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Keywords:

  • allergens;
  • IgE inhibition;
  • immunoblotting inhibition;
  • parasites

Abstract

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

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.

The nematode Anisakis simplex commonly parasitizes marine invertebrates as well as fish and sea mammals. Its life cycle comprises the egg, several larval stages, and the adult nematode. Encapsulated larvae of the third stage are common in the viscera and musculature of many fishes (1). The larva infects fish of many species and over a broad geographic area (2); thus, eating raw or undercooked infected fish can lead to infestation with the larva and cause severe gastrointestinal symptoms (anisakiasis) (3). Several studies have suggested that A. simplex can act as a food allergen and induce IgE-mediated reactions, mostly urticaria and angioedema, but also anaphylactic reactions (4–8). Two case reports have shown A. simplex to be also an occupational allergen causing asthma and conjunctivitis (9, 10). It has been assumed that even properly cooked infected fish can cause allergic symptoms, as some of the A. simplex allergens have been demonstrated to be thermostable (11). However, recent studies indicate that allergic clinical symptoms can be induced only after ingestion of a live larva, which can then secrete antigens affecting the gastric mucosa (12–15).

IgE antibodies to the parasite have been detected in high frequency among individuals without symptoms, including blood donors, controls, and atopic children (2). The incidence of IgE to A. simplex in 169 sera from blood donors at the Karolinska Hospital, Stockholm, Sweden, was 13% (not published). However, the cause of sensitization to A. simplex is not fully understood. It has been suggested that the occurrence of IgE to A. simplex in individuals not displaying allergic symptoms is due to an earlier infestation with the larvae, probably without symptoms, and is a consequence of the normal immune response to parasites (16, 17). Cross-reactivity may also be a possible explanation. Antigenic and allergenic cross-reactivity between A. simplex and other nematodes is a well-known phenomenon (18, 19). Surprisingly, allergenic cross-reactivity has also been described between A. simplex and arthropods (20). CAP FEIA and immunoblotting inhibition analysis have shown that German cockroach and red mosquito larvae (Blattella germanica and Chironomus spp.) share IgE-binding components with A. simplex (20). In a preliminary study, we found an association between sensitization to A. simplex and yet another arthropod, the nonpyroglyphid dust mite Lepidoglyphus destructor. Several species of nonpyroglyphid dust mites are important occupational allergens in rural environments (21, 22), but recent data show that IgE-mediated sensitization to these mites is found also in urban populations (23–25), suggesting that the nonpyroglyphid dust mites are of greater clinical importance than previously known. L. destructor is one of the most common species in Europe, but Acarus siro and Tyrophagus putrescentiae are also common (26–28).

The aim of this study was to investigate a possible allergenic cross-reactivity between A. simplex and A. siro, L. destructor, and T. putrescentiae and also between A. simplex and the pyroglyphid house-dust mite Dermatophagoides pteronyssinus.

Material and methods

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

Serum samples

Serum samples were obtained from farmers who had previously participated in an epidemiologic survey of respiratory allergy (22). Sixty-nine sera from subjects sensitized to at least one of the dust-mite species A. siro, L. destructor, T. putrescentiae, or D. pteronyssinus were chosen for screening of IgE antibodies to A. simplex. Two serum samples (nos. 2 and 3, Table 1) were used individually in the inhibition experiments.

Table 1.  Serum IgE values to A. simplex- and dust-mite-sensitized individuals. Values of ≥0.35 kU/l are positive results
Serum no.A. simplex (kU/l)A. siro (kU/l)L. destructor (kU/l)T. putrescentiae (kU/l)D. pteronyssinus (kU/l)Serum No.A. simplex (kU/l)A. siro (kU/l)L. destructor (kU/l)T. putrescentiae (kU/l)D. pteronyssinus (kU/l)
 117618.61.56336<0.350.79<0.350.392.4
 27.95.48.2177.837<0.350.673.2<0.35<0.35
 33.0114.92.96.438<0.350.660.420.530.82
 41.92.12.11.71239<0.350.60.690.49<0.35
 51.70.530.38<0.35<0.3540<0.350.59<0.350.440.62
 61.16.4175.85.341<0.350.450.92<0.35<0.35
 70.981.21.80.953542<0.350.44<0.350.380.52
 80.80.45<0.35<0.350.7943<0.350.44<0.35<0.3510
 90.741.3<0.350.35<0.3544<0.350.44<0.35<0.350.58
100.75.314106.245<0.350.430.580.62<0.35
110.562.93.72.61.646<0.350.36<0.35<0.351.2
120.50.990.390.93<0.3547<0.35<0.351.60.42<0.35
130.482.82.72.30.748<0.35<0.350.870.56<0.35
140.40.89<0.350.630.5449<0.35<0.350.85<0.35<0.35
15<0.354.6110.484.150<0.35<0.350.760.35<0.35
16<0.353.6116.19.451<0.35<0.350.76<0.35<0.35
17<0.352.95.24.82552<0.35<0.350.720.4311
18<0.352.90.470.972253<0.35<0.350.620.57<0.35
19<0.352.61251.854<0.35<0.350.620.53<0.35
20<0.352.11.331.655<0.35<0.350.55<0.353.4
21<0.3521.41.62.456<0.35<0.350.47<0.35<0.35
22<0.351.93.22.51657<0.35<0.350.46<0.35<0.35
23<0.351.92.73.10.6858<0.35<0.35<0.35<0.355.5
24<0.351.7<0.351.21.259<0.35<0.35<0.35<0.350.79
25<0.351.53.72.10.5960<0.35<0.35<0.35<0.353.6
26<0.351.40.731.31.961<0.35<0.35<0.35<0.352.6
27<0.351.30.840.941.762<0.35<0.35<0.35<0.351.5
28<0.351.3<0.3512763<0.35<0.35<0.35<0.354.5
29<0.351.20.981.41.564<0.35<0.35<0.35<0.351.3
30<0.351.10.550.661.165<0.35<0.35<0.35<0.351.7
31<0.3510.720.891.566<0.35<0.35<0.35<0.3513
32<0.350.98<0.35<0.350.8967<0.35<0.35<0.35<0.350.68
33<0.350.973.9<0.35<0.3568<0.35<0.35<0.35<0.354.4
34<0.350.860.921.11169<0.35<0.35<0.35<0.350.74
35<0.350.812.21.80

Two control groups were selected, one group comprising 25 farmers sensitized to common aeroallergens (birch, timothy, and mugwort), but not to mites, and the other group comprising 25 healthy, nonatopic farmers.

Extract preparation

A. simplex third-stage larvae were obtained from Allergon (Ängelholm, Sweden). The lyophilized larvae were homogenized in 0.15 M NaCl, 0.02% NaN3, 1/20 (w/v) with a pellet pestle mixer (KEBO, Stockholm, Sweden) on ice. The homogenate was rotated end-over-end at 4°C overnight. The extract was then centrifuged at 17 000 g for 30 min at 4°C, and the supernatant was finally filtered through a 0.45-μm filter (Millipore, Bedford, MA, USA). Sieved cultures of A. siro, L. destructor, T. putrescentiae, and D. pteronyssinus (Allergon) were extracted in 0.15 M NaCl, 0.02% NaN3 at 1/10 (w/v) overnight, at room temperature, and centrifuged at 6600 g, and the supernatant was filtered through an 0.8-μm filter. The protein concentration was measured with the Micro BCA Protein Assay Reagent (Pierce Chemical Company, Rockford, IL, USA). The extracts were kept at −20°C until tested.

IgE antibody measurements

IgE antibodies to A. simplex, A. siro, L. destructor, T. putrescentiae and D. pteronyssinus were measured with the Pharmacia CAP System RAST® FEIA (CAP FEIA) (Pharmacia & Upjohn Diagnostics AB, Uppsala, Sweden), according to the manufacturer's instructions. Results of ≥0.35 kU/l were considered positive.

Inhibition of IgE binding in CAP FEIA

Extracts of A. simplex, A. siro, L. destructor, T. putrescentiae, and D. pteronyssinus were diluted with phosphate-buffered saline, pH 7.4, to the final concentrations 1, 10, 100, and 1000 µg/ml and incubated with an equal volume of serum sample overnight at 4°C. Free antibodies to A. simplex, A. siro, L. destructor, T. putrescentiae, and D. pteronyssinus on solid phase were measured by CAP FEIA. Heterologous inhibitions were performed between A. simplex and each of the four mite species. All five organisms were also inhibited in the homologous system. The results are expressed in percent inhibition, calculated from a reference sample (serum incubated with diluent only).

SDS–PAGE and immunoblotting inhibition

SDS–PAGE was performed with the Mini-Protean® system (Bio-Rad, Richmond, CA, USA) and electroblotting in the Hoefer® Mighty SmallTM Electrophoresis Unit (Amersham Pharmacia Biotech, Uppsala, Sweden), according to the instructions of the manufacturers. An amount of 1 mg of A. simplex (not boiled) or mite extract (A. siro, L. destructor, T. putrescentiae, or D. pteronyssinus) in a nonreducing sample buffer was loaded onto a gradient gel (7.5–20%) and run for 45 min at 200 V. Electroblotting was then performed onto a polyvinyldienedifluoride (PVDF) membrane. After blotting, the membrane was blocked and cut into strips (2–3 mm) and incubated with a serum sample and extract of A. simplex, A. siro, L. destructor, T. putrescentiae, or D. pteronyssinus. The final dilution of the serum sample was 1/4 and the final concentration of the extract was 1 mg/ml. The allergens were visualized as described earlier (29). In brief, the strips were subsequently incubated with rabbit antihuman IgE and goat antirabbit Ig-ALP (Dako, Glostrup, Denmark). The enzyme-conjugated goat antibodies used as the second detection antibody reacted with a protein at >100 kDa in the A. simplex extract (Fig. 2).

image

Figure 2. Inhibition of IgE binding to A. simplex allergens on blotting strips. Serum no. 2 was used in (a) and serum no. 3 in (b). Lanes A and E show IgE binding without inhibition; lanes B, C, D, and F show IgE binding after inhibition with 1 mg of extract of A. siro, L. destructor, T. putrescentiae, and D. pteronyssinus, respectively. Lanes A–D and lanes E and F were obtained at two different inhibition experiments. Note that enzyme-conjugated goat antibodies used as second detection antibody reacted strongly with protein at >100 kDa in A. simplex extract, visible on all lanes.

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Inhibition of IgE binding was evaluated by scanning the PVDF strips with the Model GS-670 Imaging Densitometer, and Molecular Analyst Software, Version 2.1 (Bio-Rad). A peak diagram with reflectance versus position for all allergens was obtained for each strip. The degree of inhibition was calculated from the optical density (OD) values for each peak obtained after incubation with serum and extract and after incubation with serum only (reference value). Results are expressed as percentage of inhibition.

Statistical analysis

Correlation coefficients (rs) and statistical significance were determined with Spearman rank correlation analysis. P<0.05 was considered to be significant.

Results

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

IgE antibody measurements

Of the 69 mite-positive sera, 14 (20.3%) displayed an IgE antibody level of ≥0.35 kU/l to A. simplex (Table 1). The Spearman rank correlation between IgE values to A. simplex and each mite species gave the highest correlation to A. siro, rs=0.47 (P=0.0014, n=46). The corresponding figures for A. simplex and L. destructor were rs=0.29 (P=0.0495, n=48); for A. simplex and T. putrescentiae, rs=0.31 (P=0.0414, n=45); and for A. simplex and D. pteronyssinus, rs=0.24 (P=0.0833, n=54).

One of the 25 nonatopic controls had IgE antibodies (0.92 kU/l) to A. simplex, but none of the 25 atopic controls had such antibodies.

Inhibition of IgE binding in CAP FEIA

In CAP FEIA inhibition, all four mite extracts (A. siro, L. destructor, T. putrescentiae, and D. pteronyssinus) inhibited, to various degrees, the IgE response to A. simplex on the solid phase. Two sera, nos. 2 and 3 (Table 1), were analyzed, and a dose-dependent inhibition was observed in both sera, using each of the extracts (Fig. 1a and b). T. putrescentiae was the most potent inhibitor in one of the sera, 84% at 1 mg/ml (Fig. 1a), while A. siro achieved the strongest inhibition in the other serum (70%) (Fig. 1b). D. pteronyssinus inhibited the IgE binding to A. simplex to about the same degree in both sera, 65% and 63%, respectively, while L. destructor inhibited to a lesser extent, i.e., 20 and 58% in the two sera. Analogous experiments were performed with the mite extracts on solid phase and A. simplex extract as inhibitor of the IgE binding. Table 2 shows the inhibition results obtained with 1 mg/ml. The inhibition of IgE binding to A. siro, T. putrescentiae, and D. pteronyssinus reached about 55% to at least one of the two sera. However, A. simplex was not able to inhibit the IgE binding to L. destructor in either of the serum samples or to D. pteronyssinus in serum no. 2.

image

Figure 1. CAP FEIA inhibitions with A. simplex on solid phase: a) serum no. 2; b) serum no. 3. Inhibition curves obtained with extracts of A. simplex are indicated by open circles, those for A. siro by open squares, those for L. destructor by filled circles, those for T. putrescentiae by filled squares, and those for D. pteronyssinus by open triangles.

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Table 2.  CAP FEIA inhibitions with mite extracts on solid phase and extract of A. simplex as inhibitor. Figures were obtained with 1 mg/ml of inhibiting extract
 Serum no. 2Serum no. 3
Solid phaseA. simplex % inhibitionHomologous extract % inhibitionA. simplex % inhibitionHomologous extract % inhibition
A. siro19745794
L. destructor084074
T. putrescentiae55871575
D. pteronyssinus0885688

Blotting inhibition

In an attempt to identify the allergens that are involved in the cross-reactivity between A. simplex and the mites A. siro, L. destructor, T. putrescentiae, and D. pteronyssinus, we performed blotting inhibition with the same serum samples that were used in the CAP FEIA inhibition experiments. The mite extracts inhibited the IgE binding of both sera to several allergens in A. simplex (Fig. 2a and b). The overall inhibition results were consistent with those obtained with the CAP FEIA inhibitions, with T. putrescentiae as the most potent inhibitor of serum no. 2 (Fig. 2a) and A. siro of serum no. 3 (Fig. 2b). L. destructor was a poor inhibitor of serum no. 2, while several allergens were inhibited with serum no. 3. D. pteronyssinus inhibited in both sera the IgE binding to several allergens. At least 12 different A. simplex allergens of 17–42 kDa and several allergens of high molecular mass (>43 kDa) were inhibited to various degrees by the different mite extracts. There was a considerable difference in the degree of inhibition to some individual allergens. For example, the IgE binding to an allergen at 42 kDa was inhibited 70% by A. siro, but not by T. putrescentiae (Fig. 2b). Another allergen at 36 kDa was not inhibited by L. destructor, while it was inhibited by 82% by A. siro. Also in the other serum sample (Fig. 2a) was a 36-kDa allergen inhibited to various degrees: 70% by T. putrescentiae, 59% by D. pteronyssinus, 30% by A. siro, and 13% by L. destructor.

The capacity of A. simplex extract to inhibit the IgE binding to mite allergens on blotting strips was less pronounced (Fig. 3a and b). No inhibition to the A. siro allergens was observed in any of the two sera, and only one allergen in L. destructor was inhibited: a 13-kDa allergen (34%, serum no. 2) (Fig. 3a). However, extracts of A. simplex inhibited the IgE binding of serum no. 2 to more than 15 allergens in T. putrescentiae and at least eight allergens in D. pteronyssinus. In addition, the IgE reactivity to 16- and 17-kDa allergens in T. putrescentiae was inhibited 31% and 76%, respectively, in serum no. 3 (Fig. 3b).

image

Figure 3. Inhibition of IgE binding to different mite species on blotting strips. Serum no. 2 was used in (a) and serum no. 3 in (b). Lanes A and B) A. siro; C and D) L. destructor; E and F) T. putrescentiae; and G and H) D. pteronyssinus. First lane in each pair (A, C, E, and G) shows IgE binding without inhibition; second lane (B, D, F, and H) shows IgE binding after inhibition with 1 mg of A. simplex extract.

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Discussion

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

In this study, we used CAP FEIA and immunoblotting inhibition to evaluate allergenic cross-reactivity between A. simplex and the dust mites A. siro, L. destructor, T. putrescentiae, and D. pteronyssinus. We found that 20% (14/69) of dust-mite-sensitized subjects had IgE to A. simplex. Taken together with the fact that of the 50 control subjects nonsensitized to dust mites, only one was positive to A. simplex, this points to an association between A. simplex and dust mites, probably one of allergenic cross-reactivity. However, as comparison of IgE values between A. simplex and each mite species showed only weak correlation, the highest being to A. siro (rs=0.47, P=0.0014), the implication is that IgE antibodies to A. simplex among these subjects are not solely dependent on sensitization to dust mites.

When we assessed the allergenic cross-reactivity in CAP FEIA inhibition, all four mite species inhibited the IgE binding to A. simplex, but A. siro and T. putrescentiae were the most potent inhibitors in the two investigated sera. Conversely, A. simplex was able partly to inhibit the IgE binding to the dust mites. About 50% of the IgE binding to A. siro, T. putrescentiae, and D. pteronyssinus was inhibited in at least one of the sera. However, the nematode could not inhibit the IgE binding to the L. destructor allergens in any of the serum samples. The results clearly show that A. simplex and each of the mite species A. siro, L. destructor, T. putrescentiae, and D. pteronyssinus have common IgE-binding epitopes.

Allergenic cross-reactivity between A. simplex and other organisms has been reported by Fernández-Caldas et al. (19), who used ELISA inhibition to assess cross-reactivity between A. simplex and the anisakid nematode Hysterotylacium aduncum. The results suggest that the two species share allergenic epitopes. Pascual et al. (20) also described allergenic cross-reactivity between A. simplex and the two arthropods German cockroach and red mosquito larvae. By immunoblotting inhibition, they showed that several allergens, mainly of 30–40 kDa in all three species, were involved in the cross-reactions. They speculated that the cross-sensitization link between these disparate species was the 36-kDa muscle protein tropomyosin. Indeed, the highly conserved tropomyosin protein has been considered an important cross-sensitizing invertebrate panallergen (30). This allergen has been found in several organisms, among them the two (house) dust mites D. pteronyssinus (Der p 10) and D. farinae (Der f 10) (30). Different mite species have been shown to contain several groups of allergens with extensive homology between them (31). Therefore, we suspect that tropomyosin may also be a part of the allergen spectrum of all four mite species investigated in this study and involved in the cross-reactivity with A. simplex. In the immunoblotting inhibition experiment, we obtained inhibition to at least three different allergens in A. simplex with molecular mass close to that of tropomyosin. For example, an allergen at 36 kDa was in various degrees inhibited by all four mite extracts. A. simplex also inhibited allergens around 36 kDa in two of the mite extracts, T. putrescentiae and D. pteronyssinus. However, further inhibition experiments using native tropomyosin are needed to determine whether this protein is a cross-reacting allergen in A. simplex and dust mites.

According to the molecular mass of the inhibited allergens in A. simplex and in the dust mites, this study indicates that the allergenic cross-reactivity involves several allergens other than tropomyosin. For example, we recognized several allergens in A. simplex with high molecular mass that were inhibited by the dust mites. This is in accordance with the results of Lorenzo et al. (32), who recently have shown that a sugar epitope, an O-linked glycan on high-molecular-mass glycoproteins (139/154 kDa) in A. simplex, is an IgE-binding cross-reactive molecule.

In conclusion, we have shown that the nematode A. simplex and the dust mites A. siro, L. destructor, T. putrescentiae, and D. pteronyssinus have common allergenic epitopes. The allergenic cross-reactivity was demonstrated both by CAP FEIA and immunoblotting inhibition, identifying at least 12 different allergens that were involved. Fourteen of the 69 dust-mite-sensitized subjects were shown to have IgE antibodies in serum to A. simplex. The clinical relevance of these probably cross-reacting antibodies is not known and needs to be further investigated.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

Financial support for this study was received from the Swedish Council for Work Life Research; the Swedish Foundation for Health Care Sciences and Allergy Research; the Swedish Asthma and Allergy Association; the Swedish Society of Medicine; the Trygg-Hansa Foundation Fund; the Magn. Bergvall Foundation; the King Gustaf V 80th Birthday Foundation; the Hesselman Foundation; the Konsul Th C Bergh Foundation; Stockholm County Council; and the Karolinska Institute.

References

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References