Full text of DOI:10.1016/S0301-0546(02)79098-8

CLIN ICAL CASES
Barnacle hypersensitivity
M. C. Moreno Escobosa*, E. Alonso Lebrero*, A. Sánchez Alonso**, J. Méndez Alcalde**,
M. A. Rico Díaz***, G. García Alvárez***, and B. Bartolomé Zavala****
*
*
Allergy Department, Hospital Niño Jesús, Madrid, **Allergy Department, Hospital Río Carrión, Palencia,
**Allergy Department, Hospital Juan Canalejo, A Coruña, ****Bial-Arístegui Laboratory, Bilbao, Spain.
Allergol et Immunopathol 2002;30(2):100-103.
SUMMARY
Background: the aim of the present study is to in-
vestigate the responsible mechanism of different ad-
verse reactions suffered by five patients, aged bet-
ween six and thirty years-old, after consumption of
barnacle. The symptoms were angioedema, dysp-
nea, generalized urticaria, conjunctivitis and one of
them suffered from anaphylactic reaction. Four pa-
tients had personal atopic history.
Methods: the allergic study included prick by prick
test with raw and boiled barnacle and prick-test with
a standardized battery of shellfish and neumoaller-
gens, specific-IgE determination to barnacle, crusta-
cean and house-dust-mite and SDS-PAGE immuno-
blotting to barnacle. Even though an oral challenge
was proposed to three of the patients, they were re-
luctant to do the test and eventually the challenges
were not carried out.
INTRODUCTION
Crustaceans are a well-known source of food
allergens. Hypersensitivy reactions described as a
consequence of this seafood consumption include
oropharyngeal pruritus, urticaria, angioedema, gas-
trointestinal distress and anaphylaxis. Barnacle
(Pollicipes cornucopiae) is a type of sedentary mari-
ne crustacean with a free-swimming larvae. In adult
phase it has six pairs of thrunk limbs and the shell is
developed into a mantle. In Japan barnacles are
used as fertilizer (1). A species of 30-centimetres-
barnacle from the cirripeda genus is regarded as de-
licacy in South America and stalked barnacle is com-
monly eaten in parts of France and Northwestern
Spain. Until now, no adverse reactions have been
described in patients consuming this seafood. We
report a group of five patients who after ingestion
of barnacles presented different kinds of allergic re-
actions.
Results: prick to prick tests were positive to bar-
nacle for all of them. Specific-IgE was found in four
patients. The western blotting results showed an
IgE-binding band whose apparent molecular mass
ranged between 58 and 68 kDa.
Conclusions: barnacle could induce IgE-mediated
adverse reaction. Our study has demonstrated the
presence of an IgE-binding protein in barnacle ex-
tracts ranged between 58 and 68 kDa of molecular
mass. It has not been previously described a crusta-
cean allergen with the same molecular mass, so it
could be a specific allergen from barnacle. We belie-
ve that further study will confirm this is the case.
MATERIAL AND METHODS
Patients
Case 1. A 6-years-old girl suffered from angioede-
ma in her face, throat itching, disphagia, disphony,
dyspnea and wheezing immediately after eating bar-
nacle. She was quickly transferred to an emergency
unit where she was treated with adrenaline and me-
tilprednisolone and was kept under observation. The
symptoms disappeared within 1-2 hours without furt-
Key words: Barnacle hipersensitivity. Crustacean.
Cross-reactivity. Tropomyosin. Adverse reaction by
barnacle. Shellfish. Pollicipes cornucopiae.
Allergol et Immunopathol 2002; 30(2): 100-103

M. C. Moreno Escobosa, E. Alonso Lebrero, A. Sánchez Alonso, J. Méndez Alcalde, M. A. Rico Díaz, G. García Alvárez,
B. Bartolomé Zavala.— BARNACLE HYPERSENSITIVITY
101
her therapy. The patient had personal history of a se-
asonal rhinoconjunctivitis.
EAST (Enzyme AllergoSorbent Test). Specific IgE
determination. Specific-IgE levels were calculated
using BIAL-ARISTEGUI discs. Briefly the liofilized
allergen extracts were coupled (10 mg/mL) to the
6-mm diameter CNBr-activated paper discs and
EAST was performed with HY-TEC-EIA kit for speci-
fic-IgE, following the manufacturer’s instructions
(Hycor-Biomedical Inc.).
Case 2. A 30-years-old patient developed genera-
lized urticaria and dyspnea, 30 minutes after con-
sumption of lobster, barnacles and turbot. Some
months later, he experienced the same symptoms
after eating exclusively barnacles. He is sensitized to
house-dust-mite and pollens.
Case 3. A 26-years-old patient who noticed orop-
haryngeal and lip itching, swelling of his lips and urti-
caria around the mouth several minutes after eating
barnacle. He had also noticed oropharyngeal itching
when he inhaled the barnacle boiled steam. He suf-
fered from house-dust-mite allergy and was treated
with specificic immunotherapy.
Case 4. A 16-years-old boy who experienced lip
angioedema and oral itching, several minutes after
ingesting boiled barnacles. The symptoms disappea-
red some hours later without therapy. He had asth-
ma due to house-dust-mite.
Case 5. A 12-years-old girl who had conjunctivitis
and angioedema in her face several minutes after in-
gestion of a barnacle. The symptoms disappeared
some hours later without therapy. She has not per-
sonal atopic history.
SDS-PAGE. This technique was performed accor-
ding to the method of Laemmli (2) in a discontinuous
gel (running: 12.5 %; stacking: 4 %). The samples
were dissolved in 0.125 M HCl-Tris, pH 6,8 and were
dissociated with 0.1 % SDS and 5 % beta-mer-
captoethanol at 100 °C for 5 minutes. After electrop-
horesis, gels were stained by diffusion in 0.1 %
Coomassie Brilliant Blue R-250 dissolved in metha-
nol/acetic/distilled water (4:1:5). Destaining was also
performed by diffusion in the same mixture without
dye.
SDS-PAGE Immunoblotting. The proteins were
electrophoretically transferred to a polyvinylidene
membrane (Immobilo-P membranes) essentially as
described by Towin et al. (3). The membrane was
rinsed in PBS (Phosphate buffer 25 mM, NaCl
100 mM pH 7) and blocked with 0.05 % Tween
2
0 in PBS (1 h at 37 °C). After three washes with
PBS containing 0.02 % Tween 20, the blots were
incubated overnight with 0.5 ml of the patients’
sera. Later on, the blots were washed 3 times with
PBS and incubated with peroxidase-conjugated
rabbit immunoglobulins to human ꢀ-chains. Bound
peroxidase was detected by chemiluminescence
method according to the manufacturer’ instruc-
tions (Western Blotting ECL + Plus. Amersham Life
Science).
In vivo tests
Skin test. Prick to prick test was performed with
boiled and raw barnacle; briefly, a standardized lan-
cet was pricked into boiled and raw barnacle and im-
mediately thereafter into the skin. Skin-prick-tests
(
SPTs) were performed acording to Pepys’ method
using histamine (10 mg/ml) and saline solution 0.9%
in 50% glycerol), as positive and negative controls
(
respectively. If reaction occurred after 15 minutes,
the transverse and vertical diameter of the wheal
were measured and mean values recorded. Wheals
of 3 mm or greater were regarded as positive in the
absence of reaction to negative control. Prick to
prick tests to barnacle were performed in ten atopic
and non-atopic control population obtaining negati-
ve results.
Oral challenges were considered initially for all
patients. In one case, medical reasons advised
against the test. Other patient was reluctant to un-
dergo the test as he had developed general urticaria
and dyspnea every time he had eaten barnacle, and
finally the test was cancelled. Two other patients re-
fused and the only one who accepted initially did
not undergo it.
In vitro tests
RESULTS
Barnacle extracts. Barnacles were cut into pieces
and homogenized in Phosphate buffer 10 mM, NaCl
50 mM, pH 7 and their proteins were extracted by
magnetic stirring in the same buffer (2 h at room tem-
perature). The sample was centrifugated at 5600 g for
0 minutes and the supernatant was dialyzed against
water and later filtered and freeze dried.
The prick to prick test to barnacle produced whe-
als from four to ten millimetres of diameter. The
barnacle specific-IgE values are summarized in ta-
ble I.
The prick to prick and specific IgE values to barna-
cle were correlated in all our patients but one. Patient
number five had RAST class 0 for raw and boiled bar-
1
3
Allergol et Immunopathol 2002; 30(2): 100-103

M. C. Moreno Escobosa, E. Alonso Lebrero, A. Sánchez Alonso, J. Méndez Alcalde, M. A. Rico Díaz, G. García Alvárez,
B. Bartolomé Zavala.— BARNACLE HYPERSENSITIVITY
102
Table I
Barnacle specific IgE levels
Specific IgE levels. East.
Patient
Raw barnacle
kU/L Class
Boiled barnacle
kU/L
Class
No. 1
No. 2
No. 3
No. 4
No. 5
< 0.35
2.14
0.78
< 0.35
< 0.35
0
2
2
0
0
1.68
0.52
2.48
3.47
< 0.35
2
1
2
2
0
nacle and however SPT for raw and boiled barnacle
was positive.
Figure 1.—SDS-PAGE Immunoblotting. A, Raw barnacle extract B,
Boiled barnacle extract. Lanes 1, 2, 3, and 4: Patients’ sera (cases
1
, 2, 3 and 5 respectively). C, Negative control (pool from non ato-
The prick-tests performed with standardized pneu-
moallergens and shellfish battery, showed that four
patients had positive prick-tests against house-
dust-mite, two of them had positive prick-tests
against shellfish and cockroach and only one patient
had positive prick-tests against the three previous
allergens. The CAP-system (Pharmacia) was class
pic subjects’ sera). M, Molecular mass markers.
DISCUSSION
Barnacle is considered a delicacy in Spain, howe-
ver there are no reported cases about sensitization
by barnacle in the international literature, apart from
those reported by the authors in national meetings
2
to house-dust-mites in two patients and class 1 in
other one. This system was employed in the speci-
fic-IgE antibodies determination of clamp, mussel
and Anisakis simplex. A patient had CAP-system
class 2 for clamp, two of them had CAP-system class
(
4, 5). In the present study we describe five cases of
patients, with different ages, who suffered from ur-
ticaria and angioedema after eating barnacle.
1
and another one had CAP-system class 3. In all the
Prick to prick positive results indicate that all our pa-
tients were sensitized to both raw and boiled barnacle.
However we only found measurable specific-IgE le-
vels to boiled barnacle extract in four patients sera.
The IgE-binding components from the barnacle extract
were studied by SDS-PAGE immunoblotting, obtaining
a relevant IgE-binding zone within 58-68 kDa.
Although an allergen of similar molecular mass
patients, mussel and Anisakis simplex specific-IgE
antibodies were negative. The results are summari-
zed in table II.
The SDS-PAGE Immunoblotting results showed
IgE-binding bands whose molecular masses ranged
between 58-68 kDa and 37-39 kDa (fig. 1). This figu-
re shows the IgE-binding bands of cases 1, 2, 3 and
5
. Unfortunately, it was not possible to make a pho-
(
70 kDa) was described by Leung et al in oyster
to of case number 4 blotting.
Table II
Prick and specific-IgE values to standardized pneumoallergens and shellfish battery
Specific IgE levels. CAP system
Prick-tests
Shellfish
Patients
Mites
Clamp
Mussel
Anisakis
Mites
Cockroach
No. 1
No. 2
No. 3
No. 4
No. 5
Class 1
Class 0
Class 0
Class 2
Class 2
Class 3
Class 1
Class 1
Class 2
Class 0
Class 0
Class 0
Class 0
Class 0
Class 0
Class 0
Class 0
Class 0
Class 0
Class 0
+
+
+
–
+
+
–
–
–
+
+
–
–
–
+
+
: Positive. –: Negative.
Allergol et Immunopathol 2002; 30(2): 100-103

M. C. Moreno Escobosa, E. Alonso Lebrero, A. Sánchez Alonso, J. Méndez Alcalde, M. A. Rico Díaz, G. García Alvárez,
B. Bartolomé Zavala.— BARNACLE HYPERSENSITIVITY
103
(
Crassostea gigas) (6), this is the first time an allergen
tados del inmunoblotting muestran la presencia de
from barnacle is described. The western blotting re-
sults also showed an IgE binding band of 37-39 kDa, in
boiled and raw barnacle extracts. This is the same mo-
lecular mass of tropomyosin, a well-known and wi-
despread allergen in arthropods (7, 8) which is the cau-
se of many cases of cross-reactivity between
house-dust-mites and other arthropods (crustaceans,
insects) sensitization. Significantly most of our pa-
tients were sensitized to house-dust-mite. One of
them suffered from asthma so he had been treated
with immunotherapy with good results. All the pa-
tients have eaten all types of crustaceans and mo-
lluscs without adverse reaction, after the allergic reac-
tion by barnacle.
In summary, it can be concluded that barnacle
could induce IgE-mediated adverse reaction. Our
study demonstrates the presence of an IgE-binding
protein of 58-68 kDa in barnacle extract. This could
be a specific allergen from barnacle as no IgE-binding
band with the same molecular mass has been reve-
aled in western blotting of other mollusc and crusta-
cean extracts incubated with the patients’ sera.
However, further experiments using a larger variety
of mollusc and crustacean extracts should be carried
out to confirm this proteic band is really a specific
allergen from barnacle.
una banda fijadora de IgE, cuya masa molecular se si-
túa en el intervalo comprendido entre 58 y 68 kDa.
Conclusiones: el percebe puede producir reaccio-
nes adversas cuyo mecanismo está mediado por
IgE. Nuestro estudio demuestra la presencia de una
banda fijadora de IgE en el extracto de percebe, cuyo
peso molecular se sitúa entre los 58 y 68 kDa. Una
banda con este peso molecular no había sido descri-
ta con anterioridad como alergeno perteneciente a la
familia de los crustáceos, lo que nos hace pensar en
la posibilidad de que este alergeno pudiera ser espe-
cífico del percebe. Habrá que ampliar el estudio in-
munológico de estos casos, para poder confirmar de-
finitivamente a esta proteína como específica del
percebe.
Palabras clave: Hipersensibilidad a percebe. Alergia
a ácaros del polvo. Crustáceos. Reactividad cruzada.
Tropomiosina. Reacción adversa por percebe.
Marisco. Pollicipes cornucopiae.
Correspondence:
M .ª Carmen Moreno Escobosa
Pl. de la Constitución, 21, 2.º
04760 Berja (Almería). Spain
Tels.: + 34 950 49 03 21
+
34 630 34 55 16
E-mail: moreno.escobosa@teleline.es
RESUMEN
Caso clínico: el objetivo de este estudio es inves-
tigar el mecanismo de producción de las reacciones
adversas sufridas por cinco pacientes tras la inges-
tión de percebe. Los síntomas fueron diversos y
comprendieron desde un cuadro de angioedema, dis-
nea, urticaria generalizada o conjuntivitis, hasta una
reacción anafiláctica. Cuatro de nuestros pacientes
tenían antecedentes personales de atopia y sus eda-
des estaban comprendidas entre 6 y 30 años.
Métodos: el estudio alergológico consistió en la
realización de prick-prick test con percebe crudo y co-
cido y prick-test con una batería estándar de maris-
cos y neumoalergenos; determinación de IgE-especí-
fica a percebe, mariscos y ácaros y la realización de
SDS-PAGE inmunoblotting con extracto de percebe.
Se propuso la realización de provocación oral a tres
de nuestros pacientes, pero rehusaron someterse a
dicha prueba.
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