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- Material and methods
Background: Immunoglobulin (Ig) E-double positivity for honeybee (HB) and yellow jacket (YJ) venom causes diagnostic difficulties concerning therapeutical strategies. The aim of this study was to clarify the cause and relation of the cross-reactivity in patients with insect venom allergy.
Methods: For this purpose, 147 patients with suspected stinging insect allergy and CAP-FEIA-double positivity were investigated for specific sIgE to additional cross-reactive carbohydrate determinant (CCD)-containing allergens: timothy grass pollen, rape pollen, natural rubber latex (NRL), bromelain, and horseradish peroxidase (HRP). Sera with sIgE to NRL were further investigated with the commercially available recombinant latex allergens. Reciprocal inhibition assays with both venoms and HRP were performed.
Results: About 36 of 147 (24.5%) patients had sIgE to both venoms only. However, 111 of 147 (75.5%) additionally reacted to CCD-carrying allergens. 89 of 111 CCD-reactive sera had NRL-sIgE. In cases where inhibition experiments were performed, the NRL-sIgE binding was completely abolished in the presence of HRP. Only nine of 61 sera were positive for at least one recombinant latex allergen; all of them were negative in history and NRL-skin prick test. In 43 sera containing sIgE to CCD, HRP inhibition revealed unequivocal results: In 28 of 43 (65%) an HRP-inhibition >70% of sIgE to one venom occurred, pointing out the relevant venom. In three of 43 sIgE proved to be entirely CCD-specific.
Conclusions: Our data indicate that in cases of IgE positivity to both insect venoms supplementary screening tests with at least one CCD-containing allergen should be performed; HRP being a suitable tool for this test. In addition, subsequent reciprocal inhibition is an essential diagnostic method to specify cross-reacting sIgE results.
Up to 5% of the population in Europe and in North America show systemic reactions to hymenoptera stings, mostly honeybee (HB; Apis mellifera) and yellow jacket (YJ; Vespula germanica and Vespula vulgaris; 1). As many patients fail to identify the stinging insect, skin testing and in vitro detection of venom-specific immunoglobulin (Ig) E antibodies are the only tools to detect the culprit insect involved in the allergic reaction and are – in addition to the severity of clinical symptoms – used to select the appropriate venom for immunotherapy (2). The sera of up to 40% of patients with hymenoptera venom allergy show in vitro reactivity with both, HB and YJ venom (3–5). This IgE positivity to both hymenoptera venoms has been interpreted as true double sensitization leading to immune therapy against both venoms. However, there are other reasons for IgE-double positivity: (i) true independent sensitization (co-sensitization) to different allergens, a very rare phenomenon; (ii) immunochemical cross-reactivity because of sequence homologies between allergens from different sources; (iii) cross-reactive carbohydrate determinants (CCD) may be the major cause of in vitro-double positivity to both hymenoptera venoms; and (iv) nonspecific absorption of IgE to the allergosorbent, a phenomenon that is particularly relevant when total serum IgE is extremely elevated. CCD, however, are important antigen targets for specific sIgE-antibody binding providing at least two different IgE-binding sites. They are not only widely distributed in plants (pollen, plant food), but also are present in hymenoptera venoms, as invertebrate glycoproteins, as present in hymenoptera venoms, also bear IgE-binding α(1,3) fucose-containing CCD (6). According to Mari (7), 5% of nonallergic individuals have CCD-sIgE antibodies as well as 10% of nonpollen allergic subjects. The prevalence of CCD-sIgE has been estimated to be 10–15% in patients with grass pollen allergy (8) and increases up to more than 60% in patients with concomitant sensitization to pollen from trees, grasses and weeds (9). The prevalence in patients with allergy to HB venom is 20% (6). About one in four HB venom and one in 10 YJ venom allergics have been demonstrated to be CCD-sIgE-positive (10). Whether these CCD-sIgE antibodies have clinical relevance still remains controversial (11–13). Ebo et al. (14) demonstrated that sensitization to timothy grass and mugwort pollen was found to elicit false-positive IgE for hymenoptera venoms and vice versa.
The aims of this study were to investigate the prevalence of IgE-double positivity for both hymenoptera venoms and of CCD-sIgE antibodies as a cause for this phenomenon to clarify whether there is cross-reactivity or co-sensitization. In addition, to find a suitable screening allergen to detect CCD-sIgE antibodies, and subsequently to investigate, whether the reciprocal CAP-FEIA inhibition with both venoms and/or a CCD-containing allergen might be valuable tools.
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- Material and methods
In our study, 147 patients with hymenoptera venom allergy were sIgE-positive for both HB and YJ venom, 5.4% reported clinical relevance of their sensitization to both venoms. Egner et al. (3) described no in vitro DP patients with clinical relevance, whereas Mueller reported 8.8% (1).
Thirty-six patients were sIgE-positive to both venoms only, and six of 36 (16%) showed independent sensitization to both venoms (co-sensitization). This is significantly lower than the percentage rates of 32–67%, reported in the literature: in 50% of the sera a partial cross-reactivity could be shown in addition to sensitization to HB- and YJ-specific epitopes which is higher than previously reported ranges from 14% to 36% (21, 22, 26).
In the present study, 111 of 147 (75.5%) of the included patients showed a strong reactivity with the CCD-containing allergens. Specific IgE to both, HRP and bromelain, were found in 67%, which is in accordance with previous observations (27). The CCD-containing allergens timothy grass pollen, rape pollen, bromelain, and HRP were similarly recognized by sIgE. As the human immune system is not confronted with HRP in vivo, a molecule with seven glycan chains, this substance seems to be an applicable screening tool for CCD-sIgE. The detection of CCD-sIgE in patients’ sera is highly indicative for in vitro-double positivity for hymenoptera venom sIgE as could be shown previously and confirmed by our data (14, 27, 28). The inclusion of a CCD-screening allergen alone in the diagnostic allergy testing; however, is not sufficient to prove the sensitization to one or the other hymenoptera venom, because sensitization to venom-specific proteins is still additionally possible (27, 29) which was also confirmed by our results. As treatment success depends on the choice of the relevant venom, it is important to find the true sensitizer (22). Therefore, additional in vitro tests should be performed. The additional reciprocal inhibition with both hymenoptera venoms seems to be a valuable tool (22, 26). However, according to the results of the present study, it is only useful where patients are positive for IgE specific to both venoms only and some rare cases where HRP inhibition with CCD- sIgE-positive sera was not indicative concerning the culprit or nonculprit venom.
Horseradish peroxidase inhibition values above 70% indicating extensive cross-reactivity in sera containing CCD-sIgE mostly were in accordance with skin test results, indicating reliability of the method concerning the detection of the culprit venom. Horseradish peroxidase inhibition of about 50% for both venom sIgEs indicates a true double sensitization that in 50% is due to both, CCD and protein epitopes, the latter probably being a result of the sequence homology between the hymenoptera venom enzymes, the hyaluronidases and probably further protein epitopes. HB and YJ hyaluronidase, for example, show a sequence homology of between 47% and 57% (30). The hyaluronidases and high-molecular allergens of a molecular weight between 40–100 (HB) and 38–100 kDa (YJ), however, are the most important glycans (29, 31). Phospholipase A2, hyaluronidase, and acid phosphatase are glycosylated (32, 33). However, Western blot analysis revealed that the major CCD-sIgE response is directed against high-molecular glycoproteins (35–95 kDa; 29). Hemmer et al. (29) focused on the unglycosylated venom allergens and found that all wasp allergic patients were positive to antigen 5 in Western blot. This is in contrast to our results: 68 of 126 patients with a positive history for wasp sting were positive for antigen 5-specific IgE. Nearly a quarter of these patients (37 of 126) had not been able to identify the culprit insect. Not all vespid venom-allergic patients develop sIgE against antigen 5, and only a very small proportion of HB allergic patients have sIgE to HB melittin. As it is still unknown which and how many hymenoptera venom allergens apart from both hymenoptera hyaluronidases and HB phospholipase are glycosylated and whether their immunogenicity is due to peptides or glycans, immunoblot analysis is not helpful to clarify the clinically relevant sensitization.
So far, skin test has been considered as indicative for sensitization, which prompted a comparison between skin test and different in vitro tests. In those cases with discrepancy between intradermal test and inhibition this may be because of the fact that the intradermal test is too sensitive to be specific. That severity of reaction and skin test as well as skin test and sIgE detection did not coincide in some individuals is a well-known phenomenon in cases of IgE-mediated hymenoptera venom allergy (34). Skin tests with the venom to which the serum is only reactive because of CCD-sIgE antibodies are according to the literature often negative (29), which is in contrast to our observations. This may be because of the fact that allergens, which are clinically relevant might be different in in vitro diagnostic tests and vice versa. Alternatively, the reason for this discrepancy may be found in the way the allergens are presented: either on the solid phase or on mast cells.
Sera with sIgE positivity to CCD-containing NRL were further investigated, including recombinant NRL components and MBP as negative control. Subsequently, the in vitro results were evaluated for clinical significance. All in vitro results were clinically nonrelevant. IgE responses <0.4 kU/l are borderline and considered to be unspecific. Immunoglobulin E to recombinant NRL components are not protein specific but directed to MBP, the carrier protein, added as control protein in our test system. In addition, from our results it does not seem reasonable to include recombinant NRL components when NRL-sIgE are detected because of CCD binding, the latter being most probably the case when the concentrations of NRL-sIgE and HRP-sIgE as well as sIgE to CCD-containing screening allergens were similar. In cases where inhibition experiments were performed, the NRL-sIgE binding was completely abolished in the presence of HRP, indicating that in hymenoptera venom allergic patients sIgE reactions to NRL mostly because of CCD.
However, there were three sera in which HRP-bound IgE specific to both venoms with inhibition values of 70–100%, suggesting that all venom-specific IgE were reactive only to CCD. Two patients had suffered from a severe clinical reaction after hymenoptera sting indicating that CCD-sIgE may be involved in the development of a manifest allergic reaction. According to several publications, CCD generally are considered to be of no clinical significance, a hypothesis supported by the investigation of 1000 healthy blood donors who were investigated for sIgE to NRL (35). Until now, no convincing evidence has been reported to support clinical relevance of CCD-sIgE. Patients with antipollen sIgE that exclusively react with CCD are extremely rare. However, a mixed and simultaneous immune response of IgE antibodies in sera of hymenoptera venom allergic patients to glyco-epitopes as well as peptide epitopes in both venoms is still possible, so that even the detection of CCD-sIgE alone does not definitely exclude the simultaneous presence of antipeptide-sIgE (27, 29). So far, CCD-IgE represents one major cause for cross-reactivity and therefore limits the specificity of sIgE tests for insect venoms. However, there are reports suggesting their participation in the induction of clinical symptoms (36, 37 reviewed by Ref. 38) supporting our observation.
In conclusion, our data indicate that in cases of IgE-double positivity to both hymenoptera venoms supplementary CAP-FEIA investigations with at least one CCD-containing allergen should be performed. Horseradish peroxidase seems to be a valuable screening allergen for the detection of CCD-sIgE and is useful for reciprocal inhibition experiments. Timothy grass pollen and the corresponding recombinant components should be included in cases where patients suffer from additional pollinosis. Whereas reciprocal inhibition with both venoms in sera with sIgE positivity only to both venoms allows the detection of cross-reactivity and true double sensitization, inhibition with HRP is a valuable tool for specifying the venom sensitization in the population with CCD-IgE and should be performed in the first place before including both venoms into the reciprocal inhibition experiment. Only in those cases where sIgE to both venoms as well as to HRP were HRP-inhibited to about 50%, the latter may be valuable. In order to differentiate between true NRL allergy and in vitro positivity to CCD, HRP should be added as screening allergen. If recombinant components produced as fusion proteins are used for IgE detection as is the case for most recombinant latex components, MBP should in any case be included in the test as negative control. Since very recently, CCD reagents coupled to an ImmunoCAP matrix, based on the most general CCD glycan structure found in bromelain, MUXF3-type carbohydrate, are commercially available. According to Unger et al., this CCD was even more efficient in inhibitory experiments with sera with confirmed CCD reactivity than the MMXF3-type carbohydrate, a glycan found in HRP (39). MUXF3 CCD ImmunoCAP can be used as CCD-screening tool. As our study demonstrated the value of additional reciprocal inhibition, MUXF3 should also be available as reagent for inhibition procedures. Concerning the question which glycan preparation is the most suitable, more data need to be collected. The fact that HRP contains seven glycan units when compared to bromelain with one glycan unit, favors HRP as screening agent for the detection of sIgE to different glyco-epitopes, as demonstrated in the present investigation.
The development of recombinant hymenoptera allergens is a possible next step to be taken. From the results of this and other studies it seems to be inevitable to develop and include recombinant allergens in the diagnostic procedures to detect hymenoptera venom allergy. Regarding the fact that a lot of in vitro DP patients had an intradermal test positive to both venoms with 19% of them showing reactions to identical titers may indicate that CCDs play a role in skin test reaction as well. Whether or not CCDs are of clinical relevance still remains to be clarified.