Several allergens from<Emphasis Type="Italic"> Anisakis simplex</Emphasis> are highly resistant to heat and pepsin treatments

Ingestion of raw or undercooked fish can lead to infection with Anisakis simplex. Sensitized patients show specific IgE to proteins from this parasite. The aim of this study was to assess the frequency of specific IgE recognition directed to heat and/or pepsin-resistant allergens from A. simplex among sensitized patients. Twenty-seven patients with positive specific IgE and immunoblotting with a crude parasite extract were included in the study. Specific IgE detection against allergens resistant to boiling for 30 min and/or a pepsin digestion of an A. simplex extract was performed by immunoblotting. A total of 81% of the patients showed specific IgE to pepsin-resistant allergens and 67% had specific IgE to heat-resistant allergens. Thirty percent of patients recognized allergens after both treatments, one being the allergen detected by 75% of the patients of this group. Heat- and/or pepsin-resistant allergens from A. simplex could explain reactions and symptoms after the ingestion of well-cooked or canned fish.     

Anisakis simplex, a relevant etiologic factor in acute urticaria

Del Pozo MD, Audícana M, Diez JM, Muñoz D, Ansotegui IJ, Fernández E, García M, Etxenagusia M, Moneo I, Fernández de Corres L. Anisakis simplex , a relevant etiologic factor in acute urticaria.
Anisakis simplex , a parasite of fish and cephalopods, can induce IgE-mediated reactions. This study aimed to determine the etiologic role of A. simplex in patients affected by urlicaria/angioedema 'AE' or anaphylaxis. We studied 100 adult subjects suffering acute episodes of urticaria/AE, by anamnesis, prick tests with A. simplex and fish-mix extracts, and total and specific IgE to both A. simplex and cod. The following criteria of A. simplex allergy were considered: 1' urticaria/AE within 6 h after fish ingestion; 2' specific IgE to A. simplex; 3' positive prick test to A. simplex extract; 4' exclusion of other suspected causes. Double-blind, placebo-controlled food challenge was not carried out because ethical considerations forbid challenge with a parasite. Specific IgE to A. simplex '<0.7 kU/1' was found in 22 subjects, but only eight were diagnosed as having A. simplex allergy. Other allergens were involved in 37 patients, and 55 cases were considered idiopathic. Specific IgE to fish '<0.7 kU/1' was found in two patients, but only one was diagnosed as having fish allergy. We concluded that A. simplex is an important etiologic factor in acute urticaria. We suggest that it should be considered in cases of urticaria/AE or anaphylaxis, especially after fish ingestion.     

Characterization of allergens secreted by Anisakis simplex parasite: clinical relevance in comparison with somatic allergens

BACKGROUND: Diagnostic methods for the study of allergic reactions to Anisakis simplex (A.s.) based on whole-body extracts of the larva are clearly insufficient. OBJECTIVES: To study the allergenicity of the proteins secreted by the parasite. Comparison with somatic antigens and determination of their clinical importance in allergic patients were also addressed. METHODS: An excretory/secretory (E/S) extract was produced by culturing third-stage A.s. larvae. It was used to perform immediate skin tests and to determine specific IgE in 10 patients diagnosed with allergy to A.s. Both tests were compared with the results obtained with the whole-body extract (somatic (S)). The molecular weight (MW) of their allergens was determined by immunoblotting, and a single-blind placebo-controlled oral challenge with E/S proteins was performed. Finally, allergens' resistance to gastric pepsin and acid pH was explored. RESULTS: A.s. larvae secreted allergens more potent than those present in the S extract. The skin prick test wheal area produced by E/S molecules and the absorbance obtained in the determination of specific IgE with these allergens (ELISA) were 5.8 times bigger than those obtained with S extract. MW allergens of 72 and 56 kDa in E/S extracts and those of 56, 48 and 43 kDa in S extract were recognized by more than 50% of the patients. Partial cross-reactivity between them was revealed by immunoblotting inhibition studies. Oral challenge with E/S extract (up to 479 microg) was negative in all the patients. Treatment of E/S proteins with gastric pepsin inhibited the binding of the E/S allergens for specific IgE. The acid pH did not affect the overall binding of IgE to E/S extract. It decreased by 15.23% and 19.96% at pH 4 and 2, but the difference was not statistically significant. CONCLUSION: A.s. secretes allergens more potent than somatic antigens and should be used in the diagnostic procedures. These allergens are inactivated by the pepsin, which supports the theory that live larva is necessary to induce an allergic reaction in most of the patients.     

Western blot analysis of water-soluble wheat flour (Triticum vulgaris) allergens.

Water-soluble wheat flour allergens were analyzed using the western blot technique. The sera of 130 bakers (42 with wheat flour allergy, 88 without allergy) were analyzed for IgE, IgG, and IgG4 binding towards wheat flour allergens. Three major allergens with molecular weights of 47, 17, and 15 kD were identified (Tri v Bd 47, Tri v Bd 17, and Tri v Bd 15; IUIS allergen nomenclature) by their ability to bind IgE from approximately 50% of the allergic sera. The specificity of the IgG was mostly directed against three high molecular weight components (134, 122, and 98 kD). In contrast, IgE and IgG4 was found frequently against the major allergens. 40% of healthy donors versus 12% of the asthmatic bakers had no detectable IgG4 against wheat flour extract. The biological activity of the water-soluble wheat flour components has been shown by their ability to induce histamine release from basophil leukocytes obtained from patients with bakers' asthma. The release of histamine from basophil leukocytes significantly correlated with the presence of IgE antibodies against wheat flour components of 47, 17 and 15 kD. A regulatory role of wheat flour specific IgG4 in allergic diseases could not be evaluated by western blot analysis.     

Incidence of sensitivity to Anisakis simplex in a risk population of fishermen/fishmongers.

BACKGROUND: Anisakis simplex, a fish and cephalopodes parasite, can cause either gastrointestinal symptoms or allergic reactions in humans on eating/handling contaminated fish. OBJECTIVE: The aim of our study was to determine the capacity of Anisakis simplex to induce specific IgE production and allergic reactions following eating and handling fish in a population at risk. METHODS: We determined the levels of total IgE, specific IgE, and eosinophil count in 28 fishermen/fishmongers (group A) and 15 healthy donors (group B). A skin prick test (SPT) with extracts from Anisakis and the most common species of fish in our country, were also carried out. RESULTS: Specific IgE to Anisakis were found in 14 subjects of group A (13 of them had a positive SPT to the same extract) and none of group B (only one subject had a positive SPT). The SPT with fish extracts was positive in 4 patients of group A but in none of group B. Subjects in group A with specific IgE to Anisakis showed higher total IgE levels and eosinophil counts compared with either other individuals of the same group or to those of group B. CONCLUSIONS: These results indicate that fishermen/fishmongers are a population at risk for Anisakis simplex sensitization and suggest that this kind of sensitization should also be investigated not only in subjects like fishermen/fishmongers who live in countries where fish is likely to be contaminated with Anisakis simplex parasites, but also in those who handle fish for other reasons.     

Allergenic cross-reactivity between the nematode Anisakis simplex and the dust mites Acarus siro, Lepidoglyphus destructor, Tyrophagus putrescentiae, and Dermatophagoides pteronyssinus

Background: The nematode Anisakis simplex is a common parasite on fish and other seafood. It is considered to be a food allergen and to induce IgE-mediated reactions. Allergenic cross-reactivity between A. simplex and other nematodes has been reported, as has cross-reactivity with arthropods: red mosquito larvae and German cockroach. We have here studied the allergenic relationship between A. simplex and four different dust-mite species.
Methods: Serum samples collected from 69 farmers allergic to dust mites were analyzed for IgE to A. simplex by CAP FEIA. Allergenic cross-reactivity between A. simplex and dust mites was studied in two of the sera by CAP FEIA and immunoblotting inhibition.
Results: We found that 14/69 farmers had detectable levels of IgE antibodies to A. simplex . The IgE response in CAP FEIA to A. simplex was inhibited to various degrees in the two studied sera by extracts of the dust mites Acarus siro , Lepidoglyphus destructor , Tyrophagus putrescentiae , and Dermatophagoides pteronyssinus . In the reverse inhibition experiment, extract of A. simplex inhibited the response in both sera to A. siro and T. putrescentiae , but not to L. destructor . The IgE binding to D. pteronyssinus was inhibited in one of the two sera. In blotting inhibition experiments, the IgE binding to several allergens in A. simplex was inhibited by each of the four mite extracts, especially by A. siro and T. putrescentiae , which completely inhibited the IgE binding to several allergens.
Conclusions: The results show allergenic cross-reactivity between several allergens in A. simplex and four dust-mite species. The clinical significance of this cross-reactivity remains to be evaluated.     

Characterization of allergens of Anisakis simplex

BACKGROUND: Anisakis simplex is an intestinal parasite of sea mammals. The larvae infect crustaceans, cephalopods and fish. Humans may consume A. simplex third stage larvae (L3) when eating infected raw or under-cooked fish. Consumed larvae cause an inflammatory reaction when they penetrate the digestive mucosa. The larvae or their secretory/excretory products can sensitize humans and induce an immunoglobulin E (IgE)-mediated allergic reaction. This parasite is now being implicated in numerous cases of allergic reactions after eating fish. The purpose of this study was to evaluate the allergenicity of proteins present in an extract of the third stage larva. METHODS: Rabbit antiserum raised to A. simplex somatic extract (L3) was reacted by crossed immunoelectrophoresis (CIE) with the same somatic extract. Crossed radioimmunoelectrophoresis (CRIE) was also performed by incubating CIE gels first in the sera of 13 individuals with positive immunoCAP to A. simplex and then in radiolabeled anti-human IgE. RESULTS: Twelve to 16 antigen-antibody precipitin peaks were visualized on Coomassie blue stained CIE gels in which somatic extract was reacted with somatic-antiserum. Autoradiography of CRIE gels showed that 18 different proteins bound IgE in patient sera. Individual patients had serum IgE directed at two to 10 different allergens. Five of these allergens were recognized by >/=50% of the patients. No allergen was recognized by every patient and no patient had serum IgE directed at all 18 allergens. CONCLUSION: Somatic extracts of A. simplex L3 larva contain a large number of allergenic molecules and there is significant variability between patients in their sensitivity and reactivity to these allergens.     

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.     

The diversity of allergens involved in bakers' asthma

Sera from 35 individuals with suspected allergies to inhaled flour were screened for the presence of immunoglobulin E (IgE) specific for wheat-flour proteins. Sera from nine asthmatic bakers with high wheat RAST scores were selected for further study with the aim of purifying the allergen(s) involved in bakers’ asthma and related conditions. However, each of the different techniques applied–ion exchange chromatography, preparative isoelectric focusing and the electrophoretic transfer, or‘Western blotting’ technique, showed that serum IgE from different individuals have markedly different specificities and bind to numerous wheat proteins. When three purified wheat proteins were tested–wheat germ agglutinin; a fraction purified using a concanavalin-A affinity column and a putative trypsin inhibitor–all were identified as allergens for some but not all of the allergic bakers.     

Prevalence of and risk factors for IgE sensitization to Anisakis simplex in a Spanish population

BACKGROUND: The number of allergic reactions to A. simplex reported in Spain has increased dramatically in the last decade. Nevertheless, there have been no studies of the prevalence of and possible risk factors for IgE sensitization to this parasite, possibly because suitably specific diagnostic methods have only recently become available. The objective was to investigate the prevalence of and risk factors for IgE sensitization to A. simplex in Galicia, a region of northwestern Spain with a population of about 3 million and high average fish consumption (78.5 g/person per day). METHODS: The study was performed with a random sample of 2801 healthy blood donors distributed in 53 geographic areas, proportional to the density of donors. IgE sensitization to A. simplex was tested by a capture ELISA method that has proved to be the most specific method currently available. RESULTS: The results showed a total of only 12 positive subjects, of whom five also showed IgG1 sensitization. All positive subjects and 101 randomly selected seronegative subjects were then included in a case-control study of risk factors for sensitization to A. simplex, based on a telephone interview about fish consumption (especially raw and undercooked fish). All seropositive subjects (but only 25% of seronegative subjects) reported consumption of undercooked fish or homemade raw-fish products. CONCLUSIONS: Our results strongly suggest that sensitization to A. simplex is caused only by live larvae, and not by allergens contained in fish tissues, and that ingestion of homemade boquerones (anchovies [Engraulis encrasicholus] in vinegar), and to a much lesser extent of undercooked fish, are the main risk factors for IgE sensitization to Anisakis in this region.     

Exposure to wheat allergen and fungal α-amylase in the homes of bakers

BACKGROUND: Few data are available on exposure to occupational allergens in dwellings occupied by inhabitants with occupational exposure to allergens. In small bakeries working and living often takes place in the same building. It is possible that allergens from the bakery can be transported into the homes of the bakers, via the clothes or shoes of the baker. OBJECTIVE: The aims of this study were to investigate exposure to occupational allergens, wheat and fungal alpha-amylase in the homes of bakers, and evaluate potential determinants of exposure. Sensitization in family members to occupational allergens was investigated in a small preliminary survey. METHODS: Floor dust samples were collected in the homes of 34 bakers. Levels of wheat and fungal alpha-amylase allergens were determined in an extract of the dust samples. Blood samples were collected from bakers and their family members to determine the prevalence of sensitization to occupational allergens. RESULTS: The concentration of wheat and alpha-amylase allergens ranged from 38.9 to 172.4 microgeq/m(2) (GM), to 10.5-76.7 ngeq/m(2) (GM). Higher levels of dust and allergens were measured when the house could be reached directly through the bakery, and in houses with textile floor covers. Higher concentrations were also measured when bakers brought their work clothes and shoes into the house and when textiles from the bakery were laundered at home. Some family members appeared to be sensitized to wheat flour and alpha-amylase, but it cannot be excluded that they became sensitized because of their incidental presence in the bakery. CONCLUSIONS: Occupational allergens can be found in house dust from the homes of bakers and levels are associated with hygienic behaviour and distance to the bakery.     

Skin test and RAST responses to wheat and common allergens and respiratory disease in bakers

Interrelationships between skin and humoral tests for immediate hypersensitivity to wheat and indicators of respiratory disease were examined in 176 male bakers. Skin tests were assessed by measuring the diameter of the weal resulting from prick innoculation of allergen extract and circulating allergen-specific IgE by radioallergosorbent test (RAST). Fifteen per cent of subjects showed positive skin-prick test responses to wheat extracts. These subjects demonstrated an increased prevalence of respiratory symptoms and of measurable bronchial responsiveness to methacholine. Thirty per cent of subjects had positive skin test responses to common allergens but negative responses to whole wheat. Compared to subjects with no positive skin test responses they had an increased prevalence of bronchial responsiveness to methacholine but a similar prevalence of respiratory symptoms. There was a significant association between skin test responses to whole wheat and skin test responses to common allergens suggesting that bakers with pre-existing sensitivity to common allergens are at increased risk of developing wheat flour sensitization. There was no significant difference between skin-prick test and RAST responses to wheat, water-soluble wheat protein and common allergens. Both tests showed similar relationships with indices of respiratory disease. The associations between skin test and RAST responses to wheat extracts and indices of respiratory disease was stronger for the water-soluble wheat proteins than for other wheat grain extracts. These results suggest that immediate hypersensitivity to wheat flour is important in the development of non-specific bronchial hyperreactivity in bakers and that the water-soluble fractions of wheat flour are the most important allergenic components. It agrees with in vitro results showing that water-soluble wheat proteins bind IgE in sensitive bakers more closely than other grain extracts.     

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.     

Characterization of allergenic components of rye and wheat flour (Secale, Triticum vulgaris) by western blot with sera of bakers: their effects on CD23 expression

The allergenic components of water-soluble rye flour extract were studied by immunoblotting. Sera from 100 bakers were analyzed for their IgG, IgG4 and IgE binding pattern. Two allergens with molecular weights of 35 and 14 kD were detected. Previously, the major allergens of wheat flour extract were identified. The wheat flour components at a MW of 15/17 kD and the rye flour component at a MW of 14 kD were purified and isolated. The modulation of the low affinity receptor for IgE (Fc epsilon RII/CD23) on monocytes by separated allergenic components was studied. Depending on the allergen concentration the CD23 expression on isolated cells increased after stimulation with the rye flour component (MW 14 kD). The combined addition of the rye flour component (14 kD) with IL-4 induced a significant CD23 expression as compared to IL-4 alone.     

A phage display system for the identification of novel Anisakis simplex antigens

Anisakis simplex has been recognized as an important cause of disease in man and as a foodborne allergen source. Actually, this food-borne was recently identified as an emerging food safety risk including allergenic symptoms. This parasite contains a large variety of allergenic proteins enforcing the necessity to detect new allergens. Commonly, these efforts have been focused on the developing of cDNA libraries, where virtually all expressed mRNAs are present, by using immunoreactive patient serum or polyclonal antibodies. Phage display system is an alternative strategy which permits the physical binding of the genotype with the phenotype, since the products are expressed by the phage on its surface, thereby allowing more efficient selection. In this work we have constructed two libraries in the pJuFo phage, obtaining a primary titer of around 103 cfu/ml and an amplified titer of the order of 1013 cfu/ml whereas the insert sizes varied from 0.35 to 1.2kb. Both libraries were subsequently analyzed by enrichment with polyclonal antibodies to an A. simplex extract and immunoreactive sera from patients with a clinical history of allergy to this parasite. Finally, 30 clones were scrutinized detecting several Anisakis candidate antigens. Actually, one protein, belongs to the fructose-1,6-bisphosphatase family, was found in 34% of scrutinized clones revealing as a promising novel A. simplex allergen. Phage display technology has to date not yet been applied to the identification of new A. simplex allergens, and the present work opens up new avenues to the understanding of the Anisakis allergenic process.     

Purification and cDNA cloning of a new heat-stable allergen from Anisakis simplex

The nematode Anisakis simplex is a representative parasite for marine animals and occasionally causes not only anisakiasis but also allergic reactions in sensitized subjects. Besides the known allergens, a number of unidentified allergens have been suggested to still exist in A. simplex. In this study, a new heat-stable allergen of 15kDa (named Ani s 8) was purified from the third stage larvae of A. simplex by gel filtration on Sephacryl S-300, anion-exchange HPLC on Mono Q and reverse-phase HPLC on TSKgel Phenyl-5PW RP. Analysis by fluorescence ELISA showed that 7 of 28 Anisakis-allergic patients had elevated serum levels of IgE to Ani s 8. On the basis of the determined partial amino acid sequence, the complete sequence of Ani s 8 (composed of 150 amino acid residues) was elucidated by cDNA cloning, in which as many as 32 homologs of the cDNA encoding 10 isoforms of Ani s 8 were detected. Ani s 8 shares amino acid sequence homology (up to 36%) with several members of the SXP/RAL-2 protein family, including Ani s 5 (15kDa) previously identified as an A. simplex allergen. Inhibition ELISA data demonstrated the IgE cross-reactivity between Ani s 8 and Ani s 5.     

Baker’s asthma: Still among the most frequent occupational respiratory disorders

Background: Baker’s asthma and rhinitis are among the most frequent occupational respiratory disorders. Objective: The aim of the study was to evaluate the frequency of work-related symptoms and the clinical relevance of sensitization to allergens in screened and symptomatic bakers. Methods: Eighty-nine bakers participating in a screening study and 104 bakers filing a claim for compensation were examined with regard to occupational and clinical case history, lung function parameters, and sensitization to bakery allergens by skin prick tests, specific IgE analyses, and inhalative challenge tests. Results: A high prevalence of respiratory disorders, abnormal lung function parameters, and sensitization to bakery allergens exists. Most frequently, bakers with workplace-related respiratory symptoms showed sensitization to wheat flour (64%), rye flour (52%), soy bean flour (25%), and α-amylase (21%). The correlation between these sensitizations and asthma case history and inhalative challenge test responses was significant. However, approximately 29% of the bakers with respiratory symptoms showed no sensitization to these bakery allergens, whereas 32% of the sensitized bakers in the screening group had no workplace-related symptoms. Atopic status defined by skin prick test sensitization to common allergens or elevated total IgE levels was found to be a risk factor for the development of sensitization to bakery allergens and respiratory symptoms. On the other hand, there is evidence for an increased frequency of elevated total IgE as the result of occupational allergen exposure because respective findings were observed in bakers without symptoms. Conclusion: Sensitization to bakery allergens seems to be the main cause of baker’s asthma and rhinitis but cannot explain the asthma case history in each case. Further methods are required to objectively assume irritative pathomechanisms. Our findings indicate the necessity for an improved primary prevention of exposure to inhalative noxae in bakeries. (J Allergy Clin Immunol 1998;102:984-97.)     

Allergen discs prepared from nitrocellulose: detection of IgE binding to soluble and insoluble allergens

Nitrocellulose discs, 6 mm in diameter, are suggested as an alternative to cyanogen bromide-activated paper for the coupling of allergens in radio- or enzyme-linked assays to estimate allergen-specific IgE in serum. The preparation of allergen discs with nitrocellulose is simple, involving 3 steps: (a) drying allergen extract onto disc, (b) soaking discs in a 3% solution of bovine serum albumin, and (c) washing out the buffer. Very similar results (r = 0.94) were obtained using either paper or nitrocellulose discs for radioallergosorbent testing of sera from 15 bakers using allergens from mites, ryegrass pollen or wheat grain. The amount of protein (as radiolabelled albumin) actually bound to either type of disc or microtitre trays was similar, and low (5% of the protein applied for each media). However, when the protein was applied to nitrocellulose in 1% KOH up to 70% of it was bound. This solvent permitted better evaluation of IgE binding to insoluble allergens such as glutenin, which proved to be the most allergenic wheat-grain fraction tested by this method for a group of 9 bakers.     

Biological activity of IgE specific for cross-reactive carbohydrate determinants

BACKGROUND: The clinical relevance of IgE specific for cross-reactive carbohydrate determinants (CCDs) has been a matter of controversy. Until now, no convincing experiments have been performed to test the biologic significance of individual multivalent allergens that carry multiple carbohydrate epitopes. OBJECTIVE: We sought to contribute to the understanding of the role of CCD-specific IgE antibodies and to study whether CCD-specific IgE antibodies are able to activate basophils using different multivalent glycoproteins as antigens. METHODS: Purified natural tomato beta-fructofuranosidase (nLyc e 2) and rLyc e 2.02 expressed in Escherichia coli were compared by means of histamine release tests. In addition, native and deglycosylated horseradish peroxidase and a neoglycoprotein consisting of a defined glycopeptide (Manalpha1-6[Xylbeta1-2]Manbeta1-4GlcNAcbeta1-4[Fucalpha1-3]GlcNAc) coupled to BSA were used in histamine release assays using stripped basophils from normal donors resensitized with IgE from CCD-reactive patients with food allergy to tomato. RESULTS: Ten CCD-positive and 2 CCD-negative sera from patients with tomato allergy underwent histamine release testing by using the glycoproteins and nonglycosylated controls as antigens, respectively. All sera induced histamine release with tomato extract (up to 100%), confirming the allergic status of the donors. Four of the CCD-positive sera induced releases ranging from 12% to 82% with all of the glycoproteins but not with the nonglycosylated or monovalent controls. All other sera showed no response or only very weak response to the glycoproteins. CONCLUSION: Approximately one third of the CCD-positive sera from patients with tomato allergy have biologically relevant CCD-specific IgE antibodies. Therefore the general claim that CCD-specific IgE is clinically irrelevant has to be reconsidered critically. Hence IgE specific for CCDs should be taken into consideration in the diagnosis and therapy of certain allergies. In the subgroup of patients sensitized to CCDs, the use of natural allergens should be preferred over the use of recombinant allergens expressed in prokaryotic organisms.     

Anisakis simplex: from obscure infectious worm to inducer of immune hypersensitivity

Infection of humans with the nematode worm parasite Anisakis simplex was first described in the 1960s in association with the consumption of raw or undercooked fish. During the 1990s it was realized that even the ingestion of dead worms in food fish can cause severe hypersensitivity reactions, that these may be more prevalent than infection itself, and that this outcome could be associated with food preparations previously considered safe. Not only may allergic symptoms arise from infection by the parasites ("gastroallergic anisakiasis"), but true anaphylactic reactions can also occur following exposure to allergens from dead worms by food-borne, airborne, or skin contact routes. This review discusses A. simplex pathogenesis in humans, covering immune hypersensitivity reactions both in the context of a living infection and in terms of exposure to its allergens by other routes. Over the last 20 years, several studies have concentrated on A. simplex antigen characterization and innate as well as adaptive immune response to this parasite. Molecular characterization of Anisakis allergens and isolation of their encoding cDNAs is now an active field of research that should provide improved diagnostic tools in addition to tools with which to enhance our understanding of pathogenesis and controversial aspects of A. simplex allergy. We also discuss the potential relevance of parasite products such as allergens, proteinases, and proteinase inhibitors and the activation of basophils, eosinophils, and mast cells in the induction of A. simplex-related immune hypersensitivity states induced by exposure to the parasite, dead or alive.