María Luisa Caballero

Occupational immunologic contact urticaria from pine processionary caterpillar (Thaumetopoea pityocampa): experience in 30 cases.

Cutaneous lesions caused by the pine processionary caterpillar Thaumetopoea pityocampa (TP) are frequent in pinewood areas. In the present study, 30 patients diagnosed with occupational immunologic urticaria from this caterpillar were included. Immediate hypersensitivity was demonstrated by performing prick and IgE‐immunoblotting tests. Workers were grouped according to their common tasks. Occupations at risk of exposure to TP were pine‐cone collectors/woodcutters (14), farmers/stockbreeders (8), other forestry personnel (4), construction workers (2), residential gardeners ( 1 ) and entomologists ( 1 ). Besides contact urticaria, angioedema (60%), papular lesions of several days of evolution (30%) and anaphylactic reactions (40%) were also detected. The most frequently detected molecular weight bands by immunoblot were 15 (70%), 17 (57%) and 13 kDa (50%). The appearance of isolated bands corresponds with the least serious cases. Only 8 subjects had bands higher than 33 kDa, which was present in the 3 most severe cases of anaphylactic reactions. By presenting these cases, we wish to offer the largest series reported so far of occupational immunologic contact urticaria caused by TP. We include the first cases described in certain occupations, some of them not directly related to forestry work. Pine‐cone or resin collectors, woodcutters, farmers and stockbreeders were the most frequently and severely affected workers.

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.

Isolation and characterization of Tha p 1, a major allergen from the pine processionary caterpillar Thaumetopoea pityocampa

Background: Pine processionary caterpillars induce dermatitis by a toxic–irritative mechanism. The existence of true allergic reactions to allergens from these caterpillars has been recently demonstrated by positive immediate skin prick tests and specific IgE determination by immunoblotting using crude larval extracts. The aim of this work was to purify allergens from the crude larval extract in order to characterize IgE‐binding proteins from these caterpillars.

Specific IgE determination to Ani s 1, a major allergen from Anisakis simplex, is a useful tool for diagnosis.

BACKGROUND The most serious limitation of the serodiagnosis of parasitoses is the occurrence of cross-reactions. OBJECTIVE The possible use of Anisakis simplex major allergen Ani s 1 for diagnosis. PATIENTS AND METHODS Forty-nine non-fish-allergic patients with Anisakis simplex hypersensitivity, 21 patients without allergic episodes suffering intestinal anisakiasis with obstruction of the intestinal lumen, and 10 unrelated sera as a control were included in this study to determine specific immunoglobulin (Ig)E and IgG to Anisakis simplex major allergen Ani s 1 by immunoblotting. RESULTS Eighty-six percent of patients with Anisakis simplex hypersensitivity showed specific IgE directed to Ani s 1. Identical result was obtained for IgG detection in this group. Among patients with intestinal anisakiasis, 86% showed specific IgE, but only 29% had specific IgG (P < 0.001, two-tailed Fisher exact test). One of the 10 control subjects was positive both for IgE and IgG (P < 0.001). CONCLUSIONS Determination of specific IgE directed to Anisakis simplex major allergen Ani s 1 is a useful tool for the diagnosis of hypersensitivity and intestinal anisakiasis. Further, measurement of specific IgG directed to Anisakis simplex major allergen Ani s 1 is only valid for Anisakis simplex allergy.

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.

Unsuspected Lupin Allergens Hidden in Food

Background: Lupin is a herbaceous plant from the legume family whose seed allergens usually have cross-reaction with peanut. Lupin flour is used in human nutrition because of its high nutritional and functional qualities. Aims: The aim of this work was to detect non-specified lupin proteins contained in several manufactured foods. Methods: Serum from a patient suffering anaphylactic episodes after ingestion of a certain brand of cookies and with oral allergy syndrome after eating chicken bouillon was used as a tracer. Lupin seeds and commercial food extracts were analyzed by SDS-PAGE, immunoblotting and immunoblotting inhibition. Lupin extract allergenicity after thermal processing was also analyzed. Results: A lupin allergen with a molecular weight close to 14 kDa was detected in extracts from cookies, a chicken bouillon cube and a chicken dehydrated soup. Conclusions: The presence of unsuspected, hidden non-specified lupin sources in food labeling was demonstrated. According to the results of this study, it is important for food-allergic patients that food labels should declare all the components irrespective of their quantity.

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.

High Prevalence of Seropositivity to a Major Allergen of Anisakis simplex, Ani s 1, in Dyspeptic Patients

ABSTRACT Finding evidence of anisakidosis requires invasive methods. We have developed a serological assay based on the detection of an immunoglobulin E (IgE) specifically directed against Ani s 1 protein, a major parasite allergen of Anisakis simplex, which has shown a high level of accuracy in the diagnosis of anisakidosis. We used this tool to determine the prevalence of anti-Ani s 1 IgE in dyspeptic patients and to investigate if its seropositivity could be related to epidemiological factors other than raw fish consumption. A total of 174 dyspeptic patients who submitted to upper digestive tract endoscopy were studied. Specific IgE against Ani s 1 was determined by immunoblotting. Quantitative information on smoking, alcohol consumption, and fish consumption as well as a history of gastric surgery was recorded. Twenty-four (13.8%) patients were seropositive for Ani s 1 protein. The seroprevalence of anti-Ani s 1 IgE increased with age in patients who were less than 62 years old (P = 0.047). Seropositivity to Ani s 1 was associated with the consumption of fish in vinegar (P < 0.001), raw fish (P = 0.001), and smoked fish (P = 0.007). There was no relationship between anti-Ani s 1 IgE seropositivity and the number of cigarettes smoked (P = 0.098) or alcohol intake (P = 0.179). Five patients had undergone previous gastric surgery, and three of those patients were seropositive for Ani s 1 (P = 0.019). In multivariate analysis, the consumption of fish in vinegar (P = 0.006), raw fish (P = 0.017), and smoked fish (P = 0.002) and a history of gastric surgery (P = 0.005) were independent factors associated with anti-Ani s 1 IgE detection. In conclusion, at present, anisakidosis might frequently be underdiagnosed, and it might have a clinical role in patients with upper dyspeptic symptoms. Uncooked-fish ingestion and previous gastric surgery were associated with seropositivity for Ani s 1 protein.