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Hypersensitivity to banana in latex-allergic patients. Identification of two major banana allergens of 33 and 37 kD
Hypersensitivity to banana in latex-allergic patients. Identification of two major banana allergens of 33 and 37 kD M F Delbourg, PhD*; L Guilloux, PhD*; D A Moneret-Vautrin, MD†; and G Ville, PhD*
Background: Allergy to banana often occurs in patients sensitized to latex. The spectrum of IgE-mediated responses to latex allergens is more and more documented but banana allergens and epitopes shared by these two allergens have not yet been characterized, even though the existence of cross-reacting IgE antibodies has been demonstrated. Objective: The purpose of this study was to assess the relationship between banana hypersensitivity and latex allergy in a population of 19 latex-allergic patients and to identify allergenic components in banana and the common structures shared with latex. Methods: The in vivo study was conducted in our outpatient department in patients with well-documented latex allergy. Clinical histories were evaluated and skin prick tests were performed with banana and latex extracts. IgE responses from 19 patients were investigated by means of CAP RAST assay and SDS PAGE immunoblotting. Epitopes shared by banana and latex were investigated by means of immunoblotting experiments. Results: Eight of 16 patients (50%) reported symptoms after eating bananas and banana skin prick tests were positive in 5 of 14 patients (36%). Banana RAST results were positive in 12 of the 19 patients (63%). In immunoblot experiments, 17 of the 19 patients (89%) exhibited specific banana IgE antibodies and 16 allergenic components were identified with molecular weights ranging from 17 to 128 kD. Two were considered as major allergens: 33 kD was detectable in 15 of 19 sera (88%) and 37 kD in 13 of 19 sera (76%). Inhibition studies by preincubation of two individual sera with banana or latex extract demonstrated the complete disappearance of IgE binding on banana blotted allergens. Conclusion: This study confirms the “latex-fruit syndrome” already described by Blanco et al. Two major allergens were revealed in banana at 33 and 37 kD and the presence of more than ten common components with latex was observed. Ann Allergy Asthma Immunol 1996;76:321– 6.
INTRODUCTION Cross-reacting allergies between unrelated species have been known for many years.1–3 Rubber latex allergy, for example, is often associated with allergy to fruits such as avocado, ba* Unite´ de Radioanalyse; Institut Pasteur de Lyon; Av Tony Garnier, 69 365; Lyon Cedex 07; France. † Department of Medicine “D”; Clinical Immunology and Allergology; Hopital Central, 29; Av du Mare´chal de Lattre de Tassigny; 54 035 Nancy Cedex; France. Received for publication August 1, 1995. Accepted for publication in revised form October 18, 1995.
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nana, chestnut, kiwi, papaya, and peaches.4 –11 M’Raihi et al have for the first time evidenced the association between banana and latex allergens using RAST inhibition assays.12 Numerous studies relating to latex hypersensitivity and latex allergens have been published,13–17 whereas banana antigenic and allergenic components have not been well characterized. Recently, an allergen of approximately 30 kD was identified in both latex and fruit extracts (banana, avocado).18 Our study was undertaken to assess the relationship between banana hypersensitivity and latex allergy in a population of 19
latex-allergic patients and to identify banana proteins and potential allergens by means of SDS PAGE and Western blot experiments. Common structures shared by the two allergens were investigated by means of immunoblotting inhibition assays. We have previously published preliminary results on banana antigens and allergens in abstract form.19,20 MATERIALS AND METHODS Patients Sera from 19 patients (17 women and 2 men) referred to the outpatient department (Department of Medicine “D”; Clinical Immunology and Allergology; Hoˆpital Central; Nancy, France) with well-documented latex allergy were selected for this study. All patients gave their informed consent to participate in the study. They were skin tested with two latex extracts: an ammoniated natural rubber latex emulsion prepared as previously described,17 and a commercial extract of ammoniated latex 1:100 wt/vol from Stallerge`nes SA (Fresnes, France). Symptoms to latex are summarized in Table 1. Most patients were healthcare workers [surgeon (1), dental assistant (1), nurses (8), and laboratory workers (4)]. The others were cook (1), hairdresser (1), housewife (1), and office workers (2). Sixteen of the 19 patients were asked whether they had symptoms after eating bananas and the prick ⫹ prick technique21 with a freshly peeled banana was performed. Control skin prick tests were performed with codeine phosphate (9%) as positive control and the negative control was saline. The criterion of positivity was a 3-mm mean diameter wheal over the negative control. Sera
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Table 1. General Characteristics of the 19 Latex-Allergic Patients Characteristics
Number
Total Male/Female Atopic % Positive skin prick test to ammoniated natural rubber latex % Positive skin prick test to commercial latex extract % Symptoms caused by latex Asthma Contact urticaria Rhinoconjunctivitis Pruritus Edema Erythema Anaphylaxis Conjunctivitis Eczema Rhinitis
19 2/17 65 81
83
7 7 5 4 4 3 2 1 1 1
of five subjects with negative clinical histories were used as control group. Total and specific IgE to banana and latex were measured with the CAP RAST system (Pharmacia, Uppsala, Sweden). Values higher than 0.35 kU/L were considered positive. Allergenic Extracts Used in SDS PAGE and in Western Blot Experiments Banana was pulverized and stirred for two hours in NH4HCO3, 125 mmol/L, pH 8, EDTA 1 mmol/L at 4°C. The mixture was centrifuged for 30 minutes at 10,000 g at 4°C. The supernatant was filtered on Whatman filter N. 4 and 40. Ammonium sulfate was then added in the proportion of 29.5 g/100 mL of liquid, and the solution was stored at 4°C during the night. After centrifugation (10,000 g, 30 min, 4°C), the precipitate was resuspended in water and the solution was dialyzed overnight at 4°C in water across a 3.5-kD cut-off membrane. The dialysate was filtered on 0.45-m filter, the pH was adjusted to 7 and the banana solution was freeze dried.
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Ammoniated natural rubber latex, kindly provided by Safic Alcan (Paris, France), was centrifuged for 30 minutes at 20,000 g. The aqueous phase containing the soluble proteins was separated from the upper rubber phase, centrifuged several times at 20,000 g until no more rubber coagulate formed, filtered on 0.45 m and 0.22-m filters (Millipore, Bedford, Ma, USA) and used in Western blot inhibition experiments. Protein concentrations of the different extracts were measured by the BCA Protein Assay Reagent (Pierce, Rockford, Chemical Co, IL, USA) according to the manufacturer’s instructions (30 minutes at 37°C, with bovine serum albumin as standard protein). The extracts had the following protein concentrations: latex 2.5 mg/mL and banana 0.6 mg/mL. SDS PAGE and IgE Immunoblotting SDS PAGE was carried out as described by Laemmli22 on a vertical electrophoresis mini-Protean II system (Bio Rad Laboratories, Richmond, Ca, USA) using 12% homogeneous gels (8 ⫻ 7.3 cm). Samples were heated at 100°C for five minutes in sample buffer containing 1%  mercaptoethanol. Twenty-four microliters of a 0.6 mg/mL protein solution of banana was applied to each well. Electrophoresis was performed for 45 minutes at a constant voltage of 150 V. Then, electrophoresed proteins were stained using an ammoniated silver staining procedure23 or electrotransferred. Molecular weights of the proteins under investigation were determined by comparing their electrophoretic mobility with the protein standards of known molecular weight, ranging from 6.5 to 220 kD (Amersham, Buckinghamshire, UK). Protein transfer to nitrocellulose (Schleicher & Schu¨ell, Dassel, Germany) was carried out as described by Towbin et al.24 After blotting, the membranes were incubated with blocking buffer PBS/BSA 3%, for one hour at 37°C prior to immunodetection. Sera diluted 1:5 in blocking buffer were added to the strips and
were incubated overnight at room temperature with continuous shaking. Strips were then washed five times for five minutes with washing buffer (0.05% Tween/PBS). Bound IgE antibodies were detected by incubating each strip with 125I rabbit anti-human IgE (Pharmacia, Uppsala, Sweden) diluted 1:2 in blocking buffer for four hours. Autoradiography was performed at ⫺80°C for three to seven days with Kodak Xomat films (Kodak, Heidelberg, Germany). Strips were also processed with the pool of control subjects’ sera. Immunoblotting Inhibition To determine the apparent molecular weight of proteins involved in crossreactivity, immunoblot inhibitions were performed with banana blotted allergens and different concentrations of banana and latex extracts. Inhibition assays were done for patient no. 11 and no. 12. Sera were diluted 1:10 and preincubated with increasing concentrations of banana (from 0 to 200 g/ mL), of latex (from 0 to 650 g/mL) or with 450 g/mL of honey bee venom extract as negative control (Pharmalgen, Pharmacia) during four hours under shaking. Immunoblots were then performed as described above with the preincubated sera. Bound IgE was detected with anti-human 125I labeled IgE. RESULTS Patients Table 2 summarizes the symptoms elicited by banana ingestion, the skin prick test and the in vitro results of the 19 latex-allergic patients. Briefly, 8 of 16 patients (50%) developed symptoms after eating bananas. Banana induced reactions including gastrointestinal disorders, pruritus, erythema, edema, rhiniconjunctivitis, and asthma. Banana skin prick tests were positive in 5 of the 14 patients tested (36%). Positive specific IgE against latex was found in all patients and banana CAP RAST results were positive in 12 of 19 (63%). All control subjects had negative skin prick tests and CAP RAST results to banana and latex.
ANNALS OF ALLERGY, ASTHMA, & IMMUNOLOGY
Banana Proteins and Allergens The silver stained profile of banana proteins is illustrated in Figure 1 and shows predominant components of apparent molecular weight located at: 14, 19 –21, 29.5, 31.6, 33, 36, 40, 55, and 60 kD. Banana immunoblot results were positive in 17 of 19 patients (89%), and apparent molecular weight of allergens ranged from 17 to 128 kD. IgE binding occurred mainly at 19 kD (4/19, 21%), at 33 kD (15/ 19, 88%), at 37 kD (13/19, 76%), at 39 kD (4/19, 21%), and at 40 kD (5/19, 26%). Two different patterns were obtained: for sera no. 2 and no. 11, respectively 11 and 16 IgE binding proteins were detected, whereas for the other sera, fewer allergens were found (from 1 to 5). Table 3 summarizes the results of Western blotting and Figure 2 shows the IgE binding pattern for 9 patients on banana blotted allergens. The control group demonstrated no reactivity against banana proteins. Immunoblotting Inhibition Banana immunoblotting inhibition was done for the two patients having
higher levels of specific IgE to banana or latex (no. 11 and no. 12). Patient no. 11 with the highest banana RAST value (7.9 kU/L) recognized 16 different proteins on banana blotted extracts. In homologous inhibitions, IgE binding was suppressed with 80 g/mL of banana protein extract. A concentration of 130 g/mL of latex proteins preincubated with the serum, first inhibited the binding of IgE on seven banana peptides (17, 19, 29.5, 31, 33, 91 and 105 kD) and weakened the others. With a higher concentration of 650 g/mL of latex proteins, the whole response was extinguished (Fig 3a). In patient no. 12 with the highest latex RAST (above 100 kU/L), IgE binding occurred at 33 and 37 kD. Banana and latex both inhibited IgE binding with quantities of respectively, 160 g/mL and 15 g/mL of proteins (Fig 3b). In two cases, IgE reactivity against banana was completely inhibited by homologous and latex extract. Nonspecific inhibition was excluded in control studies with honey bee venom allergen.
Table 2. Symptoms Elicited by Ingestion of Banana, Banana Skin Prick Test, and In Vitro Results of 19 Latex-Allergic Patients Patients No 1 2 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
Specific IgE to
Total IgE, kU/L
Latex
Banana
2200 nd 34 403 nd nd 1139 181 nd 1300 780 24 nd nd 1250 16 nd nd 376
15 2.0 2.1 1.75 20 0.63 13 6.6 5.6 1.5 ⬎100 21 8.6 5.3 29 4.8 3.0 16 2.0
5.1 5.0 0.35 ⬍0.35 2.6 0.64 0.66 0.41 0.36 7.9 1.7 0.36 ⬍0.35 0.42 ⬍0.35 ⬍0.35 ⬍0.35 ⬍0.35 ⬍0.35
Skin Prick Test to Banana
Symptoms* Caused by Banana
⫺ nd ⫺ ⫺ ⫺ ⫺ ⫹ ⫹ ⫺ nd nd ⫹ ⫹ nd nd ⫺ ⫹ ⫺ ⫺
⫺ * ⫺ ⫺ ⫺ ⫺ G RC, Ed * ⫺ P, Er G, U,A,C P G ⫺ RC G * ⫺
* G, gastrointestinal disorders; RC, rhinoconjunctivitis; Ed, edema; P, pruritus; Er, erythema; U, urticaria; A, asthma; C, conjunctivitis; nd, not done; and *, unknown.
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Figure 1. SDS PAGE analysis of banana proteins (12% polyacrylamide gel, silver staining).
DISCUSSION In our study, 50% of patients reported symptoms after eating bananas, which agrees with a previous study of Ma¨kinen-Kiljunen who found that 52% of patients with latex allergy had food allergy to bananas.25 Extending their investigation to other fruits (avocado, chestnut, kiwi, and papaya) Blanco et al6 found that 52% of latex allergic patients had multiple sensitivities, 28% of them having hypersensitivity to banana. Lavaud et al18, studying 17 latexallergic patients reported ten subjects (58%) with an associated fruit allergy (avocado or/and banana). Banana skin prick tests carried out in 14 patients showed five positive cases allowing us to find an 83% concordance with the clinical diagnosis (5/6, Table 2). Blanco et al6 obtained a similar result (80%, with native fruits). CAP RAST results to banana were positive in 12 of our 19 latex-allergic patients (63%); in the Finnish study,25 specific IgE to banana was revealed with commercial CAP RAST in 26 of 47 patients with latex allergy (55%). Lavaud et al using Phadebas RAST
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Table 3. Molecular Weight of Banana Allergens Recognized by the 19 Latex-Allergic Patients Apparent Molecular Weight, kD
Patients No.
128
105
95
91
77
72
66
61
53–51
40
39
37
33
31
29.5
19
17
1 2 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
⫺ ⫹ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫹ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺
⫺ ⫹ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫹ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺
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evidenced specific IgE to banana in 7 of 17 latex allergic patient (41%), whereas, using the same test, de Corres et al4 found only one positive patient on 8. In the same way, Ross et al26 using ELISA experiments, failed to find specific IgE to banana in 12 patients with latex allergy. The discrepancies between these studies may be due to the different solid phases and allergenic extracts used, and point up, as in the case of latex allergen, the lack of standardization of banana allergens. The main constituents of banana are starch, sugars, and cellulose, while banana protein content is low.27 After selective precipitation of proteins with ammonium sulfate and quantification with BCA protein assay, we found 0.03 g of proteins for 100 g of fruit. Adisa et al28 obtained 2.2 to 2.5 g of proteins for 100 g of fruit on the total pulp by the Kjeldahl method. Our lower values may be explained by only measuring soluble proteins in supernatant after discarding a viscous pellet. By SDS PAGE we observed at least 15 different protein bands in banana with molecular weights ranging from 6.5 kD to more than 100 kD with intense staining located at 14, 19 –21, 29.5, 31.6, 33, 36, 40, 55, and 60 kD. Ba-
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Figure 2. IgE binding patterns for nine patients on banana blotted allergens.
nana proteins are less studied than latex ones; nevertheless, some of them have been purified by gel filtration or characterized with SDS PAGE. Wadee et al29 found less than ten proteins after Coomassie blue staining with molecular weights ranging from 14 to 67 kD. In Lavaud’s study, banana proteins of apparent molecular weight of 14, 25, 30 to 35, and 68 to 75 kD were reported.18 Konishi et al30 have purified with
gel filtration two ␣-D-glucosidases with a respective molecular weight of 70 and 42 kD, that are involved in starch and maltooligosaccharide metabolism. Koshte et al31 for their part have characterized a mannoside binding lectin of molecular weight 13 kD. Recently, 1-aminocyclopropane-1-carboxylate oxidase (ACC oxidase) from banana fruit has been purified. This enzyme is involved in the biosynthesis
ANNALS OF ALLERGY, ASTHMA, & IMMUNOLOGY
Figure 3a. Inhibition of banana immunoblots. Serum from patient 11 was preincubated with banana, latex, or honey bee venom extract before immunoblotting procedure. Lane 1: no inhibiting extract, lane 2: 80 g/mL of banana, lane 3: 130 g/mL of latex, lane 4: 650 g/mL of latex, and lane 5: 450 g/mL of honey bee venom allergen. Figure 3b. Inhibition of banana immunoblots. Serum from patient 12 was preincubated with banana, latex, or honey bee venom before immunoblotting procedure. Lane 1: no inhibiting extract, lane 2: 160 g/mL of banana, lane 3: 15 g/mL of latex, lane 4: honey bee venom allergen.
of ethylene, an important plant hormone during the ripening of climacteric fruits. The molecular mass of the purified enzyme was estimated to be 40 kD by gel filtration and 36 kD by SDS PAGE.32
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In our Western blot experiments, 17 of the 19 sera studied displayed IgE binding to banana proteins. We obtained two different kinds of pattern (for sera no. 2, no. 11 and for the others), and in opposition to Lavaud et
al’s results, this difference is not associated with the clinical histories. Indeed, our group of patients clinically allergic to both banana and latex (patients no. 8, 9, 12, 13, 15, 17, 18) did not exhibit a different IgE pattern than patients allergic to latex only. We highlighted two allergens with high frequency: one at 33 kD (88%) and the other at 37 kD (76%). Allergens of similar apparent molecular weight have been found in two other studies: one of apparent molecular weight of 30 kD shared by avocado, latex, and banana that could be hevamine18; and one of molecular weight of 30 kD found in peach, mandarin, strawberry, guava, and banana.29 In our study, the allergen of apparent molecular weight of 37 kD found in 13 of 19 cases could correspond to the banana ACC oxidase (molecular weight 36 kD) recently purified. This one shares common epitopes with the tomato ACC oxidase.32 Furthermore, ACC oxidase activity has been studied in dicotyledonous fruits such as melon, avocado, apple, pear, and winter squash.32 It would be interesting to search for an ACC oxidase activity in Hevea brasiliensis latex because Hevea trees produce ethylene and this hormone is known to stimulate the rubber production.33 The existence of enzymes devoted to biochemical synthesis or hydrolysis in plants (eg, ACC oxidase for ethylene biosynthesis, sucrose synthase and sucrose phosphate synthase for saccharose biosynthesis,33,34 phosphatase, phosphorylase, protease, glucosidases, etc) could explain the cross-reactivities observed between different fruits and between latex and fruits. Immunoblot inhibition studies on banana immunoblots were performed with serum no. 11 which exhibited the strongest and diversified response and with serum no. 12 which was representative of the general sera responses. Our inhibition studies allowed us to find a complete inhibition by both banana and latex extracts. The complete inhibition observed for serum no. 11 (including the protein located at 37 kD) showed that more than ten allergens are involved in latex and banana
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cross-reactivity, which agrees with Ma¨kinen-Kiljunen et al previous results.35 In conclusion, this study confirms the “latex-fruit syndrome” already described by Blanco et al.6 Two major allergens were revealed in banana with apparent molecular weight of 33 and 37 kD and the existence of more than ten common components with latex was observed. Our current research is directed towards extending the analysis of proteins located in the zone of 33 to 37-kD and to compare them with ACC oxidase and latex allergens already sequenced (heveine, hevamine, and prenyltransferase.36,37 ACKNOWLEDGMENTS We thank Christine Juillet for her excellent technical assistance and Ge´rard Joly for photography.
10.
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Request for reprints should be addressed to: M F Delbourg, PhD Unite´ de Radioanalyse Institut Pasteur de Lyon Av Tony Garnier 69 365 Cedex 07 France
ANNALS OF ALLERGY, ASTHMA, & IMMUNOLOGY
Background: Allergy to banana often occurs in patients sensitized to latex. The spectrum of IgE-mediated responses to latex allergens is more and more documented but banana allergens and epitopes shared by these two allergens have not yet been characterized, even though the existence of cross-reacting IgE antibodies has been demonstrated. Objective: The purpose of this study was to assess the relationship between banana hypersensitivity and latex allergy in a population of 19 latex-allergic patients and to identify allergenic components in banana and the common structures shared with latex. Methods: The in vivo study was conducted in our outpatient department in patients with well-documented latex allergy. Clinical histories were evaluated and skin prick tests were performed with banana and latex extracts. IgE responses from 19 patients were investigated by means of CAP RAST assay and SDS PAGE immunoblotting. Epitopes shared by banana and latex were investigated by means of immunoblotting experiments. Results: Eight of 16 patients (50%) reported symptoms after eating bananas and banana skin prick tests were positive in 5 of 14 patients (36%). Banana RAST results were positive in 12 of the 19 patients (63%). In immunoblot experiments, 17 of the 19 patients (89%) exhibited specific banana IgE antibodies and 16 allergenic components were identified with molecular weights ranging from 17 to 128 kD. Two were considered as major allergens: 33 kD was detectable in 15 of 19 sera (88%) and 37 kD in 13 of 19 sera (76%). Inhibition studies by preincubation of two individual sera with banana or latex extract demonstrated the complete disappearance of IgE binding on banana blotted allergens. Conclusion: This study confirms the “latex-fruit syndrome” already described by Blanco et al. Two major allergens were revealed in banana at 33 and 37 kD and the presence of more than ten common components with latex was observed. Ann Allergy Asthma Immunol 1996;76:321– 6.
INTRODUCTION Cross-reacting allergies between unrelated species have been known for many years.1–3 Rubber latex allergy, for example, is often associated with allergy to fruits such as avocado, ba* Unite´ de Radioanalyse; Institut Pasteur de Lyon; Av Tony Garnier, 69 365; Lyon Cedex 07; France. † Department of Medicine “D”; Clinical Immunology and Allergology; Hopital Central, 29; Av du Mare´chal de Lattre de Tassigny; 54 035 Nancy Cedex; France. Received for publication August 1, 1995. Accepted for publication in revised form October 18, 1995.
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nana, chestnut, kiwi, papaya, and peaches.4 –11 M’Raihi et al have for the first time evidenced the association between banana and latex allergens using RAST inhibition assays.12 Numerous studies relating to latex hypersensitivity and latex allergens have been published,13–17 whereas banana antigenic and allergenic components have not been well characterized. Recently, an allergen of approximately 30 kD was identified in both latex and fruit extracts (banana, avocado).18 Our study was undertaken to assess the relationship between banana hypersensitivity and latex allergy in a population of 19
latex-allergic patients and to identify banana proteins and potential allergens by means of SDS PAGE and Western blot experiments. Common structures shared by the two allergens were investigated by means of immunoblotting inhibition assays. We have previously published preliminary results on banana antigens and allergens in abstract form.19,20 MATERIALS AND METHODS Patients Sera from 19 patients (17 women and 2 men) referred to the outpatient department (Department of Medicine “D”; Clinical Immunology and Allergology; Hoˆpital Central; Nancy, France) with well-documented latex allergy were selected for this study. All patients gave their informed consent to participate in the study. They were skin tested with two latex extracts: an ammoniated natural rubber latex emulsion prepared as previously described,17 and a commercial extract of ammoniated latex 1:100 wt/vol from Stallerge`nes SA (Fresnes, France). Symptoms to latex are summarized in Table 1. Most patients were healthcare workers [surgeon (1), dental assistant (1), nurses (8), and laboratory workers (4)]. The others were cook (1), hairdresser (1), housewife (1), and office workers (2). Sixteen of the 19 patients were asked whether they had symptoms after eating bananas and the prick ⫹ prick technique21 with a freshly peeled banana was performed. Control skin prick tests were performed with codeine phosphate (9%) as positive control and the negative control was saline. The criterion of positivity was a 3-mm mean diameter wheal over the negative control. Sera
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Table 1. General Characteristics of the 19 Latex-Allergic Patients Characteristics
Number
Total Male/Female Atopic % Positive skin prick test to ammoniated natural rubber latex % Positive skin prick test to commercial latex extract % Symptoms caused by latex Asthma Contact urticaria Rhinoconjunctivitis Pruritus Edema Erythema Anaphylaxis Conjunctivitis Eczema Rhinitis
19 2/17 65 81
83
7 7 5 4 4 3 2 1 1 1
of five subjects with negative clinical histories were used as control group. Total and specific IgE to banana and latex were measured with the CAP RAST system (Pharmacia, Uppsala, Sweden). Values higher than 0.35 kU/L were considered positive. Allergenic Extracts Used in SDS PAGE and in Western Blot Experiments Banana was pulverized and stirred for two hours in NH4HCO3, 125 mmol/L, pH 8, EDTA 1 mmol/L at 4°C. The mixture was centrifuged for 30 minutes at 10,000 g at 4°C. The supernatant was filtered on Whatman filter N. 4 and 40. Ammonium sulfate was then added in the proportion of 29.5 g/100 mL of liquid, and the solution was stored at 4°C during the night. After centrifugation (10,000 g, 30 min, 4°C), the precipitate was resuspended in water and the solution was dialyzed overnight at 4°C in water across a 3.5-kD cut-off membrane. The dialysate was filtered on 0.45-m filter, the pH was adjusted to 7 and the banana solution was freeze dried.
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Ammoniated natural rubber latex, kindly provided by Safic Alcan (Paris, France), was centrifuged for 30 minutes at 20,000 g. The aqueous phase containing the soluble proteins was separated from the upper rubber phase, centrifuged several times at 20,000 g until no more rubber coagulate formed, filtered on 0.45 m and 0.22-m filters (Millipore, Bedford, Ma, USA) and used in Western blot inhibition experiments. Protein concentrations of the different extracts were measured by the BCA Protein Assay Reagent (Pierce, Rockford, Chemical Co, IL, USA) according to the manufacturer’s instructions (30 minutes at 37°C, with bovine serum albumin as standard protein). The extracts had the following protein concentrations: latex 2.5 mg/mL and banana 0.6 mg/mL. SDS PAGE and IgE Immunoblotting SDS PAGE was carried out as described by Laemmli22 on a vertical electrophoresis mini-Protean II system (Bio Rad Laboratories, Richmond, Ca, USA) using 12% homogeneous gels (8 ⫻ 7.3 cm). Samples were heated at 100°C for five minutes in sample buffer containing 1%  mercaptoethanol. Twenty-four microliters of a 0.6 mg/mL protein solution of banana was applied to each well. Electrophoresis was performed for 45 minutes at a constant voltage of 150 V. Then, electrophoresed proteins were stained using an ammoniated silver staining procedure23 or electrotransferred. Molecular weights of the proteins under investigation were determined by comparing their electrophoretic mobility with the protein standards of known molecular weight, ranging from 6.5 to 220 kD (Amersham, Buckinghamshire, UK). Protein transfer to nitrocellulose (Schleicher & Schu¨ell, Dassel, Germany) was carried out as described by Towbin et al.24 After blotting, the membranes were incubated with blocking buffer PBS/BSA 3%, for one hour at 37°C prior to immunodetection. Sera diluted 1:5 in blocking buffer were added to the strips and
were incubated overnight at room temperature with continuous shaking. Strips were then washed five times for five minutes with washing buffer (0.05% Tween/PBS). Bound IgE antibodies were detected by incubating each strip with 125I rabbit anti-human IgE (Pharmacia, Uppsala, Sweden) diluted 1:2 in blocking buffer for four hours. Autoradiography was performed at ⫺80°C for three to seven days with Kodak Xomat films (Kodak, Heidelberg, Germany). Strips were also processed with the pool of control subjects’ sera. Immunoblotting Inhibition To determine the apparent molecular weight of proteins involved in crossreactivity, immunoblot inhibitions were performed with banana blotted allergens and different concentrations of banana and latex extracts. Inhibition assays were done for patient no. 11 and no. 12. Sera were diluted 1:10 and preincubated with increasing concentrations of banana (from 0 to 200 g/ mL), of latex (from 0 to 650 g/mL) or with 450 g/mL of honey bee venom extract as negative control (Pharmalgen, Pharmacia) during four hours under shaking. Immunoblots were then performed as described above with the preincubated sera. Bound IgE was detected with anti-human 125I labeled IgE. RESULTS Patients Table 2 summarizes the symptoms elicited by banana ingestion, the skin prick test and the in vitro results of the 19 latex-allergic patients. Briefly, 8 of 16 patients (50%) developed symptoms after eating bananas. Banana induced reactions including gastrointestinal disorders, pruritus, erythema, edema, rhiniconjunctivitis, and asthma. Banana skin prick tests were positive in 5 of the 14 patients tested (36%). Positive specific IgE against latex was found in all patients and banana CAP RAST results were positive in 12 of 19 (63%). All control subjects had negative skin prick tests and CAP RAST results to banana and latex.
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Banana Proteins and Allergens The silver stained profile of banana proteins is illustrated in Figure 1 and shows predominant components of apparent molecular weight located at: 14, 19 –21, 29.5, 31.6, 33, 36, 40, 55, and 60 kD. Banana immunoblot results were positive in 17 of 19 patients (89%), and apparent molecular weight of allergens ranged from 17 to 128 kD. IgE binding occurred mainly at 19 kD (4/19, 21%), at 33 kD (15/ 19, 88%), at 37 kD (13/19, 76%), at 39 kD (4/19, 21%), and at 40 kD (5/19, 26%). Two different patterns were obtained: for sera no. 2 and no. 11, respectively 11 and 16 IgE binding proteins were detected, whereas for the other sera, fewer allergens were found (from 1 to 5). Table 3 summarizes the results of Western blotting and Figure 2 shows the IgE binding pattern for 9 patients on banana blotted allergens. The control group demonstrated no reactivity against banana proteins. Immunoblotting Inhibition Banana immunoblotting inhibition was done for the two patients having
higher levels of specific IgE to banana or latex (no. 11 and no. 12). Patient no. 11 with the highest banana RAST value (7.9 kU/L) recognized 16 different proteins on banana blotted extracts. In homologous inhibitions, IgE binding was suppressed with 80 g/mL of banana protein extract. A concentration of 130 g/mL of latex proteins preincubated with the serum, first inhibited the binding of IgE on seven banana peptides (17, 19, 29.5, 31, 33, 91 and 105 kD) and weakened the others. With a higher concentration of 650 g/mL of latex proteins, the whole response was extinguished (Fig 3a). In patient no. 12 with the highest latex RAST (above 100 kU/L), IgE binding occurred at 33 and 37 kD. Banana and latex both inhibited IgE binding with quantities of respectively, 160 g/mL and 15 g/mL of proteins (Fig 3b). In two cases, IgE reactivity against banana was completely inhibited by homologous and latex extract. Nonspecific inhibition was excluded in control studies with honey bee venom allergen.
Table 2. Symptoms Elicited by Ingestion of Banana, Banana Skin Prick Test, and In Vitro Results of 19 Latex-Allergic Patients Patients No 1 2 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
Specific IgE to
Total IgE, kU/L
Latex
Banana
2200 nd 34 403 nd nd 1139 181 nd 1300 780 24 nd nd 1250 16 nd nd 376
15 2.0 2.1 1.75 20 0.63 13 6.6 5.6 1.5 ⬎100 21 8.6 5.3 29 4.8 3.0 16 2.0
5.1 5.0 0.35 ⬍0.35 2.6 0.64 0.66 0.41 0.36 7.9 1.7 0.36 ⬍0.35 0.42 ⬍0.35 ⬍0.35 ⬍0.35 ⬍0.35 ⬍0.35
Skin Prick Test to Banana
Symptoms* Caused by Banana
⫺ nd ⫺ ⫺ ⫺ ⫺ ⫹ ⫹ ⫺ nd nd ⫹ ⫹ nd nd ⫺ ⫹ ⫺ ⫺
⫺ * ⫺ ⫺ ⫺ ⫺ G RC, Ed * ⫺ P, Er G, U,A,C P G ⫺ RC G * ⫺
* G, gastrointestinal disorders; RC, rhinoconjunctivitis; Ed, edema; P, pruritus; Er, erythema; U, urticaria; A, asthma; C, conjunctivitis; nd, not done; and *, unknown.
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Figure 1. SDS PAGE analysis of banana proteins (12% polyacrylamide gel, silver staining).
DISCUSSION In our study, 50% of patients reported symptoms after eating bananas, which agrees with a previous study of Ma¨kinen-Kiljunen who found that 52% of patients with latex allergy had food allergy to bananas.25 Extending their investigation to other fruits (avocado, chestnut, kiwi, and papaya) Blanco et al6 found that 52% of latex allergic patients had multiple sensitivities, 28% of them having hypersensitivity to banana. Lavaud et al18, studying 17 latexallergic patients reported ten subjects (58%) with an associated fruit allergy (avocado or/and banana). Banana skin prick tests carried out in 14 patients showed five positive cases allowing us to find an 83% concordance with the clinical diagnosis (5/6, Table 2). Blanco et al6 obtained a similar result (80%, with native fruits). CAP RAST results to banana were positive in 12 of our 19 latex-allergic patients (63%); in the Finnish study,25 specific IgE to banana was revealed with commercial CAP RAST in 26 of 47 patients with latex allergy (55%). Lavaud et al using Phadebas RAST
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Table 3. Molecular Weight of Banana Allergens Recognized by the 19 Latex-Allergic Patients Apparent Molecular Weight, kD
Patients No.
128
105
95
91
77
72
66
61
53–51
40
39
37
33
31
29.5
19
17
1 2 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
⫺ ⫹ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫹ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺
⫺ ⫹ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫹ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺
⫺ ⫹ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫹ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺
⫺ ⫹ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫹ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺
⫺ ⫹ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫹ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺
⫺ ⫹ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫹ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺
⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫹ ⫺ ⫺ ⫺ ⫹ ⫺ ⫺ ⫺ ⫺ ⫺
⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫹ ⫺ ⫺ ⫺ ⫹ ⫺ ⫺ ⫺ ⫺ ⫺
⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫹ ⫺
⫺ ⫹ ⫺ ⫺ ⫺ ⫺ ⫹ ⫺ ⫺ ⫹ ⫺ ⫺ ⫺ ⫹ ⫺ ⫺ ⫺ ⫹ ⫺
⫺ ⫹ ⫺ ⫺ ⫺ ⫺ ⫹ ⫺ ⫺ ⫹ ⫺ ⫺ ⫺ ⫹ ⫺ ⫺ ⫺ ⫺ ⫺
⫹ ⫹ ⫹ ⫺ ⫹ ⫹ ⫹ ⫹ ⫺ ⫹ ⫹ ⫹ ⫺ ⫺ ⫹ ⫹ ⫹ ⫺ ⫺
⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫺ ⫺ ⫹ ⫹ ⫹ ⫺ ⫺
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⫺ ⫹ ⫺ ⫺ ⫺ ⫺ ⫹ ⫺ ⫺ ⫹ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫹ ⫺ ⫺
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evidenced specific IgE to banana in 7 of 17 latex allergic patient (41%), whereas, using the same test, de Corres et al4 found only one positive patient on 8. In the same way, Ross et al26 using ELISA experiments, failed to find specific IgE to banana in 12 patients with latex allergy. The discrepancies between these studies may be due to the different solid phases and allergenic extracts used, and point up, as in the case of latex allergen, the lack of standardization of banana allergens. The main constituents of banana are starch, sugars, and cellulose, while banana protein content is low.27 After selective precipitation of proteins with ammonium sulfate and quantification with BCA protein assay, we found 0.03 g of proteins for 100 g of fruit. Adisa et al28 obtained 2.2 to 2.5 g of proteins for 100 g of fruit on the total pulp by the Kjeldahl method. Our lower values may be explained by only measuring soluble proteins in supernatant after discarding a viscous pellet. By SDS PAGE we observed at least 15 different protein bands in banana with molecular weights ranging from 6.5 kD to more than 100 kD with intense staining located at 14, 19 –21, 29.5, 31.6, 33, 36, 40, 55, and 60 kD. Ba-
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Figure 2. IgE binding patterns for nine patients on banana blotted allergens.
nana proteins are less studied than latex ones; nevertheless, some of them have been purified by gel filtration or characterized with SDS PAGE. Wadee et al29 found less than ten proteins after Coomassie blue staining with molecular weights ranging from 14 to 67 kD. In Lavaud’s study, banana proteins of apparent molecular weight of 14, 25, 30 to 35, and 68 to 75 kD were reported.18 Konishi et al30 have purified with
gel filtration two ␣-D-glucosidases with a respective molecular weight of 70 and 42 kD, that are involved in starch and maltooligosaccharide metabolism. Koshte et al31 for their part have characterized a mannoside binding lectin of molecular weight 13 kD. Recently, 1-aminocyclopropane-1-carboxylate oxidase (ACC oxidase) from banana fruit has been purified. This enzyme is involved in the biosynthesis
ANNALS OF ALLERGY, ASTHMA, & IMMUNOLOGY
Figure 3a. Inhibition of banana immunoblots. Serum from patient 11 was preincubated with banana, latex, or honey bee venom extract before immunoblotting procedure. Lane 1: no inhibiting extract, lane 2: 80 g/mL of banana, lane 3: 130 g/mL of latex, lane 4: 650 g/mL of latex, and lane 5: 450 g/mL of honey bee venom allergen. Figure 3b. Inhibition of banana immunoblots. Serum from patient 12 was preincubated with banana, latex, or honey bee venom before immunoblotting procedure. Lane 1: no inhibiting extract, lane 2: 160 g/mL of banana, lane 3: 15 g/mL of latex, lane 4: honey bee venom allergen.
of ethylene, an important plant hormone during the ripening of climacteric fruits. The molecular mass of the purified enzyme was estimated to be 40 kD by gel filtration and 36 kD by SDS PAGE.32
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In our Western blot experiments, 17 of the 19 sera studied displayed IgE binding to banana proteins. We obtained two different kinds of pattern (for sera no. 2, no. 11 and for the others), and in opposition to Lavaud et
al’s results, this difference is not associated with the clinical histories. Indeed, our group of patients clinically allergic to both banana and latex (patients no. 8, 9, 12, 13, 15, 17, 18) did not exhibit a different IgE pattern than patients allergic to latex only. We highlighted two allergens with high frequency: one at 33 kD (88%) and the other at 37 kD (76%). Allergens of similar apparent molecular weight have been found in two other studies: one of apparent molecular weight of 30 kD shared by avocado, latex, and banana that could be hevamine18; and one of molecular weight of 30 kD found in peach, mandarin, strawberry, guava, and banana.29 In our study, the allergen of apparent molecular weight of 37 kD found in 13 of 19 cases could correspond to the banana ACC oxidase (molecular weight 36 kD) recently purified. This one shares common epitopes with the tomato ACC oxidase.32 Furthermore, ACC oxidase activity has been studied in dicotyledonous fruits such as melon, avocado, apple, pear, and winter squash.32 It would be interesting to search for an ACC oxidase activity in Hevea brasiliensis latex because Hevea trees produce ethylene and this hormone is known to stimulate the rubber production.33 The existence of enzymes devoted to biochemical synthesis or hydrolysis in plants (eg, ACC oxidase for ethylene biosynthesis, sucrose synthase and sucrose phosphate synthase for saccharose biosynthesis,33,34 phosphatase, phosphorylase, protease, glucosidases, etc) could explain the cross-reactivities observed between different fruits and between latex and fruits. Immunoblot inhibition studies on banana immunoblots were performed with serum no. 11 which exhibited the strongest and diversified response and with serum no. 12 which was representative of the general sera responses. Our inhibition studies allowed us to find a complete inhibition by both banana and latex extracts. The complete inhibition observed for serum no. 11 (including the protein located at 37 kD) showed that more than ten allergens are involved in latex and banana
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cross-reactivity, which agrees with Ma¨kinen-Kiljunen et al previous results.35 In conclusion, this study confirms the “latex-fruit syndrome” already described by Blanco et al.6 Two major allergens were revealed in banana with apparent molecular weight of 33 and 37 kD and the existence of more than ten common components with latex was observed. Our current research is directed towards extending the analysis of proteins located in the zone of 33 to 37-kD and to compare them with ACC oxidase and latex allergens already sequenced (heveine, hevamine, and prenyltransferase.36,37 ACKNOWLEDGMENTS We thank Christine Juillet for her excellent technical assistance and Ge´rard Joly for photography.
10.
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Request for reprints should be addressed to: M F Delbourg, PhD Unite´ de Radioanalyse Institut Pasteur de Lyon Av Tony Garnier 69 365 Cedex 07 France
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