Rosa Rodriguez-Perez

Cloning and expression of Ani s 9, a new Anisakis simplex allergen.

The larvae of the nematode Anisakis simplex parasitize seafood. When people eat raw or undercooked parasitized fish, they can suffer anisakiasis, an important immune human response to parasitic infection of the gastrointestinal tract. Even more, allergic manifestations like angioedema, urticaria or anaphylaxis can occur in sensitized patients. The aim of this work was to clone Ani s 9-cDNA and overproduce this recombinant allergen in Escherichia coli. The finding of this allergen was an unexpected result of a PCR using degenerate primers designed to amplify Ani s 5. The complete cDNA for Ani s 9 was obtained by RACE-PCR, cloned and sequenced. Expression of recombinant allergen was performed in E. coli. Immunodetection and immunoblot inhibition assays tests were carried out with sera from Anisakis allergic patients. The recombinant Ani s 9 (rAni s 9) is a protein of 147 amino acids. By immunoblot inhibition assay, it was located as a 14 kDa band present in a crude extract of the parasite. This new allergen is heat stable and is present in excretory/secretory products. Ani s 9 belongs to the SXP/RAL-2 family and shares amino acid sequence identity of 60% with As-14, an Ascaris suum allergen. Five of thirty-six Anisakis allergic patients (13.8%) were positive to rAni s 9 and natural Ani s 9 by immunodetection. In conclusion, Ani s 9 is a new allergen in Anisakis allergy and it has been cloned and successfully expressed in E. coli.

Sensitization to serum albumins in children allergic to cow's milk and epithelia

Patients with persistent milk allergy and specific immunoglobulin E (IgE) to bovine serum albumin (BSA) have a greater risk of rhinoconjunctivitis and asthma because of animal dander. To prove the cross‐reactivity between serum albumin (SA) of different mammals in milk, meat, and epithelia and determine if heat treatment of meats decrease the allergenicity of albumins. The study was performed using SDS‐PAGE and IgE‐immunoblotting using sera from eight patients sensitized to milk, BSA, and animal danders. Sera from non‐allergic and only animal dander allergic subjects served as a control. With one exception, all patients’ sera recognized SA in different meats (beef, lamb, deer, and pork), epithelia (dog, cat, and cow), and cows milk. Some patients even were only sensitized to SA in meat and epithelia. Danders’ allergic only recognized other proteins in epithelia but not SA. No patients reacted to SA from heated meat extracts. Serum albumin is an important allergen involved in milk, meat, and epithelia allergy. The first contact with SA was through cows milk and patients developed sensitization to epithelia SA even without direct contact with animals. Patients with both BSA and cows milk allergy must avoid raw meats and furry pets.

Sensitization to the fish parasite Anisakis simplex: clinical and laboratory aspects

Hypersensitivity to Anisakis simplex is a worldwide medical problem. The parasite larvae die after freezing or cooking, but the tolerance of sensitized subjects to eating frozen fish remains a matter of controversy with contradictory findings. The aim of this study was to test if intolerance to properly cooked/frozen fish was due to the recognition of a particular allergen. Sixty-four patients with Anisakis simplex sensitization were studied by an IgE multiblot using simultaneously five different antigenic extracts. The antigens tested were a crude extract, excretory/secretory allergens, a heated extract, and two gradient ethanol precipitates of the crude extract. Intolerance was reported by 20% of the patients and was not related to the detection of any special allergen, nor to total or specific IgE levels. Intolerant patients only reported a higher frequency of digestive symptoms than the patients who tolerated fish ingestion. The most sensitive immunoblot antigen source was the 50–66% ethanol fraction of a crude extract (10× concentrated) that was found to be positive in 100% of the samples. Interestingly, 95% sensitivity in the IgE-immunoblot assay could be achieved using only two allergens, Ani s 1 and Ani s 4. Allergens from the dead larvae remain a problem for 20% of the sensitized subjects. The use of a fractionated and concentrated crude extract improved the sensitivity of the immunoblot assay.

Gal d 6 Is the Second Allergen Characterized from Egg Yolk

Only one allergen from the egg yolk, alpha-livetin (Gal d 5) has been described thus far. A new egg yolk allergen was detected studying 27 egg allergic patients. The study was performed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and IgE-immunoblotting and IgE-immunoblotting-inhibition assays. An egg yolk extract was fractioned by reverse-phase high-performance liquid chromatography (RP-HPLC), and the new allergen detected was characterized by N-terminal amino acid analysis. A total of 5 of the 27 patients (18%) detected a yolk allergen of an apparent molecular weight of 35 kDa by SDS-PAGE. Heating and reduction treatments did not affect its allergenicity, although digestion with simulated gastric fluid diminished the IgE-binding capacity of the allergen. The N-terminal amino acid sequence corresponded with the YGP42 protein, a fragment of the vitellogenin-1 precursor. Thus, a second egg yolk allergen has been described and designated Gal d 6 by the World Health Organization (WHO)/International Union of Immunological Societies (IUIS) Allergen Nomenclature Subcommittee.

Ani s 10, a new Anisakis simplex allergen: Cloning and heterologous expression ☆

Anisakiasis is a human disease caused by accidental ingestion of larval nematodes belonging to the Anisakidae family. Anisakiasis is often associated with a strong allergic response. Diagnosis of A. simplex allergy is currently carried out by test based on the IgE reactivity to a complete extract of L3 Anisakis larvae although the specificity of these diagnostic tests is poor. Improving the specificity of the diagnostic test is possible using purified recombinant allergens. A new Anisakis allergen, named Ani s 10, was detected by immunoscreening an expression cDNA library constructed from L3 Anisakis simplex larvae. The new allergen was overproduced in Escherichia coli; it is a protein of 212 amino acids and it was localized as a 22 kDa protein band in an ethanol fractionated extract from the parasite. Ani s 10 has no homology with any other described protein, and its sequence is composed by seven almost identical repetitions of 29 amino acids each. A total of 30 of 77 Anisakis allergic patients (39%) were positive both to rAni s 10 and natural Ani s 10 by immunoblotting. The new allergen could be useful in a component-resolved diagnosis system for Anisakis allergy.

Isolation of Ani s 5, an excretory–secretory and highly heat-resistant allergen useful for the diagnosis of Anisakis larvae sensitization

Allergens Ani s 1 and Ani s 4 have demonstrated their utility for the diagnosis of the sensitization to larvae of the genus Anisakis. The aim was to determine the number of patients with compatible clinical history, who did not recognize Ani s 1 and Ani s 4, and characterize the allergens responsible for their sensitization. Eighty-four patients were studied by CAP and immunoglobulin E (IgE) immunoblotting. The 12% of the patients recognized allergens different from Ani s 1 and Ani s 4, being half sensitized to a heat-resistant 15-kDa allergen, which was isolated by ethanol fractionation, followed by a hydroxyapatite chromatography and a reversed-phase high-performance liquid chromatography and identified by its amino terminal sequence as Ani s 5. A total of 41 of the 84 patients studied (49%) showed specific IgE to Ani s 5 that was detected among the excretory–secretory products and immunohistochemically located at the excretory gland, ventriculus, and the luminal surface of the intestinal epithelium of the 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.

Occupational Sensitization to Fungal Enzymes Used in Animal Feed Industry

Background: Industrial enzymes cause the increasing prevalence of occupational hypersensitivity. Our objective was to study workers occupationally exposed to fungal enzymes in 2 animal feed factories to determine if the sensitization originated in the enzymes or was caused by the microorganism used to produce the enzymes. Methods: Eighty-six consenting workers were studied by skin prick tests with extracts from the enzymatic products handled in their factories. Positive workers were then studied by IgE immunoblotting and basophil activation was measured by flow cytometry. Results: Eight of the 86 workers analysed (9%) tested positive and were more frequently sensitized to phytase from Trichoderma and Peniophora. Glucanase and α-amylase from Bacillus amyloliquefaciens did not cause sensitization in any worker. No cross-reactions were observed between Trichoderma and Peniophora sp. phytases. Workers were sensitized to the product that they handled. Conclusions: Fungal enzymes cause occupational hypersensitivity in animal feed industries. Immunoblotting and basophil activation are useful to evaluate the effects of handling enzymes as part of the medical surveillance of enzyme-exposed workers. We describe Peniophora sp. 6-phytase as a new allergen and enzymes from Trichoderma as strong sensitizers.

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.

Characterization of occupational sensitization by multiallergen immunoblotting in workers exposed to laboratory animals

BACKGROUND Studies have estimated that 10% to 23% of workers exposed to laboratory animals report symptoms of laboratory animal allergy. OBJECTIVES To determine the level of occupational sensitization in workers exposed to laboratory animals and to develop a diagnosis system based on a multiallergen IgE immunoblot. METHODS A total of 75 workers exposed to laboratory animals were initially studied with skin prick tests performed with animal epithelia extracts. The workers with suspected occupational disease and positive skin prick test results were further studied with the ImmunoCAP system to determine specific IgE levels to urine and epithelia allergens and with multiallergen IgE immunoblotting to detect specific IgE levels to epithelia allergens and bovine serum albumin. RESULTS Twenty of the 75 workers were studied with ImmunoCAP and multiallergen IgE immunoblotting. Nine were polysensitized and 3 were sensitized to only one animal. The results obtained by ImmunoCAP and multiallergen IgE immunoblotting were concordant except for in 3 workers, who had low or negative values of specific IgE determined by ImmunoCAP but positive allergen detections by immunoblotting. On the basis of the results of the study and the clinical symptoms related by workers, 16% were diagnosed as having occupational allergy. CONCLUSIONS Multiallergen immunoblotting by means of a unique test offers a graphic representation of sensitization to the different animals to which workers are exposed, providing additional information on the clinical symptoms caused by the involved allergens. The results presented suggest that this system can improve the diagnosis of laboratory animal allergy by obtaining a sensitization profile for each exposed worker.