Shinshu Med J, 71⑵：99～107, 2023

Clinical Study of Pollen-food Allergy Syndrome Estimated by
Double-blind, Placebo-controlled Food Challengesof Ten Apple Cultivars
Yumi Koike1）, Naoko Okishima2）＊, Mai Tokunaga1）
Katsuhiko Kobayashi3） and Yuji Inaba4）1） Department of Allergology, Nagano Children’s Hospital2） Department of Health and Nutrition Science, Faculty of Human Health Science, Matsumoto University
3） Kobayashi Otorhinolaryngology Clinic4） Life Science Research Center, Nagano Children’s Hospital

Background : The number of patients with pollen-food allergy syndrome (PFAS) has increased globally. Apples
are a major causative food for PFAS. Varied reports have demonstrated the usefulness of skin prick test (SPT)for the diagnosis of PFAS, besides differences in symptom expression among apple cultivars, thus the diagnosis
of PFAS remains unclear.
Purpose : To investigate the clinical features of apple-induced PFAS by performing a double-blind placebocontrolled food challenge (DBPCFC) using different apple cultivars on patients with apple-induced PFAS.
Method : DBPCFC was performed for six patients with apple-induced PFAS using 10 apple cultivars. We
measured the degree of symptoms using the Visual Analog Scale (VAS). Further, we assessed the correlations of
Mal d 1 and Bet v 1-specific IgE levels and SPT findings to the VAS.
Results : Three of six patients (50 ％) were VAS-positive for two apple cultivars, one patient each (17 ％) was
positive for three, four, and five apple cultivars. The correlation between SPT findings and VAS was insignificant (p＝0.103). The VAS displayed a positive relationship with Mal d 1 and Bet v 1 (r＝0.5 and r＝0.84, respectively).
Conclusion : It is necessary to perform DBPCFC with multiple apple cultivars to diagnose PFAS. The SPT was
not useful in diagnosing PFAS ; however, Bet v 1-specific IgE levels may be advantageous. This novel clinicalstudy of PFAS assessed multiple Japanese apple varieties, therefore, our findings can serve as a baseline for
future studies. Shinshu Med J 71 : 99―107, 2023
(Received for publication October 3, 2022 ; accepted in revised form December 26, 2022)
Key words : pollen-food allergy syndrome, skin-prick test, visual analog scale, oral food challengeApple, a frequently consumed fruit, is a major trig-

Ⅰ Introduction

ger allergen for PFAS. The typical symptoms of

The number of patients with hay fever has in1）

PFAS vary from oral and throat mucosal symptoms

creased worldwide, including Japan . This increase

to birch pollinosis. Mal d 1 is the causative protein ofincludes an upsurge in the number of patients diag-allergy symptoms in patients with apple-induced

2）-4）

PFAS ; it induces a reaction by cross-reactivity with

nosed with pollen-food allergy syndrome (PFAS).

Bet v 1, the major birch pollen allergen5）-8）. PFAS＊

Corresponding author : Naoko Okishima
Department of Health and Nutrition Science,
Faculty of Human Health Science, Matsumoto University,
2095-1 Niimura, Matsumoto, Nagano 390-1295, Japan
E-mail : naoko.okishima@t.matsu.ac.jp

No. 2, 2023

often develops in school-age children and adults, andtheir quality of life gets impaired because they areunable to eat frequently consumed foods ; it is difficult to expect a spontaneous remission9）-13）.
99

Koike・Okishima・Tokunaga et al.

The diagnosis of PFAS is difficult because the(SD), Sun-Tsugaru (ST), Akibae (Aki), Ourin (O),symptoms are predominantly oral, besides a lack of

Sennshu (Se), Toki (To), Shinano-hoppe (SH), Aika no

objective symptoms. Skin prick tests (SPTs) arekaori (AK), and Sun-fuji (SF). The DBPCFC consist-

14）

generally considered useful for diagnosing PFAS .

ed of apple and cabbage smoothies, which followed

However, SPTs alone may not be diagnostic in actual

the protocol of the Model II pita bread study byclinical practice and some reports reveal that there isHansen et al.16）, such that the protocol could be ap-

a relationship between SPT and PFAS symptoms15）.

plied to patients with wheat allergies as well. The

Clinicians need to perform an oral food challengesmoothies were prepared using 25 g of apples, 50 g

(OFC), which entails the ingestion of an allergen-of cabbage, 35 ml of apple juice, 2.5 ml of lemon juice,2）

containing food, to observe symptom occurrence .

and two teaspoons of sugar. Based on the Japanese

Because the symptoms of PFAS are subjective, astyle of apple consumption, the apple peels were re-single-blind OFC may generate false-positive results

moved before preparing the smoothie. To mask thein the presence of psychogenic thus, lacks diagnostic

taste, we added commercially processed apple juice

accuracy16）. Further, few reports elucidate a double-(trade name : Apple Clear, Harada Dairy) with inacti-

blind placebo-controlled food challenge (DBPCFC)

vated Mal d 1. The placebo was prepared using

15）
17）

comprising numerous apple varieties. Applesimilar ingredients as the apple smoothie, but with-; therefore,out adding fresh apples. The patients ingested four

DBPCFC should be performed on several apple vari-

to five smoothies (three–four apple smoothies andeties for an accurate diagnosis.

one placebo smoothie) per OFC. OFCs were conduct-

varieties differ in their antigenicity

17）
-19）

Thus, we aimed to perform DBPCFC on 10 of theed thrice across different days to permit the patients

28 apple varieties whose Mal d 1 mRNA expression

to be challenged with 10 apple cultivars. For patients

levels were analyzed by our coauthors

20）

and whose

under antihistamine medications, we discontinued

antigenicity was expected to vary based on thethe medication 3 days before performing the OFC.

results. Further, we intended to examine the rela-

Co-investigators who were not involved in the OFC

tionship between OFC results and Mal d 1- and Bet

randomized the smoothies in the food challenge. Thev 1-specific IgE levels.

interval between the ingestion of each smoothie was ≥Ⅱ Methods
Ａ

Study participants

30 min to ensure sufficient time for the symptoms todisappear in participants who developed them afteringesting a smoothie. The smoothies were preparedWe recruited patients who visited the Nagano

immediately before the OFC. If the DBPCFC couldChildren’s Hospital between September 2019 andnot be conducted on a similar day, the apples wereJanuary 2020, had a known allergy to raw apple, andstored in a freezer at －18 ℃ to ensure consistency

tested positive for birch pollen-specific IgE ( ≥ 0.35

in the Mal d1 content21）.

kUA/L). Six participants (all female, median age 24.5

We used the Visual Analog Scale (VAS), which

years, range 10-45 years) who had previously pro-measures the intensity of clinical symptoms, to eval-vided written informed consent were included in thisuate the intensity of symptoms experienced by eachstudy.

patient ; the intensity ranged from 0 (none) to 100

Ｂ Double-blind placebo-control food challenge(strongest). The patients were provided a sheet of(DBPCFC)

paper with a 100 mm line drawn on it : the left end

The DBPCFC was conducted during the hay fever

represented “no symptoms (＝0),” whereas the rightseason (September 2019 to January 2020) character-end represented “worst symptoms (＝100).” The pa-

ized by a good supply of apples in the market. We

tients were requested to indicate their degree of pain

used 10 apple cultivars from the Nagano Prefecture,on that line upon exhibiting symptoms. The rater

including the Shinano-piccolo (SP), Shinano-dolce

assessed the results and assigned them a numerical100

Shinshu Med J. 71

Clinical study of PFAS by DBPCFC

We performed the Chi-square test to compare thevalue. OFC positivity was defined as a VAS＞0.
Ｃ

number of patients with a positive VAS score andSkin prick test (SPT)The SPT was performed on the volar side of thethose who were SPT positive. The Spearman’s rank

forearm using a bifurcated needle (Tokyo MI Compa-

correlation coefficient was used to evaluate the spe-

ny, Tokyo, Japan) and the prick-to-prick method.

cific IgE levels, VAS, VAS positivity rate, and Mal d1

We performed SPTs on the day of the OFC and thelevels. The level of statistical significance was set at a

day before. Histamine dihydrochloride (10 mg/ml)

p-value ＜0.05 (one-tailed). GraphPad Prism 9 for(Torii Pharmaceutical Co., Ltd., Tokyo, Japan) was

Windows (GraphPad Software Inc., La Jolla, CA,

used as a positive control, whereas a scratch extractUSA) was used for all statistical analyses. The age(Torii Pharmaceutical Co., Ltd., Tokyo, Japan) was

and specific IgE values are presented as the medianused as a negative control solution. We measured(range).

wheal diameter 15 min following pricking. A positiveＦ

result was defined as a wheal diameter ≥ 3 mm than

Ethical considerations
This study was approved by the Nagano Children’sthat of the negative control22）23）.

Hospital Institutional Review Board (ApprovalＤ

No. : 31-28, Approval date : September 03, 2019). All

Laboratory Data

study participants or their legal guardians provided

We measured the specific IgE to apples, birch pollen,

written informed consent.

Mal d a, Mal d 3, and Bet v 1 using the ImmunoCAP®
assay system (Thermo Fisher Scientific/Phadia, Up-Ⅲ

psala, Sweden). The specimens were collected on theＡ

day of the first OFC and the day before. A positive

Results

Study participants

result was defined as an IgE level ≥ 0.35 kU/l. The

Six patients with apple-induced PFAS participated

OFC and SPT results were compared to determine

in this study. Table 1 summarizes their characteristics.

the usefulness of the SPT for diagnosing PFAS.

All patients were female, with a median age of 24.5

Ｅ

(10-45 years) years. All patients had allergic rhinitis

Statistical analyses
Table 1

Characteristic of the study participants

1

2

3

4

5

6

Sex

F

F

F

F

F

F

Age (y)

10

12

14

35

42

45

24.5

Age at onset of
PFAS (y)

9

7

10

12

14

14

11

Allergic factors

AR

AR

AR

AR

AR

AR

Birch pollen(UA/ml)

34.2

＞100

7.62

3.82

2.33

3.75

5.72

Cedar pollen(UA/ml)

10.1

87.4

37.4

9.16

0.98

9.66

9.41

peach,
kiwi fruit,
cherry,tomato,strawberry,loquat,pear,
orangepeach,
cherry,pear

peach,
cherry,
pear,
grape

Specific IgE

Case

Foods other thanapples that cause
PFAS

Medianpeach,
kiwi fruit,
cherry,strawberry,loquat,pear

AR ; allergic rhinitis ; IgE, immunoglobulin E ; and PFAS, pollen-food allergy syndrome
No. 2, 2023

101

Koike・Okishima・Tokunaga et al.
Table 2

Specific IgE and VAS of DBPCFC

Specific IgECase

VAS of DBPCFC

Mal d 1UA/ml

Mal d 3UA/ml

Bet v 1
UA/ml

SP

SD

1

13.3

＜0.1

26.2

90

0

2

90.9

＜0.1

＞100

0

0

0

85

3

1.21

＜0.1

2.61

15

35

0

4

0.85

＜0.1

2.79

10

0

5

2.29

＜0.1

5.97

0

6

3.34

＜0.1

4.59

0

ST

Aki

O

Se

To

SH

AK

90

0

0

50

5

0

25

0

0

0

0

0

10

10

0

5

15

0

0

10

5

0

0

0

10

10

0

SF

Positivemean

rate (％) VAS score
50

90

0

40

41.3

0

0

20

25

0

0

0

50

10

0

0

0

0

20

7.5

10

0

0

0

30

10

SP, Shinano-piccolo ; SD, Shinano-dolce ; ST, Sun-Tsugaru ; Aki, Akibae ; O, Ourin ; Se, Sennshu ; To,
Toki ; SH, Shinano-hoppe ; AK, Aika no kaori ; SF, Sun-fuji ; IgE, immunoglobulin E ; DBPCFC, doubleblind placebo-controlled food challenge ; and VAS, Visual Analog Scale.

as a comorbidity. The patients were positive for cedar

tween the Mal d1-specific IgE levels and the meanpollen, with a level of 9.41 (0.98-87.4) UA/ml. Four of

VAS score for the apples for which the patientsthe six patients consumed PFAS-causing foods other

tested OFC-positive (r＝0.5 and p＝0.27) ; however,than apples. None of the patients had consumed any

the correlation was insignificant (Fig. 1).
Meanwhile, we identified a significant positive cor-antihistamines for 3 days prior to the OFCs.
Ｂ

DBPCFC (VAS) and specific IgE levels

relation between the Bet v1-specific IgE levels andTable 2 summarizes the DBPCFC results (VAS)

the mean VAS values for the apples for which theand specific IgE levels of each patient for birch andpatients tested OFC-positive (r＝0.84 and p＝0.09)

apple. Patient 1 could not undergo all food challenge(Fig. 2).

tests because of the symptom intensity ; the patientＣ

Correlations between SPT and VAS

participated in the OFCs for four apple cultivars (SP,Fig. 3 depicts the relationship between the SPT

SD, Se, and To). All patients were VAS-positive forresults and VAS scores according to the apple culti-

at least two apple cultivars and VAS-negative forvars.

several others. All patients were VAS-negative for

ST and To with large wheal diameters ranging

the placebo. They were Mal d 3-negative (Table 2).

from 7 mm to 9 mm had low VAS scores ranging from

The median Mal d 1 level was 2.82 (0.85-90.9) kU/l,

0 to 5, Se, Aki, and SP with wheal diameters ranging

whereas the median Bet v 1 level was 5.28 (2.61-

from 3 to 5 mm had high VAS scores ranging from> 100) kU/l. We observed a correlation between the

85 to 90, thus indicating the overall variations. We

Mal d 1- and Bet v 1-specific IgE levels (r＝0.89 andobserved VAS positivity and SPT positivity in 12/18

p＝0.024).

subjects (VAS-positive and SPT-positive/VAS-

Table 2 depicts the VAS positivity rate according

positive, 67 ％ ), whereas VAS negativity and SPT

to the apple cultivars. The VAS positivity rates were

negativity were observed in 21/37 subjects (VAS-

50 ％ (3/6) for SP, 17 ％ (1/6) for SD, 20 ％ (1/5) for

negative and SPT-negative/VAS-negative, 57 ％) (pST, 80 ％ (4/5) for Aki, 40 ％ (2/5) for O, 50 ％ (3/6)＝0.103).

for Se, 50 ％ (3/6) for To, 0 ％ (0/5) for SH, 20 ％ (1/5)
for AK, and 0 ％ (0/5) for SF (Table 2, Fig 1).
１．Relationship between specific IgE levels and theⅣ

Discussion

In this study, patients with apple-induced PFAS

VAS

underwent DBPCFC of 10 different apple varieties.

We observed a moderate positive correlation be-

Previous studies performed single-blind OFC on pa-

102

Shinshu Med J. 71

Clinical study of PFAS by DBPCFC

Fig. 1 Correlation between Mal d1-specific IgE levels and the average Visual Analog Scale (VAS) in patients
who are oral food challenge-positive to the apple cultivars
The horizontal axis denotes the log Mal d1-specific IgE level, whereas the vertical axis denotes the average
VAS. The straight line that rises steadily to the right is the regression line. A positive insignificant correlation
has been observed (r＝0.5, p＝0.27).

Fig. 2 Correlation between Bet v1-specific IgE levels and the average Visual Analog Scale (VAS) in patients
who are oral food challenge positive to the apple cultivars
The horizontal axis denotes the log Bet v1-specific IgE levels, whereas the vertical axis denotes theaverage VAS. The straight line that rises steadily to the right is the regression line. A significant and positive
correlation has been observed (r＝0.84, p＝0.09).
No. 2, 2023

103

Koike・Okishima・Tokunaga et al.

Fig. 3 Correlations between the skin prick test (SPT) and the Visual Analog Scale (VAS)
The wheal diameter (mm) resulting from the SPT is presented on the horizontal axis, whereas the VAS ispresented on the vertical axis. Twelve/18 types are VAS-positive and SPT-positive (VAS positive and SPT
positive/VAS positive, 67 ％), whereas 21/37 types are VAS-negative and SPT-negative (VAS negative and
SPT negative/VAS negative, 57 ％).
SP, Shinano-piccolo ; SD, Shinano-dolce ; ST, Sun-Tsugaru ; Aki, Akibae ; O, Ourin ; Se, Sennshu ; To,
Toki ; SH, Shinano-hoppe ; AK, Aika no kaori ; and SF, Sun-fuji ;tients with PFAS15）. However, PFAS is primarily awere less likely to contain allergens ; our recipe cansubjective symptom, and single-blind studies are not

be used as a reference for DBPCFC in the future. In

reliable for determining the presence of symptoms

this study, OFC-positive and OFC-negative cultivars

with OFC. This necessitates modifying the morpholo-

differed among the participants. However, the reli-

gy of apples to perform DBPCFC. Limited studies

ability of our study is supported by the DBPCFC

have focused on DBPCFC using apples, all of whichapproach instead of a single-blind approach and the

16）
17）

were conducted in Europe

16）

con-

absence of symptoms with placebo ingestion. There-

ducted a study by hiding apple pieces inside pita

fore, this study demonstrated the potential of validity

bread and adding cabbage to mask the texture of theof DBPCFC using apple smoothies.

. Hansen et al.

apples. However, patients with wheat allergies cannot

Scattered reports have used DBPCFC for patientsconsume pita bread, thus, we performed the DBPCFC

with apple-induced PFAS in Europe, nonethelessusing apple smoothies following this recipe. Further-

these reports used only two to four apple varieties15）17）.

more, the conversion of apples into smoothies en-

In the present study, we performed DBPCFC on nu-

sured that the apples did not maintain their original

merous apple varieties15） selected from the co-authors’form such as a pita bread, thus creating a higher

previous report20） and evaluated it using a more rig-mask effect. Suzanne et al. mixed apples and yogurt

orous approach. Previous results have revealed dif-to prepare the challenge food in their study. Dairy

ferences in symptom appearance among apple vari-allergies are the second most prevalent allergies ineties. Vlieg-Boerstra et al. used four apple cultivars15）

Japan24）. Conversely, the smoothies used in this study

in an OFC. Despite a significant difference in the104

Shinshu Med J. 71

Clinical study of PFAS by DBPCFC

symptom intensity, the VAS score (range : 0-80) var-

the diagnosis of PFAS, despite being considered gen-ied significantly according to the cultivar. An OFC

erally useful for the same29）. The OFC results were

for the diagnosis of apple-induced PFAS is usuallyconsistent with SPT results in another report6）. The

performed on a single variety, however, the resultspresent study, as well as previous reports that men-of Vlieg-Boerstra et al. suggested that an OFC ontioned the SPT is not useful for the diagnosis offewer varieties may generate false-negative results

PFAS15）30）, indicated that the SPT cannot be used toor even the removal of other varieties from the dietmake a definitive diagnosis of PFAS. The results ofthat can be consumed without symptoms, despite

the present OFC on numerous apple varieties sug-

being OFC-positive. Further, considering no differ-gested that the SPT is unlikely to be a criterion forence in VAS for each of the original apple variety

the diagnosis of PFAS in the future, besides the need20）

crosses , it is necessary to study more cases of indi-

to incorporate the use of an OFC with multi-cultivar

vidual apples.

apples.

The mechanism of PFAS is due to IgE cross-This study had some limitations. First, it had areactivity between fruit allergens and pollen aller-

small sample size. PFAS often manifests in children

gens. First, the patient is sensitized to pollen, follow-

after school age. This study comprised 10 apple culti-

ing which fresh fruits and vegetables come into

vars, thus, the patients were admitted for several

contact with the sensitized site, the oral mucosa,

days to undergo assessments, which suggested thatcausing a type I allergic reaction. However, the fruit

several patients with PFAS could not participate be-allergens involved in PFAS are unstable to digestion,

cause of school or work. Despite a positive correla-thus, lose their antigenicity during digestion, result-tion between Bet v 1-specific IgE levels and VAS in

ing in fewer organ symptoms outside the orophar-six cases, we included outliers in case 1. We ob-

ynx

25）
-27）

. Bet v 1-related protein is a type of infec-

served a weak positive correlation (r＝0.58, p＜0.001),

tion-specific protein produced by plants to protect

excluding this outlier and examining the five cases.

themselves from physical stresses such as invasion

Researchers should investigate the correlation be-

by infectious microorganisms and insect damage. It

tween Mal d1 levels and VAS scores in a larger pa-

27）

is also found in birch pollen . It is cross-antigenictient sample. The absence of difference in VAS forwith Bet v 1, the major allergen of birch pollen, andeach of the original apple variety crosses warrants

13）

Mal d 1, an allergen found in apple . In Europe, Mal

investigating higher cases of individual apples. Cur-d 3 reportedly cause systemic symptoms as therently, we are conducting a study with additionalcausative protein pf apple-induced PFAS, unlike Mal

cases and will report its results in the future. Second,d 1. In the present study, Mal d 3-specific IgE levels

the reproducibility of the OFC has not been com-

1）
27）
28）

were measured prior to OFC, and all patients

pletely established. Bet v 1 may be a predictor ofwere negative, thus allowing a safe OFC. We ob-

symptom appearance in apple PFAS. However, it isserved an insignificant but positive correlation be-difficult to predict the difference in symptom appear-

tween Mal d 1-specific IgE levels and VAS, and aance among different apple varieties in each case. In

significantly positive correlation between Bet v

this study, patients with apple-induced PFAS could

1-specific IgE levels and VAS, thereby suggesting

consume several apple varieties without symptom

Bet v 1-specific IgE levels may be useful for theappearance, which necessitates, further studies on

diagnosis of apple-induced PFAS. Our results arehigher apple varieties, including a similar variety

highly novel because few studies have examined thefrom different harvest years and other apple varieties.

correlation between specific IgE levels and VAS inpatients with DBPCFC, despite several reports on
the correlation between SPT and VAS.
Our results revealed that SPT was not useful forNo. 2, 2023

Ⅴ

Conclusion

In summary, the DBPCFC was more accurate thana single-blind OFC in case of subjective PFAS symp105

Koike・Okishima・Tokunaga et al.

toms. However, DBPCFC is labor intensive. The SPT

tic accuracy of PFAS.

was not useful in the diagnosis of apple-induced

Ⅵ

PFAS ; however, Bet v 1-specific IgE levels may be

Acknowledgment

considered a diagnostic aid. The results obtained by

The authors would like to thank Dr. Yasuhito

performing DBPCFC on multiple apple varieties re-

Kondo from the Fujita Health University Bantane

vealed differences in the appearance of PFAS symp-

Hospital. This study was funded by the Japan Healthtoms, which suggests the importance for examining

Foundation and Nagano Children’s Hospital Clinicalagainst multiple varieties for improving the diagnos-

Medical Research Grant.

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Ann Allergy Asthma Immunol 99 : 522-525, 2007

（2022. 10. 3 received ; 2022. 12. 26 accepted）

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