani s 9, we induced experimental allergic airway inflammation in a mouse model by repeated intranasal administration of the allergens. Both recombinant proteins (rAni s 1 and rAni s 9) elicited increased airway hyperresponsivity, airway infiltration by inflammatory cells (especially eosinophils), bronchial epithelial cell hyperplasia, all of which are characteristic of allergic airway inflammation. These allergens significantly increased the levels of Th2-related cytokines (IL-4, IL-5, IL-13, and IL-25) and Th17 related cytokines (IL-6 and IL-17) in both splenocytes and airway (except IL-17 in airway by rAni s 9). OVA-specific IgE and total IgE were increased in rAni s 1 and rAni s 9 treated mice as compared with controls treated with OVA alone. In addition, these two allergens induced gene expression of thymic stromal lymphopoietin (TSLP) and IL-25 (initiators of the Th2 response), as well as CXCL1 (initiator of the Th17 response) in mouse lung epithelial cells. In conclusion, repeated intranasal treatments with rAni s 1 and rAni s 9 induced airway inflammation in mice by elevating of Th2 and Th17 responses in the lung.

Relationships between IgE/IgG4 epitopes, structure and function in Anisakis simplex Ani s 5, a member of the SXP/RAL-2 protein family.

BACKGROUND: Anisakiasis is a re-emerging global disease caused by consumption of raw or lightly cooked fish contaminated with L3 Anisakis larvae. This zoonotic disease is characterized by severe gastrointestinal and/or allergic symptoms which may misdiagnosed as appendicitis, gastric ulcer or other food allergies. The Anisakis allergen Ani s 5 is a protein belonging to the SXP/RAL-2 family; it is detected exclusively in nematodes. Previous studies showed that SXP/RAL-2 proteins are active antigens; however, their structure and function remain unknown. The aim of this study was to elucidate the three-dimensional structure of Ani s 5 and its main IgE and IgG4 binding regions. METHODOLOGY/PRINCIPAL FINDINGS: The tertiary structure of recombinant Ani s 5 in solution was solved by nuclear magnetic resonance. Mg2+, but not Ca2+, binding was determined by band shift using SDS-PAGE. IgE and IgG4 epitopes were elucidated by microarray immunoassay and SPOTs membranes using sera from nine Anisakis allergic patients. The tertiary structure of Ani s 5 is composed of six alpha helices (H), with a Calmodulin like fold. H3 is a long, central helix that organizes the structure, with H1 and H2 packing at its N-terminus and H4 and H5 packing at its C-terminus. The orientation of H6 is undefined. Regarding epitopes recognized by IgE and IgG4 immunoglobulins, the same eleven peptides derived from Ani s 5 were bound by both IgE and IgG4. Peptides 14 (L40-K59), 26 (A76-A95) and 35 (I103-D122) were recognized by three out of nine sera. CONCLUSIONS/SIGNIFICANCE: This is the first reported 3D structure of an Anisakis allergen. Magnesium ion binding and structural resemblance to Calmodulin, suggest some putative functions for SXP/RAL-2 proteins. Furthermore, the IgE/IgG4 binding regions of Ani s 5 were identified as segments localized on its surface. These data will contribute towards a better understanding of the interactions that occur between immunoglobulins and allergens and, in turn, facilitate the design of novel diagnostic tests and immunotherapeutic strategies.

Cross-reactivity between Anisakis spp. and wasp venom allergens.

BACKGROUND: Anisakiasis is caused by the consumption of raw or undercooked fish or cephalopods parasitized by live L3 larvae of nematode Anisakis spp. Larvae anchor to stomach mucosa releasing excretion/secretion products which contain the main allergens. It has been described that nematode larvae release venom allergen-like proteins among their excretion/secretion products. We investigated potential cross-reactivity between Anisakis and wasp venom allergens. METHODS: Two groups of 25 patients each were studied: wasp venom- and Anisakis-allergic patients. Sera from patients were tested by ImmunoCAP, dot-blotting with recombinant Anisakis allergens and ADVIA-Centaur system with Hymenoptera allergens. Cross-reactivity was assessed by IgE immunoblotting inhibition assays. Role of cross-reactive carbohydrate determinants (CCDs) was studied by inhibition with bromelain and periodate treatment. RESULTS: A total of 40% of wasp venom-allergic patients had specific IgE to Anisakis simplex and 20% detected at least one of the Anisakis recombinant allergens tested. Likewise, 44% of Anisakis-allergic patients had specific IgE to Vespula spp. venom and 16% detected at least one of the Hymenoptera allergens tested. Wasp venom-allergic patients detected CCDs in Anisakis extract and peptide epitopes on Anisakis allergens rAni s 1 and rAni s 9, whereas Anisakis-allergic patients only detected CCDs on nVes v 1 allergen from Vespula spp. venom. The only Anisakis allergen inhibited by Vespula venom was rAni s 9. CONCLUSIONS: This is the first time that cross-sensitization between wasp venom and Anisakis is described. CCDs are involved in both cases; however, peptide epitopes are only recognized by wasp venom-allergic patients.

Anisakis allergy component-resolved diagnosis: clinical and immunologic differences between patients from Italy and Spain.

BACKGROUND: Anisakissimplex is the main organism responsible for the zoonotic disease anisakiasis which follows the ingestion of live larvae present in raw or undercooked marine fish. Clinical features include severe epigastric pain, frequently accompanied by severe allergic reactions. We investigated the prevalence of immunoglobulin E (IgE) specific for 5 Anisakis allergens in Italian patients sensitized or allergic to the parasite. The results were compared with those obtained previously in a similar Spanish population. PATIENTS AND METHODS: We conducted a descriptive, cross-sectional validation study. Asymptomatic Anisakis-sensitized subjects (15 Italian and 17 Spanish) and Anisakis allergic-patients (42 Italian and 35 Spanish) were studied by ImmunoCAP, Western-blotting with nAni s 4 and dot-blotting with rAni s 1, rAni s 5, rAni s 9 and rAni s 10. RESULTS: Anisakis IgE CAP classes 1 or 2 were associated with a high probability of asymptomatic sensitization (66.7%) while CAP classes 4 or above, were associated with a very high probability of allergy to Anisakis (95.2%). The most frequently detected allergen among Italian and Spanish allergic patients was Ani s 1. All of the Spanish patients versus 76.2% of the Italian patients recognized at least one of the allergens tested. Patients suffering from gastrointestinal symptoms only were significantly more frequent among the Italians whereas the Spanish presented more frequently with urticaria, angioedema or anaphylaxis. CONCLUSIONS: Anisakis hypersensitivity shows different immunological patterns in different European countries. Allergen component diagnosis might help us to better understand this complex entity. Anisakis-specific IgE levels may have moderate prognostic significance.

A rat model of intragastric infection with Anisakis spp. live larvae: histopathological study.

Anisakiasis is a fish-borne parasitic disease caused by consumption of raw or undercooked fish or cephalopods parasited by Anisakis spp. third stage larvae. The pathological effects of the infection are the combined result of the mechanical action of the larva during tissue invasion, the direct tissue effects of the excretory/secretory products released by the parasite, and the complex interaction between the host immune system and the Anisakis antigens. The aim of this study was to develop an experimental model of infection with Anisakis spp. live larvae in rats, useful to study the acute and chronic histopathological effects of the Anisakis infection. Sprague-Dawley rats were subjected to esophageal catheterization to place larvae directly into the stomach. Reinfections at different intervals after the first infection were preformed. Live larvae were found anchored to the mucosa and passing through the wall of the stomach and showed a strong resistance being able to stay alive at different sites and at the different pH. Migration of larvae from the stomach to other organs out of the gastrointestinal tract was also observed. The histopathological study showed the acute inflammatory reaction, with predominance of polymorphonuclear eosinophils and a mild fibrotic reaction. The model of infection described is valid to study the behavior of the larvae inside the host body, the histopathological changes at the invasion site, and the effects of the repeated infections by ingestion of live larvae.

Anisakis simplex recombinant allergens increase diagnosis specificity preserving high sensitivity.

BACKGROUND: So far, the frequency of Anisakis simplex-specific IgE antibodies has been determined by skin prick tests (SPTs) and the ImmunoCAP system. These commercial methods have good sensitivity, but their specificity is poor because they use complete parasite extracts. Our aim was to determine the frequency of sensitization to A. simplex using recombinant Ani s 1, Ani s 3, Ani s 5, Ani s 9 and Ani s 10 and to evaluate these allergens for diagnosis, comparing their performance with the commercial methods. PATIENTS AND METHODS: We conducted a descriptive, cross-sectional validation study performed in an allergy outpatient hospital clinic. Patients without fish-related allergy (tolerant patients, n = 99), and A. simplex-allergic patients (n = 35) were studied by SPTs, ImmunoCAP assays and detection of specific IgE to A. simplex recombinant allergens by dot blotting. RESULTS: SPTs and ImmunoCAP assays were positive in 18 and 17% of tolerant patients, respectively. All A. simplex-allergic patients had positive SPTs and ImmunoCAP assays. Specific IgE against at least one of the A. simplex recombinant allergens tested was detected in 15% of sera from tolerant patients and in 100% of sera from A. simplex-allergic patients. Detection of at least one A. simplex recombinant allergen by dot blotting and ImmunoCAP assay using complete extract showed a diagnostic sensitivity of 100% with both methods. However, the specificity of dot blotting with A. simplex recombinant allergens was higher compared with ImmunoCAP (84.85 vs. 82.83%). CONCLUSIONS: There are 15% of tolerant patients with specific IgE against important A. simplex allergens. The recombinant allergens studied here increase the specificity of A. simplex diagnosis while keeping the highest sensitivity. A. simplex recombinant allergens should be included with A. simplex allergy diagnostic tests to improve their specificity.