Allergenicity of casein containing chalk in milk allergic schoolchildren

Ann Allergy Asthma Immunol 110 (2013) 335e339

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Allergenicity of casein containing chalk in milk allergic schoolchildren Carlos H. Larramendi, MD *; Francisco M. Marco, MD, PhD y, z; Mónica Llombart, MD x; Ana de la Vega, MD {; Eusebi Chiner, MD jj; José Luis García-Abujeta, MD *; and José Miguel Sempere, MD, PhD z * Sección

de Alergia, Hospital Marina Baixa, Villajoyosa, Alicante, Spain R&D Department, Immunal S.A.U. Madrid, Spain z Departamento de Biotecnología, Universidad de Alicante, Spain x Sección de Neumología, Hospital Marina Baixa, Villajoyosa, Alicante, Spain { Servicio de Pediatría, Hospital IMED Levante, Benidorm, Alicante, Spain jj Sección de Neumología, Hospital San Juan de Alicante, San Juan de Alicante, Alicante, Spain y

A R T I C L E

I N F O

Article history: Received for publication November 11, 2012. Received in revised form February 3, 2013. Accepted for publication February 7, 2013.

A B S T R A C T

Background: Nondietary exposure to milk proteins may be a risk for children who do not outgrow milk allergy by school age. Objective: To study the allergenicity of casein containing chalk. Methods: A 6-year-old, milk allergic child developed asthma and rhinoconjunctivitis while in school. The suspected cause was dust-free chalk containing casein. To study the relationship of dust-free chalk containing casein with asthma and rhinoconjunctivitis, 13 additional milk allergic patients were studied: 3 school-aged children, 8 preschool-aged infants, and 2 children with outgrown milk allergy. Skin tests and/or specific IgE with chalk and casein were performed. A chalk use test was performed in older children. Milk allergens contained in chalk were characterized by sodium dodecyl sulfateepolyacrylamide gel electrophoresis, immunoblot, and IgE inhibition experiments. Results: All school-aged, milk allergic children were exposed to chalk and reported symptoms attributed to chalk exposure. The skin test result to chalk was positive in 5 of 12 cases, and the specific IgE test result was positive in all 12 study participants in which it was performed. Casein strongly inhibited the binding of IgE to chalk. Chalk sodium dodecyl sulfateepolyacrylamide gel electrophoresis showed proteins with molecular weight similar to caseins. Immunoblot demonstrated strong binding of IgE to chalk in a blurred pattern and a band at 30 kDa, inhibited by casein. The chalk challenge test result was positive in 2 school-age children who had a positive skin test result to chalk. Their symptoms improved after avoidance of chalk in the school. In 2 other cases in which the challenge test result was negative, chalk was reintroduced without problems. Conclusion: Inhalation of chalk dust containing casein can induce asthma symptoms in milk allergic patients. Hidden and nondietary sources of exposure should always be considered in food allergic patients. Ó 2013 American College of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

Introduction Allergy to cow’s milk protein (CMP) is one of the most important causes of food allergy in children. Although most cases of milk allergy resolve in the first years of life before patients go to school,1 some patients do not outgrow their sensitivity and become milk allergic school children. Besides the dietary hazards posed by the school diet and classmates’ foodstuff, other risks may be present at school for milk allergic patients. The source of milk proteins is not always food

Reprints: Carlos H. Larramendi, MD, Sendero de la Barrina 7, 03581 L’Alfàs del Pi, Alacant, Spain; E-mail: [email protected]. Drs Larramendi and Marco equally contributed to this work. Disclosures: Authors have nothing to disclose.

because it may be found in many other settings, including stationery products, such as paper, ink, or glue.2 Provided that milk proteins retain their allergenic potential in the manufactured finished product, the use of these products may cause allergic reactions in sensitized patients. As a genuine food allergen, the natural route of exposure to milk is ingestion; nevertheless, milk proteins (casein and whey proteins) and casein derivatives may give rise to respiratory symptoms by inhalation as has been documented in previous case reports of occupational exposure.3e7 The role of CMPs as hidden food allergens is well known,8 but the hazards of nonalimentary uses of CMPs have not been well emphasized. We had the opportunity to study the case of a milk allergic patient who developed an acute allergic reaction by exposure to a modified form of casein contained in a therapeutic

1081-1206/13/$36.00 - see front matter Ó 2013 American College of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.anai.2013.02.006

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iron preparation.9 Several months after this initial study, the child reported experiencing asthma attacks while in class. The investigation of a possible causative factor in the school setting pointed to the use of chalk as a potential trigger because there was a warning released by the Spanish Society of Food and Latex Allergic Patients (http://www.aepnaa.org/) about the presence of cow’s milk in chalk.10 This suggestion led us to study the potential role of milk proteins contained in chalk as a trigger of allergic reactions in children allergic to cow’s milk.

(Table 1). After oral consent was obtained, chalk extract was additionally tested in the routine workup for CMP allergy by SPT (on children older than 8 months) or in vitro tests. A serum sample was obtained for in vitro experimental tests. A minor wheal diameter of at least 3 mm, compared with the saline control, was defined as a SPT positive reaction. For analysis, patients were arbitrarily stratified into 3 groups based on their school age, corresponding to possible exposure to nonpowdered chalk. Study groups included 4 school-aged children (including the index case) classified as exposed, 2 children in the playschool (among 3- and 4-years-old) and 6 preschool-aged children (younger than 3 years) classified all as nonexposed, and 2 control children having outgrown their allergy but still sensitized to CMP (Table 1).

Methods The study was performed in a group of 14 children allergic to CMP. The clinical and analytical characteristics of these patients are summarized in Table 1. The study was approved by the local bioethics committee.

Chalk Extracts Chalk (Giotto Robercolor) was purchased at a local store. Proteins contained in chalk were extracted overnight at 50% (wt/vol) in 0.1 M sodium hydroxide. Extracts were centrifuged at 10,000g, dialyzed against purified water with 3,500-Da membranes (Pierce, Rockford, Illinois), filter sterilized (Millipore, Bedford, Massachusetts), and freeze-dried. Chalk extract was used for SPTs and in vitro assays. For SPTs, the extract was adjusted at a concentration of 0.2 mg/mL of total protein (Bradford method) in phosphatebuffered saline containing 50% phenolated glycerin.

Index Case The index case was a 6-year-old boy allergic to CMP who 2 years earlier had developed a systemic reaction after the first dose of an iron compound containing casein.9 At the beginning of his third school year, the patient began to experience episodes of rhinoconjunctivitis, dyspnea, and wheezes at school, while in the classroom, and when exercising. No symptoms were reported at home and during the weekend. Treatment with salbutamol and montelukast only partially improved his symptoms. Parents suspected chalk to be the culprit when they discovered that lowpowder chalk may contain casein10 and that this was the chalk (Giotto Robercolor; FILA S.p.a., Milan, Italy) used at child’s school. After avoidance of this brand of chalk in the classroom, symptoms improved greatly. Symptoms completely disappeared when the chalk was withdrawn from the whole school.

Specific IgE to Casein and Chalk Specific IgE was measured by an enzyme allergosorbent test as previously described11 on cyanogen bromideeactivated paper disks. IgE titer to casein and chalk was compared with an in-house reference standard (a serum pool from patients allergic to olive pollen titrated by the CAP System FEIA; Pharmacia AB, Uppsala, Sweden). In all assays 10 normal serum samples were included as controls.

Patients To determine the allergenicity of the putative CMPs contained in chalk, an additional group of 13 patients allergic to CMP were recruited after parental consent. All children had been diagnosed as having IgE-mediated allergy to CMP on the basis of convincing clinical manifestations, skin prick tests (SPTs), and in vitro tests

SDS-PAGE, Immunoblot, and Immunoblot Inhibition Extracts were analyzed on discontinuous gels at 15% acrylamide. Protein bands were detected by silver stain12 or transferred to

Table 1 Clinical and immunologic characteristics of cases and controls at the time of the study Patient No.

b

E-1 E-2 E-3 E-4 NE-1 NE-2c NE-3 NE-4 NE-5d,e NE-6f NE-7g NE-8 OA-1 OA-2

Age

7y4m 6y2m 7y4m 10 y 1 m 4y4m 3y3m 14 m 9m 2y1m 8m 8m 18 m 6y4m 2y4m

Sex

M M F F F F F F M M F F M F

Clinical manifestations of CMP allergy

Other sensitizations

U, Rh U, AO POE, V U, V, Rh U, A, V U, V V POE, V POE, U U, AX C POE, U POE, U V

No No Egg, mites, dog Peach, Olea, Salsola Egg, mites, cat, dog, Olea No No No No Egg Egg No No Mites, Olea

Specific IgE,a kU/L Milk

ALA

BLG

Casein

10.1 16.8 8.2 3.4 45 0.7 2.2 3.7 class 2 1.2 1 78.5 2.8 13.8

2.6 <0.35 6.5 2.9 14.3 <0.35 0.85 0.07 class 0 0.37 0.36 6.6 0.8 3.3

2.5 <0.35 3.1 0.8 5.3 <0.35 0.29 2.9 class 1 0.25 1.4 8.3 1 2.4

18.1 15.6 4 1.4 28.5 0.5 1.3 2.5 class 2 1.4 0.13 91.4 0.6 1.7

Abbreviations: A, asthma; ALA, a-lactalbumin; AO, angioedema; AX, anaphylaxis; BLG, b-lactoglobulin; C, cough; E, exposed; ND, not done; NE, nonexposed; OA, outgrown allergy; POE, perioral erythema; Rh, rhinitis; U, urticaria; V, vomiting. a Results of standard in vitro tests performed by the CAP System FEIA method (Phadia AB, Uppsala, Sweden) before or at the time of study. b Index case. c Tolerant at 4 y 11 m. d Tolerant at 3 y 5 m. e Exact CAP values not available. Results expressed in classes: class 0, 0 to 0.35 kU/L; class 1, 0.35 to 0.69 kU/L; class 2, 0.7 to 3.49 kU/L. f Tolerant at 11 m. g Tolerant at 15 m.

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nitrocellulose membranes for immunoblot. Serum samples were tested at 1/5 dilution in medium containing 0.1% gelatin. Bound IgE antibodies were detected with a biotinylated mouse monoclonal antibody to human IgE (Operon, Zaragoza, Spain), followed by streptavidin-peroxidase and the ECL Plus System (Amersham, Uppsala, Sweden). In some experiments, IgE to chalk proteins was inhibited by preincubation with 25 mg of casein before immunoblot. Basal Spirometry and Methacholine Challenge Test Lung function tests were performed in the 4 school-aged children. Basal spirometry was performed according to European Respiratory Society/American Thoracic Society recommendations13 with a Vmax22 spirometer (Sensor Medics Co, Yorba Linda, California). Written consent was obtained from parents before performing challenge tests. Methacholine test was performed according to the American Thoracic Society protocol14 with methacholine chloride (Provocholine, Methapharm Inc, Brantfort, Ontario, Canada). According to the protocol, when tests were scheduled, a list of items/medications to avoid before the test was given to patients to minimize any factor that could interfere with bronchial responsiveness. Chalk Use Test Chalk use test was performed on the 4 school-aged children with chalk dust. Briefly, after a basal spirometry and clinical evaluation of eye and nose symptoms, patients came into a clean, closed, 25-m3 room and performed the usual tasks of writing and erasing on a board with a piece of chalk. Simultaneously, increasing amounts of chalk dust were dispersed in the air under a multidirectional turbulent flow, releasing doses of 1 g in the first 5 minutes, 3 g in 15 minutes, and 5 g in 30 minutes (cumulated doses of 1, 4, and 9 g, respectively). The appearance of chest, eye, or nose symptoms was monitored, and spirometry was performed after each period. The procedure finished after the last dose was released or when a decrease of 20% or greater in forced expiratory volume in 1 second (FEV1) was recorded. In the last case, a bronchodilator was applied and the test ended after the complete recovery of FEV1. A peak flow meter was provided to the patients to evaluate delayed responses, with measurements every 2 hours during daytime and on night awakenings for the next 24 hours.

337

Skin Tests and Specific IgE The chalk extract was skin tested in 12 patients, and the results were positive in 5 (2 exposed and 3 nonexposed patients). All chalk-positive patients also tested positive to casein (Table 2). All tested patients had specific IgE to casein (mean [SD], 19.4 [43.8] kU/L; range, 0.58e156.2 kU/L) and chalk (mean [SD], 13 [30] kU/L; range, 1.2e109.9 kU/L). Two serum samples from the index case (E-1), before and at school age, were available. Higher levels of IgE to casein and chalk were detected in the latter sample, indicating the existence of specific IgE to chalk before significant exposure. Immunoblot and Immunoblot Inhibition Immunoblot was performed with available sera from exposed and nonexposed patients (Fig 2A and Table 2). All positive samples produced a similar pattern of IgE binding to chalk: a blurred pattern in which a major band at approximately 30 kDa could be identified. Preincubation with casein produced a strong inhibition of IgE binding to this band (Fig2B). Lung Function and Chalk Use Tests Basal spirometry results were normal in 4 patients. Methacholine challenge test results were positive in 3 of 4 cases (Table 3). Chalk use test results were positive in 2 cases (Table 3). Patient E-1 developed rhinorrhea, sneezes, cough, and wheezes, with a 32% decrease in FEV1 after breathing a cumulative dose of 4 g. After 10 minutes of observation in a dust-free environment, the FEV1 was still 23% lower than baseline, and 200 mg of inhaled salbutamol was administered with full recovery of lung function. No delayed symptoms or changes in peak flow were observed. Patient E-2 developed cough after 35 minutes of exposure to an estimated cumulative dose of 6.5 g of chalk dust. At this time point spirometry revealed a 22% FEV1 decrease, and the procedure was stopped. After 10 minutes spirometry was unchanged, and 400 mg of salbutamol was administered with full recovery.

Results Symptoms Related to Chalk Exposure The 4 school-aged children allergic to CMP experienced respiratory symptoms while in class. The index case (E-1) experienced cough, dyspnea, wheezes, and rhinoconjunctival symptoms, patient E-2 experienced cough and expectoration, and patients E-3 and E-4, both highly atopic, reported rhinorrhea, sneezing, itching nose, and tearful eyes. In all cases symptoms improved when chalk was changed to a nonecasein-containing brand of chalk in the classroom. The degree of exposure to chalk among the 8 nonexposed infants was not known but was probably nonexistent or very occasional. Among the tolerant children, one (patient OA-10) could have been exposed to chalk while in class but probably had achieved tolerance before and did not report symptoms with chalk. Chalk Extract Conventional extraction of chalk at near neutral pH was unsuccessful (data not shown). Among several extraction methods alkaline extraction in the presence of 0.1 N sodium hydroxide produced an extract with a protein content of 0.05 mg/mL, equivalent to 0.1 mg of protein per gram of chalk. SDS-PAGE and silver staining showed protein bands at approximately 30 and 37 kDa (Fig 1).

Figure 1. Sodium dodecyl sulfateepolyacrylamide gel electrophoresis and silver stain.

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Table 2 Results of in vivo and in vitro tests Patient No.

SPT result (wheal size), mm

EAST test result, kU/L

Chalk

Casein

Chalk

Casein

E-1 E-2 E-3 E-4 NE-1 NE-2 NE-3 NE-4 NE-5 NE-6 NE-7 NE-8 OA-1 OA-2

64 54 Negative Negative 44 Positiveb Negative 54 Negative ND ND 2  2c Negative Negative

55 87 65 11  7 65 Positiveb Negative 11  7 Negative 10  8 23 2  2c 44 Negative

4.3 15.9 3.6 1.5 10.1 ND ND 3.2 1.8 1.9 1.7 109.9 1.2 1.3

11.1 9.3 9.4 2.4 41.5 ND ND 5.7 3.4 2.7 2.8 156.5 1.6 1.6

Chalk immunoblot test result Positivea Positivea ND ND ND ND ND Positivea Positivea Positivea Positivea Positivea ND ND

Abbreviations: E, exposed; EAST, enzyme allergosorbent; ND, not done; NE, nonexposed; OA, outgrown allergy; SPT, skin prick test. a Blurred pattern. b Wheal sizes not stored but greater than 3  3 mm. c SPT repeatedly inhibited (histamine positive control size <3  3 mm).

Patients E-3 and E-4 did not develop symptoms or changes in FEV1 during the test (chalk exposure for 50 minutes with a cumulative dose of 9 g). No patient developed delayed symptoms or peak flow changes after the challenge test. Discussion Unintentional exposure to milk is an important cause of morbidity among milk allergic children.15 In the present study, we identified a route of exposure to milk protein in the school setting that could be unnoticed by these children and their families. We

have demonstrated that casein present in chalk is immunoreactive and that the inhalation of chalk dust particles can induce respiratory symptoms in some children sensitized to CMP. Sensitivity to chalk proteins among CMP allergic children was evidenced by SPT positivity (approximately 50% of participants) and serum IgE (100%), both in school-aged and preschool-aged children (ie, exposed and nonexposed to chalk). The specificity of this finding was supported by a distinctive immunoblot pattern (Fig 2), showing a band at 30 kDa similar to that produced by a-caseins from cow’s milk. IgE binding to this band was strongly inhibited by casein. Furthermore, the clinical relevance of these findings was confirmed by the positive challenge test results. Exposure to chalk is common in Spanish schools,16 and caseincontaining chalk is indeed commonly used (J. M. Ramada, E-mail communication, July, 12, 2012). Although there are no specific data on the level of exposure, we hypothesize that it may increase in successive school years, being greatest in children older than 6 years. Among CMP allergic children we have found that sensitization to chalk may be clinically relevant because 2 sensitized children reporting asthma at school had positive inhalation challenge test results with chalk dust. In contrast, 2 other patients with negative chalk SPT results reported only upper respiratory tract symptoms, and the results of challenge tests with chalk were negative. These 2 patients developed no adverse events when casein-chalk was reintroduced in their classroom, suggesting the existence of nonspecific or subjective components in their symptoms. Several milk allergic infants with improbable contact with chalk also recognized both in vivo and in vitro casein contained in chalk with a similar pattern. The potential effect on health of this sensitization when exposure to chalk dusts becomes regular is not currently known. Milk proteins are used in a number of nonalimentary uses.2,17 A common problem of this product is the release of large amounts of dust during normal use that contain small particles that

Figure 2. Immunoblot with chalk extract. A, IgE binding from individual samples (E1-NE8 according to Table 1). B indicates Dilution buffer; NHS, normal nonatopic serum. B, IgE inhibition of chalk with casein. *Serum sample at the age of 4 years, before reporting symptoms. **Serum sample at the age of 6 years, after the beginning of symptoms with chalk.

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Table 3 Nonspecific and specific challenge tests in school-aged children Patient No.

E-1 E-2 E-3 E-4

Symptoms with chalk

Asthma Rhinitis/cough Rhinitis Rhinitis

PC20, mg/mLa

0.44 2.79 0.61 >25

Chalk use test Result

D FEV1, %

Cumulated dose, g

Chest symptoms

Nose/eye symptoms

Delayed response

Positive Positive Negative Negative

32 22 2 7

4 6.5 9 9

C, W C No No

Yes No No No

No No No No

Abbreviations: C, cough; DFEV1, maximum decrease in forced expiratory volume in 1 second; PC20, provocation concentration that caused a decrease in FEV1 of 20%; W, wheezes. a Methacholine test.

might contribute to respiratory problems.18,19 One of the methods used to produce low-powder chalk is the addition of casein due to its adhesive and compacting properties,17 but even this kind of chalk releases small particles that can be inhaled and reach the respiratory system.18,19 These particles may act as a carrier for any substance contained in its composition,20,21 including allergenic proteins such as casein, which may behave as a real hidden allergen on milk allergic patients and induce symptoms by inhalation. Casein has already been described as a hidden respiratory allergen when added as a stabilizer in the production of latex gloves.22 It has also been found in house dust apparently related to its use in plaster years ago.23 but the clinical relevance of this finding remains uncertain. Our observations clearly indicate that milk allergens are not destroyed by the chemical reactions occurring in the production of chalk because most patients in this study had IgE antibodies reactive to native casein and casein contained in chalk. IgE binding was generally weaker with chalk proteins than with native casein, indicating partial denaturation. Cow’s milk allergens contain conformational and linear epitopes.24 Sensitivity to the latter has been linked to persistent cow’s milk allergy.25,26 We may hypothesize that chemical reactions for the production of chalk disrupt structural motifs of proteins, leaving unaltered linear epitopes and leading to the persistence of IgE reactivity to casein extracted from chalk. The nature of the casein epitopes contained in chalk should be further investigated. Of special interest is patient E-1 (index case). This is a CMP allergic child with a previous reaction after the intake of a medicine containing casein.9 He had increasing levels of IgE to CMP after starting school activities despite strict dietary avoidance. It seems likely that the combination of strict diet and a hidden exposure to CMP (for example, in chalk) may have contributed to the maintenance or increase of his allergic sensitivity to CMP as has been suggested for other allergens.27 This effect may be an important factor influencing the course of allergy to CMP because the titer of IgE to CMP and peak antibody levels have been reported to be the main predictors for the persistence of allergy to CMP.28 Milk allergy frequently resolves in the first years of age,1,29 but although the natural history remains unclear,30 recent studies suggest that a significant number of patients do not outgrow their allergy by school age.28,31 In this group of patients, hidden exposure to casein may influence their clinical performance and prognosis. In our opinion, the risks of sustained exposure to casein-containing chalk for milk allergic schoolchildren cannot be neglected, and the effect of this exposure should be thoroughly investigated.

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