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Keywords:

  • allergen profile;
  • chestnut;
  • co-sensitization;
  • cross-reactivity;
  • natural rubber latex

Abstract

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

Background:  Chestnut and natural rubber latex (NRL) allergy are often associated in the latex-fruit syndrome.

Aim of the study:  To establish whether the concurrent NRL and chestnut IgE antibody reactivity are the results of co-sensitization or cross-reactivity.

Methods:  Sera from 19 patients with chestnut- and NRL-specific IgE were selected and tested for reactivity with recombinant (r) latex allergens. Cross-reactivity was explored by IgE-inhibition experiments using chestnut or NRL allergens as solid phase on ImmunoCAP.

Results:  IgE-antibodies were detected to rHev b 6.01 (prohevein) in 58% of the sera, to rHev b 5 in 32%, to rHev b 12 in four of 13 sera, to rHev b 7.02 and rHev b 11 in four, and to rHev b 1 in two of 19 sera. rHev b 8-IgE antibodies were found in nine sera (47%), whereas six displayed mono-sensitization to rHev b 8 with regard to our test panel. Three of 16 sera showed IgE to cross-reactive carbohydrate determinants. In most sera recognizing rHev b 5 and/or rHev b 6.01 as major allergens the IgE-reactivity to NRL remained unaffected by chestnut extract and chestnut-IgE remained unaffected by NRL extract. Conversely, in sera with rHev b 8 as dominant allergen IgE-binding to NRL was nearly completely inhibited by chestnut and vice versa. IgE-binding to rHev b 8 was abolished by chestnut extract.

Conclusions:  Although patients have concomitant IgE antibody reactivity to chestnut and NRL, cross-reactivity could be demonstrated mainly in those patients with IgE to Hev b 8 (profilin) from NRL.

Natural rubber latex (NRL) allergy has become a serious occupational and public health problem during the last decades. An important feature of NRL allergy is that sera from latex-allergic patients show a high degree of in vitro cross-reactivity to various foods, the so called latex-fruit syndrome (1–3). Especially latex hevein (Hev b 6.02) and class I chitinase (Hev b 11) with a N-terminal hevein-like domain were described as the main allergens responsible for cross-reactivity (4–7). Chestnut (Castanea sativa) is one of the main allergenic food items linked to NRL allergy. The major allergens of chestnut seed identified so far are Cas s 5 (6) a class I chitinase, Cas s 8 a member of the lipid transfer protein (LTP) panallergen family (8, 9) and a 24-kD chestnut protein showing homology to leguminous family (8). An additional candidate allergen associated with allergenic cross-reactions is profilin, which has been found as an allergen in a large variety of pollens, fruit and also latex. In patients with NRL sensitization, the latex profilin (Hev b 8) seemed to be of minor relevance (10). The aim of this study was to analyse the possible cross-reactivity or co-sensitization between chestnut and NRL allergens on the basis of single NRL allergen sensitization pattern and by IgE-inhibition experiments.

Materials and methods

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

Patients

Nineteen sera from chestnut allergic patients (P1–P19) displaying specific IgE to both chestnut (median 0.78 kU/l; range: 0.46–5.97 kU/l) and NRL (median 5.23 kU/l; range: 0.64–73.5 kU/l) were examined. The clinical features of chestnut allergy included an oral allergy syndrome (OAS) in most patients, followed by severe systemic reactions (Table 1).

Table 1.   Sensitization profile of 19 patients with chestnut- and NRL-specific IgE
Patient codeSexChestnut-induced symptomsIgE to chestnutIgE to NRLIgE to rHev b 1IgE to rHev b 5IgE to rHev b 6.01IgE to rHev b 7IgE to rHev b 8IgE to rHev b 11IgE to rHev b 12IgE to HRP
  1. Values are expressed as (kU/l); positive values are indicated in bold.

  2. M, male; F, female; n.d., not done, according to the limited amount of sera; OAS, oral allergy syndrome; HRP, horseradish peroxidase; NRL, natural rubber latex.

  3. All sera were negative to MBP.

P1MAvoidance0.4650.2<0.3545.4047.90<0.35<0.35<0.35<0.35<0.35
P2FNone1.055.76<0.3527.601.64<0.35<0.35<0.35n.d.n.d.
P3MOAS4.135.52<0.35<0.35<0.35<0.3511.60<0.35n.d.3.06
P4MUnknown1.464.91<0.3534.200.402.91<0.35<0.35n.d.n.d.
P5FAnaphylaxis8.336.30<0.35<0.3524.50<0.35<0.353.60<0.35<0.35
P6FUnknown0.772.52<0.35<0.352.40<0.35<0.35<0.35<0.35<0.35
P7MUnknown2.541.70<0.35<0.35<0.35<0.359.20<0.35n.d.n.d.
P8MOAS1.041.37<0.35<0.35<0.35<0.354.91<0.350.89<0.35
P9FAngioedema0.8618.600.4079.904.914.87<0.35<0.35<0.35<0.35
P10FOAS4.6413.80<0.35<0.35<0.350.3934.500.390.423.38
P11FOAS0.942.24<0.35<0.35<0.35<0.356.26<0.35n.d.0.48
P12FAvoidance0.672.17<0.35<0.35<0.35<0.3512.5<0.35<0.35<0.35
P13FOAS0.825.23<0.35<0.35<0.35<0.354.19<0.35n.d.<0.35
P14FAvoidance0.8617.40<0.352.118.260.63<0.351.7<0.35<0.35
P15Mnone4.020.64<0.35<0.350.82<0.35<0.35<0.351.11<0.35
P16MAvoidance4.3373.50<0.35<0.3553.00<0.3516.30<0.350.98<0.35
P17MAnaphylaxis0.4415.00<0.35<0.3517.10<0.35<0.35<0.35<0.35<0.35
P18FOAS, angioedema0.5570.103.2925.1043.30<0.35<0.351.04<0.35<0.35
P19FOAS5.971.19<0.35<0.35<0.35<0.3527.80<0.35<0.35<0.35
Reactivity for each allergen19/19 (100%)19/19 (100%)2/19 (11%)6/19 (32%)11/19 (58%)4/19 (21%)8/19 (42%)4/19 (21%)4/14 (29%)3/16 (19%)

Chestnut extracts preparation

Freshly peeled chestnuts (50 g) were minced, mixed with phosphate buffered saline (PBS; pH 7.4; containing EDTA, Pefabloc and ascorbic acid) and homogenized on ice followed by gentle stirring for 16 h at 4°C. The supernatant was collected after two centrifugation steps at 4°C for 45 min (20 000 × g; 22 000 × g). Chestnut-protein supernatants were collected and stored for further experiments at −70°C. Protein concentration was quantified according to Bradford.

Measurement of the NRL sensitization profile and IgE-inhibition

Serological studies and inhibition experiments were performed with the UniCAP100-System™ (Phadia, Uppsala, Sweden) using latex (k82) and chestnut (f299) ImmunoCAPs. Recombinant latex allergens (rHev b 1, rHev b 5, rHev b 6.01, rHev b 7.02, rHev b 8, rHev b 11 and rHev b 12) were coupled to ImmunoCAPs (MIAB, Uppsala, Sweden) and used for determination of the individual sensitization profile. All recombinant allergens were produced in E.coli in fusion with the maltose-binding protein (MBP) as a carrier protein. Therefore, the MBP ImmunoCAPs served as a negative control. In addition, horseradish peroxidase (HRP) ImmunoCAPs were used to detect IgE to cross-reactive carbohydrate determinants (CCDs). IgE concentrations >0.35 kU/l were considered positive. For IgE-inhibition studies, sera with IgE concentrations >1.0 kU/l (range: 1.0 and 5.0 kU/l) were preincubated with 10 μl of the specific inhibitor solution (1 mg/ml). Inhibition experiments were performed with NRL (k82), chestnut (f299) and rHev b 8 as solid phase allergens. Chestnut extract, latex C-serum extract and single recombinant latex allergens served as inhibitors. As control, PBS instead of inhibitor was applied.

Results

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

IgE-reactivity to chestnut, NRL and the single recombinant NRL allergens is depicted in Table 1. Specific IgE-antibodies were detected to rHev b 5 in 32% (6/19), to rHev b 6.01 in 58% (11/19), to rHev b 7.02 and to rHev b 11 in 21% (4/19) of the sera. Four of 13 sera (30.8%) were positive to rHev b 12 and only two to rHev b 1. rHev b 8-IgE antibodies were found in nine sera (47%), seven of the them were only sensitized to rHev b 8 when tested with this special allergen panel. IgE-values to rHev b 5 (median 30.9 kU/l) were the highest in comparison with the other allergens, followed by rHev b 8 (median 11.6 kU/l) and rHev b 6.01 (median 8.26 kU/l). In comparison, the mean values of IgE to rHev b 1, rHev b 7.02, rHev b 11 and rHev b 12 were clearly lower (mean values below 2 kU/l). All sera were negative to MBP. Three of 16 sera (18.8%) were positive to HRP; two of them showed IgE-reactivity only to rHev b 8 (P3, P11) and in the third one (P10) specific IgE to rHev b 8 showed the highest level. The IgE-response to HRP (median 3.06 kU/l) was much lower than to rHev b 8 (11.6 kU/l) in these sera. A clear discrimination was observed between patients with OAS in whom a predominance of IgE antibody reactivity to rHev b 8 was detected and patients suffering from systemic symptoms through chestnut ingestion, who were reactive to Hev b 5 and/or Hev b 6.01 as dominant latex allergens. Serum P16 was the only one with a dominant IgE-response to rHev b 6.01 (53 kU/l) and to rHev b 8 (16.3 kU/l) and a minor response to rHev b 12 (0.98 kU/l).

In 10 sera recognizing rHev b 5 and/or rHev b 6.01 as major allergens, only weak inhibition (15 ± 12%) of IgE-binding to NRL by chestnut extract was found (Fig. 1). Conversely, IgE-binding to NRL in seven sera with rHev b 8 as single or dominant allergen revealed a complete inhibition by chestnut (70–100%) (Fig. 1). In serum P16, displaying specific IgE to rHev b 6.01 and rHev b 8 IgE-binding was inhibited up to 39% with chestnut. Additionally, IgE-binding to chestnut was nearly completely inhibited (88 ± 17%) by latex extract in the rHev b 8-dominant sera (Fig. 2). On the other hand, no inhibition (2%) to chestnut using latex extract was detected in one serum with rHev b 6.01 as a major allergen. In the serum P16, the inhibition of chestnut-specific IgE was only 24%. Using rHev b 8 as solid phase allergen and chestnut extract as inhibitor a nearly complete inhibition (88 ± 11%) was observed (Fig. 3, only tested in sera with Hev b 8-IgE). In serum P16, IgE-binding to rHev b 6.01 was only inhibited to 28% with chestnut extract and to rHev b 8 to 88% with chestnut extract.

image

Figure 1.  Inhibition experiments of specific IgE-binding to latex (k82) on solid phase with chestnut extract (10 μg per assay). Sera dilutions are indicated. The arrows indicate the percentage of inhibition.

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image

Figure 2.  Inhibition experiments of specific IgE-binding to chestnut (f299) on solid phase with latex C-serum extract (10 μg per assay). The arrows indicate the percentage of inhibition.

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image

Figure 3.  Inhibition experiments of specific IgE-binding to rHev b 8 on solid phase with chestnut extract (10 μg per assay) and with serum P16 using rHev b 6.01 as additional solid phase. Sera dilutions are indicated. The arrows indicate the percentage of inhibition.

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Discussion

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

In patients recognizing one or both major NRL allergens rHev b 5 and rHev b 6.01 concomitant sensitizations to chestnut seemed to be an independent phenomenon (co-sensitization), whereas in patients’ sera with IgE predominantly to rHev b 8, cross-inhibition between chestnut and NRL was highly relevant. Therefore, the latex profilin Hev b 8 seemed to be involved in the cross-reactivity between chestnut and NRL. Especially the inhibition experiments with serum P16 demonstrated that rHev b 8 in contrast to rHev b 6.01 was the relevant allergen for the chestnut – NRL cross-reactivity.

In contrast to other studies, which described Hev b 8 as minor allergen in latex (10, 11), our data demonstrated a high prevalence of sensitization to rHev b 8 as shown previously by Nieto et al. (12). It is noteworthy to point out that all patients included in our study had detectable levels of specific IgE to both chestnut and latex. Nevertheless, it should be considered that the Hev b 8 concentrations in latex sources is extremely low resulting in a low capacity for IgE induction and therefore the homology among NRL and chestnut or other plant profilins can explain the high rate of positive rHev b 8-IgE in subjects primarily sensitized to pollens or fruits (1). In accordance with the latex sensitization pattern of the patients in our study, Hev b 11, the class I chitinase of NRL with a Hev b-like domain, showed only a minor relevance and was not important for cross-reactivity.

Cross-reactive carbohydrate determinants could be excluded as the reason for chestnut NRL cross-reaction in the patient group because only 19% of the tested sera exhibited IgE to CCD (determined with HRP). Additionally, the IgE responses to CCD were lower than those to the single NRL allergens implicating that CCDs, in contrast to other NRL co-sensitizations (13), were not responsible for cross-reactivity between NRL and chestnut.

In addition, there were also four patients’ sera displaying IgE antibodies to the recombinant Hevea LTP (rHev b 12). As it was recently shown by Sanchez-Monge et al. (9) that the LTP of chestnut Cas s 8 was an important allergen in sera of patients with chestnut but without latex allergy, co-sensitization as a result of the high homology between the LTPs of chestnut and NRL is probable. A similar situation was recently described by our group for rHev b 12 and the peach LTP Pru p 3 and LTPs from other fruits (14). Therefore, LTPs are relevant allergens linked to chestnut but not to latex allergy. Our results demonstrated that also in the four cases with rHev b 12-IgE, the LTP is not relevant for the cross-reactivity.

In summary, rHev b 8, rHev b 5 and rHev b 6.01 could be useful diagnostic tools in patients with IgE to both chestnut and NRL to discriminate between co-sensitization and cross-reactivity, which could implicate a number of plant food and pollen allergens.

Acknowledgment

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

This study was supported by the Kanert-Stiftung für Allergieforschung.

References

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