Acta Prediatr Scand 79: 343-348, 1990
IgE Antibodies to Pityrosporum orbiculare in Children with Atopic Diseases S. L. NORDVALL’T~ and SIV JOHANSSON’ From the ‘Department of Paediutrics, St. Goran’s Children’s Hospital, and the 2Department of Clinical Immunology, Karolinska Hospitul, Stockholm, Sweden ABSTRACT. Nordvall, S. L. and Johansson, S. (Department of Paediatrics, St. Goran’s Children’s Hospital and Department of Clinical Immunology, Karolinska Hospital, Stockholm, Sweden). IgE antibodies to Pityrosporum orbiculare in children with atopic diseases. Acta Paediatr Scand 79: 343, 1990.
IgE antibodies to Pityrosporum orbiculare and 16 other species of fungi were measured with the radioallergosorbenttest (RAST) in sera of 131 children. The children were 7-18 years old, suffered from atopic diseases and showed a varied allergic profile on a skin prick test (SPT). IgE antibodies to P. orbiculare were found in the sera of 26 of the 131 children. A higher proportion of the P. orbiculare RAST positive children than of the negative ones had current In a stepwise logistic regression analysis of SPT and RAST data, the eczema @5OO kUA had serum IgE antibodies to P . orbiculare. Twenty-four of the 26 P. orbiculare RAST positive children harboured serum IgE antibodies to other fungi. The strong relationship between atopic eczema and the occurrence of IgE antibodies to P. orbiculare indicates that these antibodies may be pathogenetically important in at least some patients with atopic eczema and this gives another dimension to this common skin disease. Key words: children, fungi, Pityrosporum orbiculare ( o d e ) , IgE, IgE antibodies, eczema.
Serum IgE levels are elevated in a majority of patients with atopic eczema, and in patients with severe dermatitis they are generally higher than in those with a mild dermatitis (1). The role of atopic allergy in the pathogenesis of atopic eczema is unclear, however, and is still much debated (2). It is well known that a large proportion of eczematous infants and children have IgE antibodies to foods and that many of them develop IgE antibodies to inhalant allergens. The pathogenetic significance for the dermatitis both of IgE antibodies to common foods and IgE antibodies to inhalants is, however, controversial (2, 3). Among the inhalant allergens pollens, house dust mites and moulds have been believed to have some relevance for atopic dermatitis (reviewed in 2). Recently we found a strong relationship between the occurrence of serum IgE antibodies to moulds and atopic eczema (4). Since it appeared that many children were sensitized to several different mould species, as a consequence either of multiple independent sensitization or of cross-reactivity between different fungi, it was reasonable to assume that these children would also harbour serum IgE antibodies to fungi colonizing the skin and that such IgE antibodies could be of pathogenetic significance for the eczema. Widespread colonization of fungi in the human skin is known only for the lipophilic yeast Pityrosporum orbiculare, which colonizes the skin of 90-100% of healthy adults (5). This investigation was undertaken to study the occurrence of serum IgE antibodies to P . orbiculare in atopic children and its possible relationship to atopic eczema and other atopic diseases. MATERIAL AND METHODS Patients. Children attending the outpatient allergy clinic of St. Goran’s Children’s Hospital, both new referrals and children undergoing regular follow-up, were investigated. In total 13 1 children aged 7-18
344 S . L. Nordvall and S . Johansson
Acta Paediatr Scand 79
years (median age 12 years), 83 boys and 48 girls, were included in the study. All of the children were suffering or had suffered from an atopic disease, namely bronchial asthma, allergic rhinoconjunctivitis, atopic eczema and/or IgE-mediated food allergy (Table 1). The major part of the investigation was performed in January-March 1987. In all patients a structured case history concerning atopic diseases was taken. The severity of asthma was scored as proposed by Engstrom (6) and it was recorded whether the patients had a history of allergic rhinoconjunctivitis, current or previous eczema, or allergy to important foods (cow's milk, hen's egg or fish etc.) that was likely to be IgE-mediated. Various questions were asked with the aim of ascertaining the diagnosis, e.g. in the case of eczema the location of the lesions. Blood samples were drawn from all children and serum was kept frozen until analysed. Skin prick tests. Skin prick tests were performed with precoated allergen lancets (Phazet, Pharmacia AB, Uppsala, Sweden). A panel of allergens was used: Dermatophagoides pteronyssinus, Dermatophagoides farinae, dog, horse, cat, timothy, Cladosporium herbarum, Alternuria alternata, birch, alder and mugwort. The results were evaluated in relation to a histamine reference equivalent to histamine hydrochloride 10 mg/ml in accordance with the Nordic guidelines (7). Pityrosporum orbiculare extract. An isolate of P . orbiculare from the American Type Culture Collection (No. 421 32) was grown at 37°C for four days on a special glucose-neopeptone agar containing olive oil (2%), monostearate (2.5 g I-') and polysorbate 80 (Tween 80) (0.2%) as described by Faergemann & Fredriksson (8), with the exception that no yeast extract was added to the substrate. The P . orbiculare cells were carefully harvested from the surface of the solid medium and a thick yeast cell suspension in sterile distilled water was obtained. After freeze-drying, the suspension was sonicated for 5 min in a phosphate buffer, pH 7.4, and extracted. Piiyrosporum orbiculare RAST discs. The P . orbiculare extract was coupled to cyanbromideactivated paper discs as previously described (9, 10). Coupling was performed with the extract in a concentration of 1/150 (w/v). This concentration was chosen after allergen titrations in which sera with high IgE antibody concentrations to Candida albicans were used. In these experiments sera with levels of IgE antibody to P . orbiculare in excess of 17.5 Phadebas RAST units (PRU)/ml were found. In control experiments neopeptone alone was coupled to paper discs. IgE antibody measurements IgE antibodies were measured by RAST (9, lo), using reagents from commercially available Phadebas RAST test kits (Pharmacia AB) with the exception of the aforementioned P . orbiculare discs, and following the recommendations of the manufacturer. In all assays a reference system was run and the results were expressed in PRU/ml. The sera were analysed in duplicate. IgE antibodies to P . orbiculare and to 16 other fungi were assayed by this method. The 16 fungi (ml-m16) were: Penicillium notatum, Cladosporium herbarum, Aspergillus fumigatus, Mucor racemosus, Candida albicans, Alternaria alternata, Botrytis cinerea, Helminthosporum halodes, Fusarium moniliforme, Stemphylium botryosum, Rhizopus nigricans, Aureobasidium pullulans, Phoma betae, Epicoccum purpurascens, Trichoderma viridae and Curvularia lunata. A detailed description of the results obtained with these 16 fungi will be published elsewhere. Control experiments with the neopeptone discs, using all patient sera, were negative.
Table 1. Current atopic disease manifestations in the children n = number of children. Figures within brackets indicate children with current IgE-mediated food allergy. IgE = geometric mean IgE for the group, in kU/I
Eczema
Asthma
Rhinoconj.
n
IgE 338 298 347 424 238 165 92 33
Both children had a history of previous eczema.
IQE antibodies to Pitvrosuorum orbiculare 345
Acta Paediatr Scand 79
Total ZgE measurements. Total serum IgE concentrations were determined by PRIST ( 1 1 , 12) (Pharmacia AB), following the recommendations of the manufacturer. Statistical analyses. The Spearman rank, Mann-Whitney and Fisher's exact tests were used and also one multivariate technique, Cox's linear logistic regression analysis (13).
RESULTS IgE antibodies to P . orbiculare were found in the sera of 26 of the 131 children (Table 2 ) . The antibody levels were generally low or moderate. In the sera of three of these 26 children the concentration of P. orbiculare IgE antibodies was above 3.5 PRU/ml (RAST class 3), 17 sera were in RAST class 2 (0.7-3.5 PRU/ml) and six were in RAST class I (0.35-0.7 PRU/ml). The three patients with the highest antibody levels all had severe current eczema, which in two of them dominated their atopic disease. A significantly higher proportion of the children who were RAST positive to P . orbiculare than of those without detectable IgE antibodies to P . orbiculare had current eczema @
IgE Antibodies to Pityrosporum orbiculare in Children with Atopic Diseases S. L. NORDVALL’T~ and SIV JOHANSSON’ From the ‘Department of Paediutrics, St. Goran’s Children’s Hospital, and the 2Department of Clinical Immunology, Karolinska Hospitul, Stockholm, Sweden ABSTRACT. Nordvall, S. L. and Johansson, S. (Department of Paediatrics, St. Goran’s Children’s Hospital and Department of Clinical Immunology, Karolinska Hospital, Stockholm, Sweden). IgE antibodies to Pityrosporum orbiculare in children with atopic diseases. Acta Paediatr Scand 79: 343, 1990.
IgE antibodies to Pityrosporum orbiculare and 16 other species of fungi were measured with the radioallergosorbenttest (RAST) in sera of 131 children. The children were 7-18 years old, suffered from atopic diseases and showed a varied allergic profile on a skin prick test (SPT). IgE antibodies to P. orbiculare were found in the sera of 26 of the 131 children. A higher proportion of the P. orbiculare RAST positive children than of the negative ones had current In a stepwise logistic regression analysis of SPT and RAST data, the eczema @5OO kUA had serum IgE antibodies to P . orbiculare. Twenty-four of the 26 P. orbiculare RAST positive children harboured serum IgE antibodies to other fungi. The strong relationship between atopic eczema and the occurrence of IgE antibodies to P. orbiculare indicates that these antibodies may be pathogenetically important in at least some patients with atopic eczema and this gives another dimension to this common skin disease. Key words: children, fungi, Pityrosporum orbiculare ( o d e ) , IgE, IgE antibodies, eczema.
Serum IgE levels are elevated in a majority of patients with atopic eczema, and in patients with severe dermatitis they are generally higher than in those with a mild dermatitis (1). The role of atopic allergy in the pathogenesis of atopic eczema is unclear, however, and is still much debated (2). It is well known that a large proportion of eczematous infants and children have IgE antibodies to foods and that many of them develop IgE antibodies to inhalant allergens. The pathogenetic significance for the dermatitis both of IgE antibodies to common foods and IgE antibodies to inhalants is, however, controversial (2, 3). Among the inhalant allergens pollens, house dust mites and moulds have been believed to have some relevance for atopic dermatitis (reviewed in 2). Recently we found a strong relationship between the occurrence of serum IgE antibodies to moulds and atopic eczema (4). Since it appeared that many children were sensitized to several different mould species, as a consequence either of multiple independent sensitization or of cross-reactivity between different fungi, it was reasonable to assume that these children would also harbour serum IgE antibodies to fungi colonizing the skin and that such IgE antibodies could be of pathogenetic significance for the eczema. Widespread colonization of fungi in the human skin is known only for the lipophilic yeast Pityrosporum orbiculare, which colonizes the skin of 90-100% of healthy adults (5). This investigation was undertaken to study the occurrence of serum IgE antibodies to P . orbiculare in atopic children and its possible relationship to atopic eczema and other atopic diseases. MATERIAL AND METHODS Patients. Children attending the outpatient allergy clinic of St. Goran’s Children’s Hospital, both new referrals and children undergoing regular follow-up, were investigated. In total 13 1 children aged 7-18
344 S . L. Nordvall and S . Johansson
Acta Paediatr Scand 79
years (median age 12 years), 83 boys and 48 girls, were included in the study. All of the children were suffering or had suffered from an atopic disease, namely bronchial asthma, allergic rhinoconjunctivitis, atopic eczema and/or IgE-mediated food allergy (Table 1). The major part of the investigation was performed in January-March 1987. In all patients a structured case history concerning atopic diseases was taken. The severity of asthma was scored as proposed by Engstrom (6) and it was recorded whether the patients had a history of allergic rhinoconjunctivitis, current or previous eczema, or allergy to important foods (cow's milk, hen's egg or fish etc.) that was likely to be IgE-mediated. Various questions were asked with the aim of ascertaining the diagnosis, e.g. in the case of eczema the location of the lesions. Blood samples were drawn from all children and serum was kept frozen until analysed. Skin prick tests. Skin prick tests were performed with precoated allergen lancets (Phazet, Pharmacia AB, Uppsala, Sweden). A panel of allergens was used: Dermatophagoides pteronyssinus, Dermatophagoides farinae, dog, horse, cat, timothy, Cladosporium herbarum, Alternuria alternata, birch, alder and mugwort. The results were evaluated in relation to a histamine reference equivalent to histamine hydrochloride 10 mg/ml in accordance with the Nordic guidelines (7). Pityrosporum orbiculare extract. An isolate of P . orbiculare from the American Type Culture Collection (No. 421 32) was grown at 37°C for four days on a special glucose-neopeptone agar containing olive oil (2%), monostearate (2.5 g I-') and polysorbate 80 (Tween 80) (0.2%) as described by Faergemann & Fredriksson (8), with the exception that no yeast extract was added to the substrate. The P . orbiculare cells were carefully harvested from the surface of the solid medium and a thick yeast cell suspension in sterile distilled water was obtained. After freeze-drying, the suspension was sonicated for 5 min in a phosphate buffer, pH 7.4, and extracted. Piiyrosporum orbiculare RAST discs. The P . orbiculare extract was coupled to cyanbromideactivated paper discs as previously described (9, 10). Coupling was performed with the extract in a concentration of 1/150 (w/v). This concentration was chosen after allergen titrations in which sera with high IgE antibody concentrations to Candida albicans were used. In these experiments sera with levels of IgE antibody to P . orbiculare in excess of 17.5 Phadebas RAST units (PRU)/ml were found. In control experiments neopeptone alone was coupled to paper discs. IgE antibody measurements IgE antibodies were measured by RAST (9, lo), using reagents from commercially available Phadebas RAST test kits (Pharmacia AB) with the exception of the aforementioned P . orbiculare discs, and following the recommendations of the manufacturer. In all assays a reference system was run and the results were expressed in PRU/ml. The sera were analysed in duplicate. IgE antibodies to P . orbiculare and to 16 other fungi were assayed by this method. The 16 fungi (ml-m16) were: Penicillium notatum, Cladosporium herbarum, Aspergillus fumigatus, Mucor racemosus, Candida albicans, Alternaria alternata, Botrytis cinerea, Helminthosporum halodes, Fusarium moniliforme, Stemphylium botryosum, Rhizopus nigricans, Aureobasidium pullulans, Phoma betae, Epicoccum purpurascens, Trichoderma viridae and Curvularia lunata. A detailed description of the results obtained with these 16 fungi will be published elsewhere. Control experiments with the neopeptone discs, using all patient sera, were negative.
Table 1. Current atopic disease manifestations in the children n = number of children. Figures within brackets indicate children with current IgE-mediated food allergy. IgE = geometric mean IgE for the group, in kU/I
Eczema
Asthma
Rhinoconj.
n
IgE 338 298 347 424 238 165 92 33
Both children had a history of previous eczema.
IQE antibodies to Pitvrosuorum orbiculare 345
Acta Paediatr Scand 79
Total ZgE measurements. Total serum IgE concentrations were determined by PRIST ( 1 1 , 12) (Pharmacia AB), following the recommendations of the manufacturer. Statistical analyses. The Spearman rank, Mann-Whitney and Fisher's exact tests were used and also one multivariate technique, Cox's linear logistic regression analysis (13).
RESULTS IgE antibodies to P . orbiculare were found in the sera of 26 of the 131 children (Table 2 ) . The antibody levels were generally low or moderate. In the sera of three of these 26 children the concentration of P. orbiculare IgE antibodies was above 3.5 PRU/ml (RAST class 3), 17 sera were in RAST class 2 (0.7-3.5 PRU/ml) and six were in RAST class I (0.35-0.7 PRU/ml). The three patients with the highest antibody levels all had severe current eczema, which in two of them dominated their atopic disease. A significantly higher proportion of the children who were RAST positive to P . orbiculare than of those without detectable IgE antibodies to P . orbiculare had current eczema @
