The use of inhibition assay in Api g 7 suspected allergy in a female patient with anaphylaxis: A case report

Abstract

The symptoms of celery allergy are mainly presented as oral allergy symptom, but there are several case reports of patients who experienced anaphylaxis. Defensin (Api g 7), as a novel allergen in celery root, was described in 2022 r. The female patient had a history of several episodes of dyspnea and cough, associated with ingestion of spice mixes containing dried celery. Up to the point of hospitalization, there were no objective tests, either sIgE or skin prick tests, that would confirm celery sensitization. During hospitalization, patient had a positive double-blind placebo-controlled food challenge with cooked celery. The patient was sensitized to mugwort defensin Art v 1. An inhibition assay with celery allergen extract was performed to prove cross-sensitization between Art v 1 and celery allergen responsible for symptoms in the patient. In conclusion, Api g 7 is an important celery allergen that can be responsible for severe reactions. Its cross-reactivity with Art v 1 is characteristic. Negative diagnostic tests with celery do not exclude Api g 7 sensitization.

Keywords

celery allergy mugwort inhibition assay defensin cross-reactivity

Introduction

Allergy to celery (Apium graveolens) is one of the most common food allergies. In Northern Europe, the prevalence in the adult population is estimated up to 0.45%.¹

The symptoms of celery allergy are mainly presented as oral allergy symptoms, but there are several case reports of patients who experienced anaphylaxis.^2,3 Celery allergy is often associated with mugwort (Artemisia vulgaris) sensitization. Usually, the clinical course of cross-sensitization in this case is related to classical pollen-food syndrome.^4,5 Celery defensin Api g 7 was described for the first time in 2021, as a missing link in the diagnosis of celery allergy.⁶ In the following article, we present an interesting case of a patient with anaphylaxis after celery, due to cross-sensitization with mugwort defensin.

Case report

The patient, 41, female, was admitted to the Clinic of Allergology, Clinical Immunology, and Internal Diseases in December 2022 for diagnosis of food allergy. The patient had a history of several episodes of dyspnea and cough, associated with ingestion of spice mixes containing dried celery. Symptoms have appeared since the year 2014. The patient eliminated celery from her diet and had several attempts at food allergy diagnosis in outpatient clinics. Up to the point of hospitalization, there were no objective tests, either sIgE or skin prick tests, that would confirm celery sensitization—all diagnostics were negative. The patient had intermitted allergic rhinitis due to Artemisia vulgaris sensitization, treated with blasting. The patient was treated with oral contraceptives and had an ovariectomy due to a cyst in 2018.

During the current hospitalization, a detailed interview was conducted. In physical examination, the patient had no abnormalities. The patient was qualified for basic diagnostics, including a skin prick test (Diater), prick-by-prick tests with fresh and cooked celery, and specific IgE assessment, using the ImmunoCap Singleplex method (Thermoscientific).⁷

The patient had dermographism; therefore, SPT was difficult to interpret but seemed negative with food allergens (milk, egg yolk, egg white, casein, peanut, hazelnut, cod, shrimp, wheat flour, soy flour, orange, and celery). In case of inhalatory, allergens positive only with mugwort allergen extract (6/6 mm) and negative with birch, hazel, alder, grass mix, dog dander, cat dander, Dermatophagoides farinae, Dermatoides pteronyssinus, and Cladosporium herbarum. Prick-by-prick tests with fresh and cooked celery were also negative. IgE specific to carrot, celery, and tomato were <0,10 kU/l (ImmunoCap Singleplex). Due to inconsistent clinical interviews with the result of the performed diagnosis, the patient had a component-resolved diagnosis—Alex 2 Allergy Explorer test.⁸ The results are presented in Table 1.

Table 1.

The concentration of specific IgE was measured using Alex 2 Allergy Explorer. Only positive results are presented in the table. sIgE to other allergen extracts and molecules available in Alex 2 Allergy Explorer were negative.

Allergen	Concentration of IgE
Total IgE	24 kU/L
Mugwort allergen extract	3,02 kUA/L
rArt v 1	7,93 kUA/L
Fel d 1	2,91 kUA/L
Can f 4	0,37 kUA/L
Oyster (extract)	9,23 kUA/L
Mussel (extract)	0,63 kUA/L
Squid (extract)	0,37 kUA/L
Other extracts and components	<0,1 kUA/L

It was found that the patient had elevated concentration of sIgE to mugwort allergen extract and mugwort defensin Art v 1. Specific IgE to all allergens typical for oral allergy syndrome available in Alex 2 Allergy Explorer—Bet v 1-like proteins, lipid transfer proteins, and profilins—were negative. Specific IgE to all available allergenic molecules of celery—Api g 1 (PR-10), Api g 2 (nsLTP), and Api g 6 (nsLTP)—were also negative.

The patient felt deep discomfort gathered with a lack of objective confirmation of her allergic symptoms, due to negative results both in the case of celery SPT and sIgE. The lack of objective results confirming celery allergy, subjective symptoms of an allergic reaction in the patients' history, and uncertainty about the actual allergen causing the symptoms caused a sense of anxiety in the patient. In cooperation with the patient, after discussing the benefits and risks, we decided to prepare the patient for a double-blind, placebo-controlled food challenge (DBPCFC) with cooked celery. Double-blind, placebo-controlled food challenge is time-consuming and may be dangerous in patients at risk of anaphylaxis, but it is a gold standard in confirming clinical symptoms after ingestion of food.⁹

The placebo sample contained boiled potatoes, espresso coffee, sugar, chicken meat, and boiled carrots. The active sample contained boiled celery (30g in total), espresso, sugar, chicken meat, and boiled carrots. In our opinion, the strong taste of sugar and coffee hid well the crucial ingredient, celery. The placebo and active sample were administered in the form of six doses of smoothie, increasing accordingly to a semi-logarithmic scale. The DBPCFC was performed under monitored conditions, for two consecutive days. The patient had no objective or subjective symptoms after all the placebo samples. After 4^th active sample and the ingestion of 15 g of cooked celery in total, the DBPCFC was found positive and was discontinued—the symptoms had a form of itching in the mouth. Dexamethasone and clemastinum were administered intravenously. After 30 min of observation, the symptoms worsened—the patient had a watery discharge from the nose and a feeling of nasal blockage. In the auscultation of the respiratory system, wheezing was found, respiratory rate of 20/min, heart rate 100/min, and blood pressure 150/90 mmHg. The patient was administered 0.5 mg of adrenaline intramuscularly with rapid improvement. In further observation, watery discharge from the nose and a feeling of nasal blockage reappeared after 6 h. The patient had another dose of dexamethasone administered with good clinical effect and the symptoms did not reemerge.

Due to positive DBPCFC with celery and suspicion of cross-reactivity with Artemisia vulgaris in this case, we decided to perform an inhibition assay. The methodology was described by us before, in the case of alpha-Gal sensitization.¹⁰ In short, we used “CAPs,” which are components of the ImmunoCAP system. CAPs are the carriers of the allergen. They are a plastic container in which a cellulose matrix coated with allergens is placed. This was the solid phase of the test—the allergen carrier. In the experiment, f85-CAP (celery) was used. The CAP was placed inside dry, clean Eppendorf tubes (volume 1500 µL). Then, 40µLof washing solution was added (Art. No. 10-9422-01) according to the manufacturer’s instructions. The CAP was transferred to a clean, dry Eppendorf (volume 1500 µL) tube and a total of 40 µL of patient serum was applied and the sealed Eppendorf tubes were incubated in two stages: first for 30 min at 37°C and then for 24 h at 2–8°C. After the incubation, the CAP was centrifuged (5000g; 15 min). After centrifugation, the CAP was discarded, and in the filtrates (of inhibited serum) Alex 2 Allergen Explorer. The results of the inhibition assay are presented in Table 2.

Table 2.

IgE inhibition test – to prove cross-reaction. Patient serum blocked on celery-coated ImmunoCAP membrane (IT-f85).

Alex 2 test results	Before IT-f85	After IT-f85	% of inhibition
tIgE (kU/L)	24	≤20
sIgE (kUA/L)
Mugwort (extract)	3,02	2,03	32,78%
Mugwort (defensin Art v 1)	7,93	1,61	79,7%
Oyster (extract)	9,23	9,59	−3,9
Mussel (extract)	0,63	0,59	6,34
Squid (extract)	0,37	-	-
Cat (Fel d 1)	2,91	2,75	5,5
Dog (Can f 4)	0,37	-	-

The percent of inhibition in the case of Art v 1, Artemisia vulgaris defensin, was as high as 79.7%. The percent of inhibition in the case of mugwort allergen extract was lower, but still evident, 32.78%. The other positive results in the Alex 2 test can be treated as a control for the inhibition test methodology od show the % of change before and after IT below 10%.

The clinical pattern of sensitization in this case, positive double-blind, placebo-controlled food challenge, with immunological results and inhibition assay, confirmed sensitization to celery, most probably due to defensin sensitization and cross-reactivity with mugwort.

In the 3 months follow-up, the patient is continuing an elimination diet with strict avoidance of celery and experienced no symptoms of food allergy.

Discussion

Mugwort inhalatory allergy and the coexistence of celery allergy are problems known for decades. Allergen components of celery (Apium graveolens) and mugwort (Artemisia vulgaris) described up to date are presented in Table 3.

Table 3.

Mugwort (Artemisia vulgaris) and celery (Apium graveolens) allergens described up to date.¹¹

Mugwort, wormwood (Artemisia vulgaris)		Celery (Apium graveolens)
Art v 1 (28 kDa)	Defensin-like protein linked to polyproline-rich region	Api g 1 (16 kDa)	Pathogenesis-related protein, PR-10, Bet v 1 family member
Art v 2 (20 kDa or 35 kDa non-red)	Pathogenesis-related protein PR-1	Api g 2 (9 kDa)	Non-specific lipid-transfer protein, type 1
Art v 3 (12 kDa)	Non-specific lipid-transfer protein type 1	Api g 3 (28 kDa)	Chlorophyll a-b binding protein, chloroplast
Art v 4 (14 kDa)	Profilin	Api g 4 (14 kDa)	Profilin
Art v 5 (10 kDa)	Polcalcin	Api g 5 (58 kDa)	FAD-containing oxidase
Art v 6 (44 kDa)	Pectate lyase	Api g 6 (7 kDa)	Non-specific lipid transfer protein type 2
		Api g 7 (12 kDa)	Defensin-like protein 1

There are several proteins in both celery and mugwort that share similarities in their structure and come from the same protein family and therefore might be responsible for cross-reactivity in this case. These include non-specific lipid-transfer protein, type 1, profilin, pathogenesis-related protein, FAD-containing oxidase, and, as described in the following case, defensin-like protein.^5,6,12

In general, cross-reacting major celery allergen Api g 1 has high homology to birch Bet v 1. Celery profilin (Api g 4) is an important allergenic compound in celery and shows high homology to birch pollen profilin, Bet v 2. These allergens are most often the cross-reactive compounds in pollen-fruit syndrome.¹³ In the presented case, only defensin-specific IgE was detected in Alex 2 Allergy Explorer, and no IgE specific to Bet v 1-like proteins or profilins were detected.

Defensin (Api g 7), as a novel allergen in celery root, was described in 2022, when Wangorsch A. et al. cloned celeriac defensin cDNA (accession# MT380846) and produced a recombinant Api g defensin. IgE-binding capacity and allergenicity of celeriac defensin were investigated by the authors in eight celeriac allergic patients, two celeriac-tolerant controls with mugwort pollen allergy, and a non-atopic control. Six of the celeriac allergic patients had positive double-blind placebo-controlled food challenge, and two were disqualified due to a history of anaphylaxis. In this study, all celeriac allergic patients were sensitized to rApi g 7. The key result was that the levels of specific IgE showed no clear correlation between rApi g 7 and celeriac extract, whereas sIgE levels to rApi g 7 and rArt v 1 correlated strongly.⁶ These results are in line with the described case, where the results of sIgE extract with celery were negative in all used immunological methods.

It is worth noticing that Art v 1 is a protein of 28 kDa, whereas Api g 7 is only 12 kDa. Although the difference is notable, the cross-reactivity between the two was previously proven by Wangorsch et al. The sequence of both Api g 7 and Art v 1 was compared in detail in Figure S2/Appendix 1 of their publication.⁶ There are different mechanisms of cross-reactivity between epitopes—some may have sequence identity, whereas others might have similar conformation.¹⁴ The case of Art v 1 and Api g 7 cross-reactivity would require further investigation.

An inhibition test was used to prove cross-reactivity between celery and mugwort defensin Art v 1. The inhibition test, in multiple forms, was used in the past to show cross-sensitization between different allergens. In our clinic, they were used to show cross-sensitization in case of, for example, sunflower seed sensitization and mugwort or mango pulp and mugwort. There are different diagnostic approaches in the case of inhibition testing, as described in previous publications, using both solid and liquid forms of allergen.^15,16 In the described case, we used the solid phase matrix of ImmunoCap with celery allergen. The results are clear, especially due to the fact, that in the Alex 2 test performed after inhibition, there is a very distinctive drop in the concentration of IgE specific to Art v 1 and mugwort allergen extract, but not in the case of sIgE cat or oyster, which results might be interpreted as a control for this methodological approach. Nevertheless, it is still considered an experimental method and one that would require further studies on larger samples.

Despite the fact that immunological diagnostic methods, including component-based multiplex assays, are becoming more affordable and available, the double-blind placebo-control food challenge remains the gold standard in the diagnosis of food allergy. There are many limitations of this diagnostic approach. It is time-consuming, requires the preparation of both active and placebo samples, and requires medical staff experienced in the treatment of anaphylaxis. One of the key problems is the correct qualification of patients for DBPCFC. The indications of oral food challenges are presented in Table 4.

Table 4.

The indications to oral food challenges.^17–20

When it is not clear which product caused the allergic reaction
Determining whether tolerance has been achieved in the case of allergies that have a tendency to resolve with time, such as allergy to eggs and milk in children
Assessing the effectiveness of pharmacological intervention (e.g., Immunotherapy, biological treatment)
Extending the diet in patients on extensive elimination diet.
In infants and young children, in whom it is often the only adequate diagnostic method, for example, in the assessment of factors exacerbating the course of skin lesions
To exclude or confirm allergy in patients whose allergy test results are inconsistent with the history of reactions

The first and last indication out of the above would be in line with the described patient. Contraindications to oral challenge include medications (e.g., steroids and antihistamines), that may affect the interpretation of the test result, exacerbation of persistent diseases (allergic or other), acute medical conditions (e.g., infection and psychosis), a history of severe anaphylactic reactions, suggests that the risk to the patient outweighs the potential diagnostic benefit of the test, and pregnancy.¹⁷ In the described case, the objective allergological diagnostic tests did not confirm celery sensitization. There were no severe anaphylactic reactions in the history of the patient and there were no other contraindications present. Despite careful qualification to DBPCFC, the patient had to have epinephrine administered. This shows the magnitude of the decision to challenge the patient. In each case, there is always the risk of anaphylaxis, and the medical site, which declares the possibility of performing oral food challenges, must be prepared for this type of complication.

The presented case report has some limitations. The main limitations include the lack of immunoblotting to confirm the binding of the patient’s serum IgE to celery extract. Another aspect that could have been taken into account would be performing not only skin prick tests but also intradermal tests with celery extract. In some cases, intradermal tests can be positive in patients with negative skin prick tests and prove IgE dependent sensitization. On the other hand, the proposed, experimental methodology, using inhibition assay, supports the clinical diagnosis based on DBPCFC.

One of the key questions is the possibility of treatment of patients with celery-related symptoms gathered with cross-sensitization with mugwort defensin. There are no definite contraindications to mugwort allergen extract-specific immunotherapy in patients that underwent anaphylaxis due to the potential cross-reactivity of food allergens with mugwort defensin. Theoretically, during immunotherapy with an allergen extract containing Art v 1, we might fear anaphylaxis. On the other hand, the large number of patients undergoing mugwort immunotherapy each year shows generally high-safety profile.^4,21 In the case of a high-risk patient, premedication with antihistamines might be considered²²

The future might bring other therapeutic options worth taking into account—there are some preliminary studies on hypoallergenic molecules that may hold immunogenic properties but have better safety profiles than crude allergen extracts.²³ Further research would be necessary on other therapeutic options for defensin-related allergy.

Conclusion

This interesting case confirms that celery defensin sensitization is an important clinical problem that may be a reason for anaphylactic reaction in sensitized patients. What is interesting, standard diagnostic methods in this type of celery sensitization may be negative, whereas during the inhibition test, it was proven that celery allergen extract binds mugwort defensin-specific IgE (Art v 1). Further research is necessary to analyze the potential benefits and risks of mugwort allergen-specific immunotherapy in patients with celery-related symptoms.

Footnotes

Author’s contributions

NUS – Concept of the article; methodology design, analysis and interpretation of data, interview with the patient, drafted, reviewed, and accepted the manuscript. Agreed to be accountable for all aspects of the work. KL – methodology design, performed immunoassay and experimental model of inhibition, reviewed and approved the manuscript. Agreed to be accountable for all aspects of the work. MG – interview with the patient, methodology design, reviewed and approved the manuscript. Agreed to be accountable for all aspects of the work. MB – methodology design, reviewed and approved the manuscript. Agreed to be accountable for all aspects of the work. ZB – supervision, methodology, reviewed and approved the manuscript. Agreed to be accountable for all aspects of the work.

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) received no financial support for the research, authorship, and/or publication of this article.

Ethical statement

ORCID iD

Natalia Ukleja-Sokolowska

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