An extended study of seroprevalence of anti-Anisakis simplex IgE antibodies in Norwegian blood donors.

During the last decade, cases of the fish parasite Anisakis simplex infection and allergy in human have increased in countries with high fish consumption. Our aim was to perform an extended seroprevalence study of anti-IgE antibodies against this parasite in Norway, one of the high fish-consuming countries. At the Department of Immunology and Transfusion Medicine and the Laboratory of Clinical Biochemistry, Haukeland University Hospital, Bergen, Norway, two main groups of anonymized serum samples were collected; the first (n = 993) from recently recruited blood donors (designated 'BDO') and the second (n = 414) from patient with total IgE levels ≥1000 kU/l (designated 'IGE+'). The sera were analysed by the ImmunoCAP(®) method for total IgE and IgE antibodies against A. simplex, house dust mite (HDM), shrimp, cod, crab, brine shrimp and shrimp tropomyosin. The A. simplex positive sera were further tested by an enzyme-linked immunosorbent assay (ELISA) method, which uses 2 recombinant (r) major allergens, rAni s 1 and rAni s 7 as target antigens. SDS-PAGE and Western immunoblotting analyses were also performed. Whereas the prevalences by ImmunoCAP(®) were 0.4% and 16.2% in the BDO and IGE+ groups, respectively, analyses with recombinant allergens showed only 0.0% and 0.2%. Cross-reactivity and immunoblotting analyses suggested that most of the ImmunoCAP(®) positive sera were probably false-positive due to cross-sensitization to shrimp and HDM. However, positivity due to other A. simplex antigens should also be considered. Compared with other high fish-consuming countries, we observed a very low seroprevalence of anti-Anisakis IgE antibodies in a Norwegian population.

Identification of new potential allergens from Nile perch (Lates niloticus) and cod (Gadus morhua).

BACKGROUND: Globalization of the food industry has led to widespread exposure to new nondomestic fish species; therefore, identification of potential allergens is necessary in order to diagnose allergic reactions. OBJECTIVE: Contact with a patient who was allergic to Nile perch (Lates niloticus) prompted us to investigate the immunoglobulin (Ig) E-reactive proteins that could be allergens of this species. METHODS: 2D gel electrophoresis was used to separate the muscle proteins of L niloticus and Gadus morhua. Immunoblotting was performed with sera from 12 patients with a history of immediate-type allergic reaction to fish and from atopic and nonatopic controls. IgE-reactive proteins were detected and identified using mass spectrometry. RESULTS: The index patient had low levels of IgE binding to parvalbumins. However, 8 putative allergens other than parvalbumin from L niloticus and 5 from G morhua were identified. Further investigation revealed cross-sensitivity to enolase 3 from L niloticus in 7 of the 12 fish-allergic individuals (58%), whereas 11 of the 12 patients (92%) were sensitized to enolase 3 from G morhua. However, atopic control patients were also sensitized to enolase 3 from L niloticus and G morhua. CONCLUSION: Identification of species-specific allergens and individual sensitization could help us to improve avoidance strategies.

IgE sensitization to the fish parasite Anisakis simplex in a Norwegian population: a pilot study.

The reports on fish parasite Anisakis simplex allergy have increased in countries with high fish consumption in the last decade. In Norway, a high consumption country, the prevalence of immunoglobulin E (IgE) sensitization to A. simplex was still unknown. Thus, our objective was to investigate the sensitization prevalence in this country. At the Haukeland University Hospital, Bergen, Norway, two main groups of surplus serum samples were collected: one from newly recruited blood donors (BDO) and the other from the Allergy laboratory (ALL) after analysing IgE and IgE antibodies. The latter was divided into three series: one containing unsorted sera and two sorted by either Phadiatop(®) ≥0.35 kU(A)/l or total IgE ≥1000 kU/l. The sera were analysed for total IgE and IgE antibodies against A. simplex, shrimp, house dust mite (HDM), cod and cross-reactive carbohydrates (CCDs). The prevalence of IgE sensitization to A. simplex was 2.0%, 2.2% and 6.6% in BDO, the unsorted and Phadiatop(®) positive serum groups, respectively. A considerable degree of cross-sensitization to shrimp and HDM is further suggested. Unspecific binding because of high total IgE or by binding to CCDs seemed to play a minor role. The prevalence of IgE sensitization to A. simplex appears to be lower in a Norwegian population than in other high fish-consuming countries, but might still be overestimated owing to cross-sensitization.

Comparison between ovalbumin and ovalbumin peptide 323-339 responses in allergic mice: humoral and cellular aspects.

Ovalbumin (OVA) is widely used in allergy research. OVA peptide 323-339 has been reported to be responsible for 25-35% of isolated BALB/c mouse T-cell response to intact OVA. An investigation of whether OVA and OVA 323-339 molecules can induce equivalent in vivo and in vitro immune responses was conducted. Eight-week-old BALB/c mice were randomly divided into three groups: OVA, OVA 323-339 and saline. On days 0, 7, 14, mice were intraperitoneally injected with 25 microg OVA or OVA 323-339 absorbed on 300 microg Alum, or saline; on days 21-23, all groups were challenged intranasally with either 20 microl of 1% OVA, 1% OVA 323-339 or saline. On day 28, after killing, splenocytes were isolated and cultured under the stimulus of each allergen or medium. Evaluated by hematoxylin/eosin and major basic protein immunohistochemical stainings, OVA and OVA 323-339 induced similar lung inflammation. Interestingly, significant serum total IgE and OVA-specific IgE were observed in OVA mice when compared to saline control. OVA 323-339 mice showed higher serum OVA-specific IgE, OVA 323-339-specific IgE, IL-4 and lower IFN-gamma similar to OVA mice. The proliferative response to OVA was found in cultured splenocytes of both OVA and OVA 323-339 mice, while the similar proliferative response to OVA 323-339 was only observed in the splenocytes of OVA 323-339-sensitized and challenged mice. Although OVA 323-339 induced a Th2-like response in the mouse model as did OVA, OVA 323-339 has clearly limited immunogenic potency to activate OVA-sensitized and challenged mice splenocytes, unlike OVA.

Airway inflammation and bronchial remodelling in toluene diisocyanate-exposed BALB/c mouse model.

UNLABELLED: Toluene diisocyanate (TDI), a highly reactive industrial chemical, is one of the leading causes of occupation-related asthma in industrialized countries. The pathogenesis of TDI-induced asthma, however, remains not fully understood, in part due to lack of appropriate animal models. Twenty five female BALB/c mice (age: 8 weeks) were randomly divided into 5 groups: Ovabumin (OVA); OVA peptide amino acid residues No. 323-339 (Pep); TDI; alum and physiological saline. Mice were intraperitoneally injected with 25 microg OVA or pep absorbed on 300 microg alum, 300 microg alum or saline on days 0, 7 and 14. For the TDI group, mice were sensitized subcutaneously with 20 microl neat TDI on day 0; 20 microl of TDI in olive oil (1:10) on days 7 and 14; on days 21-23. Then each group was challenged intranasally with 20 microl of 1% OVA, 1% Pep, 1% TDI, 10% alum and saline respectively. On day 28, mice were killed under pentothal anesthesia. The results demonstrated that neutrophil-dominant inflammation with a few eosinophil infiltration occurred in the peri-bronchial and peri-vascular regions of the lungs. This was accompanied by hyperplasia/hypertrophy of cells lining the airways and mucus production as shown by HE staining. Positive immunohistochemical MBP staining in parenchyma was also shown. Th2 cytokine IL-4 and IgE production were significant increased 5 days after last challenge while IFN-gamma level was below the detection limit. CONCLUSION: the clear elevation of IL-4 and IgE could allow to conclude a possible Th2-like dominated allergic response in TDI-exposed BALB/c mouse model.

Expression and analysis of recombinant salmon parvalbumin, the major allergen in Atlantic salmon (Salmo salar).

The parvalbumin from white muscle of Atlantic salmon was previously found to be a major allergen, and designated Sal s1. Two distinct cDNAs, 14.1 and 24.1, which comprise the entire parvalbumin-encoding regions, were cloned, revealing transcripts from two different parvalbumin genes. In the present study, the protein-coding regions of these cDNAs were subcloned into an Escherichia coli expression vector (pET-19b). Both proteins were expressed and the generated target proteins were localized in both soluble and insoluble fractions of the expression host. The recombinant products in the soluble fraction were purified using the His tag-purification system and analysed on Western blots with anti-salmon parvalbumin polyclonal rabbit sera and sera from patients allergic to fish. Both recombinant products (His10-14.1 and His10-24.1) reacted positively with salmon parvalbumin-specific immunoglobulin G (IgG) from rabbits, and with specific immunoglobulin E (IgE) from the sera of six fish-allergic patients. The allergenicity of His10-14.1 was confirmed using enzyme-linked immunosorbent assay (ELISA). The 14.1 cDNA of salmon parvalbumin was shown to be the dominant type represented in a muscle cDNA library.

Cloning of two distinct cDNAs encoding parvalbumin, the major allergen of Atlantic salmon (Salmo salar).

Allergy to fish is common in Northern Europe. Variable reactions to different fish species are usually experienced among fish allergic patients. The allergens of cod fish and particularly the major allergen parvalbumin beta (Gadus callarias) have been extensively studied in Norway. In the present communication, the white muscle parvalbumin was similarly found to be a major allergen in Atlantic salmon (Salmo salar, Sal sl). A purified salmon parvalbumin was obtained by anion exchange chromatography, gel filtration chromatography (GFC) and high-performance liquid chromatography (HPLC) of the muscle extracts. The antigenicity and allergenicity of salmon parvalbumin were confirmed using various immunologic and electrophoretic techniques. The protein is representative for several isoallergens judged by the amino acid (AA) sequence variance at certain sites in the AA sequence of CNBr cleavage peptides. Using sera from patients with cod and salmon allergy Sal sl was demonstrated to be the major allergen of Atlantic salmon, as judged by RAST- and ELISA-inhibitions and crossed radioimmunoelectrophoresis (CRIE) techniques. The protein was also demonstrated to be antigenic by the use of polyclonal cod and salmon antibodies in IgG ELISA and immunoelectrophoretic methods. Cloning of parvalbumin cDNA from Atlantic salmon was performed based on an alignment of parvalbumin AA sequences from other species. A probe was generated by PCR and used for screening a salmon muscle cDNA-library. Subcloning and sequencing of two hybridizing clones revealed transcripts from two different parvalbumin genes. The translated sequences of both clones belong to the beta-lineage of parvalbumins and include the entire coding region.