cDNA cloning, biological and immunological characterization of the alkaline serine protease major allergen from Penicillium chrysogenum

BACKGROUND: Penicillium chrysogenum (Penicillium notatum) is a prevalent airborne Penicillium species. A 34-kD major IgE-reacting component from P. chrysogenum has been identified as an alkaline serine protease (Pen ch 13, also known as Pen n 13 before) by immunoblot and N-terminal amino acid sequence analysis. METHODS: In the present study, Pen ch 13 was further characterized in terms of cDNA cloning, protein purification, enzymatic activity, histamine release and IgE cross-reactivity with alkaline serine protease allergens from two other prevalent fungal species--P. citrinum (Pen c 13) and Aspergillus flavus (Asp fl 13). RESULTS: A 1,478-bp cDNA (Pen ch 13) that encodes a 398-amino-acid alkaline serine protease from P. chrysogenum was isolated. This fungal protease has pre- and pro-enzyme sequences. The previously determined N-terminal amino acid sequence of the P. chrysogenum 34-kD major allergen is identical to that of residues 116-125 of the cDNA. Starting from Ala116, the deduced amino acid sequence (283 residues) of the mature alkaline serine protease has a calculated molecular mass of 28.105 kD with two cysteines and two putative N-glycosylation sites. It has 83 and 49% sequence identity with the alkaline serine proteases from P. citrinum and A. fumigatus, respectively. The recombinant Pen ch 13 was recovered from inclusion bodies and isolated under denaturing condition. This recombinant protein reacted with IgE antibodies in serum from an asthmatic patient and with monoclonal antibodies (PCM8, PCM10, PCM39) that reacted with the 34-kD component from P. chrysogenum. The N-terminal amino acid sequence of the purified native Pen ch 13 is identical to that determined previously for the 34-kD major allergen in crude P. chrysogenum extracts. The purified native Pen ch 13 has proteolytic activity with casein as the substrate at pH 8.0. This enzymatic activity was inhibited by phenylmethylsulfonyl fluoride or diethylpyrocarbonate. Pen ch 13 was also able to degrade gelatin and collagen but not elastin. Basophils from 5 asthmatic patients released histamine (12-73%) when exposed to the purified Pen ch 13. In ELISA (enzyme-linked immunosorbent assay) experiments, IgE for Pen ch 13 was able to compete with purified Pen ch 13, Pen c 13 or Asp fl 13 in a dose-related manner. CONCLUSIONS: These results demonstrated that the 34-kD major allergen of P. chrysogenum is an alkaline serine protease. These results also indicated that atopic patients primarily sensitized by either of these prevalent fungal species may develop allergic symptoms by exposure to other environmental fungi due to cross-reacting IgE antibodies against this protease.     

Allergic inflammation induced by a Penicillium chrysogenum conidia-associated allergen extract in a murine model

BACKGROUND: Recent evidence has shown that viable conidia from the fungus Penicillium chrysogenum induce allergic effects in mice. The present study was conducted to determine the specific allergic dose response of C57BL/6 mice to the protease extract, Pen ch, isolated from viable P. chrysogenum conidia. METHODS: Mice were treated with primary intraperitoneal (IP) injections of 10 or 100 microg of Pen ch adsorbed to alum, followed by weekly IP injections of 0.1, 1.0, or 10.0 microg Pen ch with alum for 4 weeks, and with 10.0 microg of Pen ch by intranasal (IN) inoculations the final 2 weeks before killing. RESULTS: Intraperitoneal injections of 10 and 100 microg of Pen ch for 5 weeks followed by 2 weeks of IN instillation of 10 microg induced significant increases of total serum immunoglobulin (Ig)E and IgG(1). Bronchoalveolar lavage cell counts revealed increased numbers of eosinophils and neutrophils. Histopathological examination of lungs detected perivascular inflammation by eosinophils and neutrophils and increased mucous production. CONCLUSIONS: The data presented in this study indicate that sensitization to protease allergens released by viable P. chrysogenum conidia in vivo induce a strong allergic inflammatory response in a murine model, which could have implications for people exposed to high levels of conidia of this organism.     

Molecular and structural analysis of immunoglobulin E-binding epitopes of Pen ch 13, an alkaline serine protease major allergen from Penicillium chrysogenum

BACKGROUND: Through proteomic and genomic approaches we have previously identified and characterized an alkaline serine protease that is a major allergen (88% frequency of IgE binding) of Penicillium chrysogenum (Pen ch 13). OBJECTIVE: The aim of the present study is to identify the linear IgE-binding epitopes of Pen ch 13. METHODS: IgE-binding regions were identified by dot-blot immunoassay using 11 phage-displayed peptide fragments spanning the whole molecule of Pen ch 13. The minimal epitope requirements for IgE binding were further defined with overlapping peptides synthesized on derivatized cellulose membranes using SPOTs technology. The critical residues on the immunodominant epitopes were mapped through site-directed mutagenesis. The locations of the IgE epitopes identified were correlated with a three-dimensional structure of Pen ch 13. RESULTS: IgE antibodies in 35 serum samples reacted with at least one of the 11 peptide fragments of Pen ch 13. Peptide f-2n (residues 31-61) showed a high-intensity and the highest frequency (77%) of IgE binding. The frequencies of IgE binding to peptide f-4 (residues 93-133), f-1 (residues 1-37) and f-7 (residues 168-206) were 51%, 34% and 31%, respectively. SPOTs assay narrowed down the region of IgE binding of f-2n to residues 48-55 (GHADFGGR). Three, two and one epitope(s) that are four to nine amino acids in length, within f-4, f-1 and f-7, respectively, were found. Site-directed mutagenesis of Pen ch 13 revealed that substitution of His49 and/or Phe52 on Pen ch 13 with methionine resulted in proteins with drastic loss of IgE binding in seven sera tested. Proteins with amino acid replacements at residues 15-18 (RISS), or at residues 112 (I) and 116 (D) have lower IgE-binding reactivity in one of the two patient's sera tested. Substituting residues 117 (W), 119 (V) and 120 (K) also block most of the IgE binding in one of the two patient's sera tested. In addition, replacing residues 203 (V) and 204 (D) along with a deletion at residue 206 (Y) diminished the IgE binding in two serum samples tested. A model was constructed based on the structure of P. cyclopium subtilisin protease that has >90% (256 out of 283 amino acids) sequence identity with Pen ch 13. The major epitope (GHADFGGR) on Pen ch 13 formed a loop-like structure and was located at the surface of the allergen. CONCLUSIONS: Several linear IgE-reactive epitopes and their critical core amino acid residues were identified for the Pen ch 13 allergen. The major linear IgE-binding epitope, 48GHADFGGR55, formed a loop-like structure at the surface of the allergen. Substitution of His49 and/or Phe52 with methionine significantly reduced IgE-binding to Pen ch 13. Mapping of these results on a 3D model of the allergen provides valuable information about the molecular basis of allergenicity for Pen ch 13 and for designing specific immunotherapeutics.     

Prevalence of immunoglobulin E for fungi in atopic children

BACKGROUND: The prevalence of sensitization to fungi in young atopic patients in relation to age and clinical importance is largely unknown. OBJECTIVE: The aim of this study was to investigate the prevalence of sensitization to different fungi in atopic children in relation to age and other aeroallergens. METHODS: A total of 137 atopic children (male 62%, female 38%; mean age 5 years and 9 months, range 5 months-14 years) were studied. Sera of all patients were routinely tested for total IgE and specific IgE against aeroallergens and milk. Positive sera were also tested for IgE against Alternaria alternata, Aspergillus fumigatus, Cladosporium herbarum and Penicillium chrysogenum, using the Pharmacia Enzyme CAP procedure. RESULTS: In this study in atopic children total IgE showed a significant linear relation with age, whereas specific IgE against outdoor fungi, indoor fungi and house dust mite showed significant non-linearity with age. Prevalence of specific IgE for Cladosporium ranked first, followed closely by Aspergillus and Alternaria. Calculation of the sensitization of indoor and outdoor fungi showed maximum prevalence at 7.8 years, followed by lower values at higher ages. A similar significant relation was also found for Alternaria, while this relation was not significant for the other individual fungi. Specific IgE for indoor and outdoor fungi was associated with the presence of specific IgE for aeroallergen and milk. We found that all children aged 4 years and older showed IgE for house dust mite that did not decline with increasing age. CONCLUSIONS: Sensitization to fungi is prevalent in childhood, with an age-dependent distribution reaching maximum values at 7.7-7.8 years, followed by a decline for all fungal sensitization with increasing age. The importance and relative contribution of fungal sensitization to airway disease, compared with the other allergens, remains to be established.     

Use of specific IgE in assessing the relevance of fungal and dust mite allergens to atopic dermatitis: a comparison with asthmatic and nonasthmatic control subjects

BACKGROUND: Although allergens have been implicated as aggravating factors in atopic dermatitis (AD), there is little epidemiologic data on the significance of specific IgE. OBJECTIVE: We sought to compare sensitization to dust mite and fungi between patients with AD and asthmatic and nonasthmatic control subjects. METHODS: Total IgE and specific IgE to Dermatophagoides pteronyssinus, Alternaria alternata, Aspergillus fumigatus, Candida albicans, Malassezia furfur, and Trichophyton rubrum were measured in 73 patients with moderate to severe AD. Total IgE and IgE specific for D pteronyssinus, A alternata, and M furfur were also measured in sera from 156 asthmatic and 212 nonasthmatic control subjects. RESULTS: Positive correlations were found between total IgE and IgE antibodies specific for each of the antigens. IgE specific for M furfur was observed more frequently in adults compared with children with AD (P <.01). AD sera had higher levels of total IgE and a higher prevalence of positive sera to D pteronyssinus (95% vs 42% and 17% for subjects with AD, asthmatic subjects, and nonasthmatic subjects, respectively), M furfur (53% vs 1% and 0.5%), and A alternata (49% vs 29% and 18%). Among the sera from subjects allergic to mites, the contribution of IgE specific for D pteronyssinus to the total IgE levels was similar regardless of the clinical status. CONCLUSIONS: Our results demonstrate that moderate-to-severe AD is strongly associated with sensitization to dust mite andM furfur (odds ratios, 45.6 and 132 vs pooled control sera). These results suggest that both environmental allergens and colonizing fungi contribute to the severity of disease, which is consistent with the view that mite avoidance and antifungal treatment can be beneficial in the treatment of these patients.     

Complementary DNA cloning and immunologic characterization of a new Penicillium citrinum allergen (Pen c 3)

BACKGROUND: Penicillium citrinum has been identified as the most prevalent airborne Penicillium species in the Taipei area. It is important to understand the allergenic composition of this ubiquitous fungal species. OBJECTIVE: The complementary DNA (cDNA) clone of an allergen from P citrinum was isolated and expressed in Escherichia coli as a fusion protein. mAbs were prepared with the recombinant protein as antigen. The corresponding natural allergen in the fungal extracts was identified with the mAbs. METHODS: A Uni-Zap XR P citrinum cDNA library was screened with sera from asthmatic patients. An IgE-binding cDNA clone was isolated and expressed as a glutathione-S-transferase fusion protein. The frequency of IgE binding to the expressed protein was analyzed by immunoblotting. Spleen cells from BALB/c mice immunized with the recombinant protein were fused with NS-1 cells for mAb generation. RESULTS: A P citrinum cDNA library was screened with a mixture of serum samples from 4 asthmatic patients. An IgE-binding cDNA clone was obtained and designated as PCE2. PCE2 has a 694-bp insert that contains a 167 amino acids open reading frame. The deduced amino acid sequence of the encoded protein has 82.6% (138 amino acids) identity with an Aspergillus fumigatus peroxisomal membrane protein allergen (Asp f 3). PCE2 was expressed in E coli as a fusion protein and designated as Pen c 3. Sera from 13 (46%) of the 28 Penicillium-sensitized asthmatic patients demonstrated IgE binding to Pen c 3. In addition, 11 of the 13 Pen c 3-positive serum samples have IgE immunoblot reactivity to recombinant Asp f 3. The presence of IgE cross-reactivity between Pen c 3 and Asp f 3 was also detected by immunoblot inhibition. Four of the 6 mAbs generated against Pen c 3 cross-react with Asp f 3. The presence of the corresponding 18-k natural allergens in the crude extracts of P citrinum and A fumigatus were detected by immunoblot with use of the mAbs and sera from asthmatic patients. CONCLUSION: Results obtained suggest that the peroxisomal membrane protein (Pen c 3) is an important allergen of P citrinum. PCE2 is a full-length cDNA clone encoding this allergen. In addition, the mAbs generated may be useful in standardizing the diagnostic allergenic extracts.     

Antigen characterization of major cork moulds in Suberosis (cork worker's pneumonitis) by immunoblotting

BACKGROUND: We characterized by immunoblotting the antigenicity of the most frequent fungi colonizing cork during its industrial processing, Penicillium glabrum and Chrysonilia sitophila. Penicillium glabrum is the main causative agent of Suberosis, a hypersensitivity pneumonitis of cork workers. Chrysonilia sitophila induces both IgE sensitization and occupational asthma in the wood processing industry. METHODS: Serum-specific IgG, IgG4 and IgE to P. glabrum and C. sitophila from nine cork workers with hypersensitivity pneumonitis (HP) and seven with asthma (four with occupational asthma) were analysed by immunoblotting. RESULTS: Both HP and asthmatic patients' sera showed immunoreactivity to several proteins resolved in the specific immunoblot strips. The frequency of specific IgG recognition to 12-13.5 and 33 kDa proteins of P. glabrum was significantly higher in HP patients. The sera of HP patients had significantly higher specific IgG recognition to 16 and 51-55 kDa proteins of C. sitophila. There was no specific IgE recognition in the sera of HP or asthmatic patients to both fungi. CONCLUSIONS: Different patterns of antibody reactivity to P. glabrum and C. sitophila are seen in cork workers with hypersensitivity pneumonitis or asthma. The 12-13.5 and 33 kDa proteins of P. glabrum and the 16 and 51-55 kDa proteins of C. sitophila may be major antigens in Suberosis.     

IgE-mediated sensitization to mould allergens among patients with allergic respiratory diseases in a desert environment

BACKGROUND: The importance of fungal allergens in the development of allergic diseases in a desert environment is uncertain. This study evaluated the prevalence of IgE sensitization to moulds among patients with allergic respiratory diseases in Kuwait - a desert country. METHODS: A total of 810 patients (male:female ratio 1.4) with a mean age of 32.3 years (range 2-76 years) with extrinsic asthma or allergic rhinitis were studied. Sera from the patients were tested by the CAP-RAST method for specific IgE to 6 fungi (Penicillium, Cladosporium, Aspergillus, Candida, Alternaria and Helminthosporium). For comparison house dust mite and Bermuda grass were also assessed. RESULTS: The overall positivity to at least one mould was 20.9%. Among 120 matched control subjects, the value was 5. 8%. The value was much higher among patients with asthma alone (45. 8%) or both asthma and rhinitis (28.3%) than those with rhinitis alone (11.8%; p < 0.001). Asthmatic children had the highest sensitization rate (66.0% in the 7- to 12-year age group), which declined sharply with age. Among asthmatics, Candida and Aspergillus had the highest sensitization rates (23.1 and 21.3%, respectively), followed by Helminthosporium (18.8%), Cladosporium (15.9%), Alternaria (14.6%) and Penicillium (13.9%). The values for mite and Bermuda grass were 41.2 and 54.6%, respectively. Among asthmatic children, severe asthma was significantly more frequent among mould-positive (51.6%) than mould-negative patients (17.5%; p < 0. 0001). CONCLUSIONS: Even in this desert environment, sensitization to moulds is quite common among patients with allergic respiratory diseases, with a striking preponderance among children with asthma. Mould allergy could also be an important factor determining asthma severity in this environment.     

Dynamic evolution of serum immunoglobulin E to airborne allergens throughout childhood: results from the Multi-Centre Allergy Study birth cohort

Background Allergic rhinoconjunctivitis and asthma evolve dynamically throughout childhood. Yet, data on the evolution of serum levels of IgE antibodies against airborne allergens throughout the first decade of life are scarce.
Objective To describe the patterns of new and persistent sensitization against airborne allergens including remission from birth to 10 years of age and the long-term clinical outcomes up to the age of 13 years.
Methods In 273 children from the Multi-Centre Allergy Study, a German birth cohort, IgE levels were determined against airborne allergens ( Dermatophagoides pteronyssinus , cat and dog dander, birch and grass species pollens) at 2, 5, 7, and 10 years of age (ImmunoCAP, Phadia); allergic rhino-conjunctivitis and asthma were ascertained at the 13 years of age through a standardized questionnaire (International Study of Asthma and Allergies in Childhood).
Results The prevalence of sensitization to each allergen increased steadily throughout childhood, and a hierarchy of sensitization prevalence (grass>birch>mites>cat>dog) was maintained from 5 years of age onwards. A mono-sensitization state was relatively short (measurable half-life=3 years) as additional sensitizations were acquired frequently, and relatively soon after the first one. Remission of weak sensitization (UNICAP classes 1–2) was also quite frequent, especially before 5 years of age. By contrast, stronger IgE responses (>3.5 kU/L) were invariably persistent. Early sensitization was associated with a higher tendency for poly-sensitization at 10 years of age and allergic rhino-conjunctivitis and/or asthma at 13 years of age.
Conclusions IgE responses against airborne allergens undergo dynamic changes throughout childhood, with a high frequency of new sensitization or remission. The long-term persistence and the clinical impact of IgE responses are affected by the intensity of IgE sensitization and the age of its onset.     

Enumeration and detection of aerosolized Aspergillus fumigatus and Penicillium chrysogenum conidia and hyphae using a novel double immunostaining technique

The identification of collected airborne unicellular fungal conidia and hyphae using nonviable techniques is subjective and an imprecise process. Similarly, to determine whether an individual is allergic to a particular genus requires a separate immunodiagnostic analysis. This study demonstrates the development of a novel double immunostaining halogen assay, which enables (1) the simultaneous identification of collected airborne fungal conidia and hyphae of Aspergillus fumigatus and Penicillium chrysogenum using monoclonal antibodies and (2) the demonstration of patient-specific allergy to the same particles using human serum IgE. The results demonstrate that when conidia were ungerminated the binding of antibodies was homogeneous and localized in close proximity around the entire conidia for both species. However, when conidia were germinated, the proportion expressing antigen increased (P < 0.0001) for both species and the sites of binding of the two antibodies changed with double immunostaining restricted to the hyphal tips for A. fumigatus, in addition to the sites of germination for P. chrysogenum. The described immunoassay has the potential to identify fungal particles in personal environmental air samples, provided species-specific monoclonal antibodies are available, while simultaneously demonstrating allergic sensitization to the same particles by co-staining the samples with the patient's own serum. Such an immunoassay can use those fungi that the patient is actually exposed to and potentially avoids many problems associated with extract variability based on the performance of current diagnostic techniques for fungal allergy.     

Pen ch 13 allergen induces secretion of mediators and degradation of occludin protein of human lung epithelial cells

BACKGROUND: Alkaline serine proteases from six prevalent airborne Penicillium and Aspergillus species have been identified as a group of major allergens (group 13). After entering human airways, the allergens are in initial contacts with respiratory epithelial cells. The purpose of this study is to investigate interactions between the Pen ch 13 allergen from P. chrysogenum and human lung epithelial cells. METHODS: A549 cells, 16HBE14o- cells and primary cultures of human bronchial epithelial cells (HBEpC) were exposed to purified Pen ch 13 and mediators released into culture supernatants were assayed with enzyme-linked immunosorbent assay (ELISA) kits. Cleavage of occludin in 16HBE14o- cells was analysed by immunofluorescent staining of whole cells and immunoblot analysis of whole cell extracts. Fragments generated by incubating Pen ch 13 and a synthetic peptide carrying the occludin sequence were analysed by mass spectrometry. RESULTS: Pen ch 13 induced productions of prostaglandin-E2 (PGE2), interleukin (IL)-8 and transforming growth factor (TGF)-beta1 by A549 cells, 16HBE14o- cells and primary cultures of HBEpC. The protease activity of Pen ch 13 is needed for the induction of PGE2 IL-8, TGF-beta1 and cyclo-oxygenase (COX)-2 expression. A tight junction protein occludin of 16HBE14o- cells can be cleaved by Pen ch 13 at Gln202 and Gln211 which are within the second extracellular domain of the protein. Conclusion: Pen ch 13 may contribute to asthma by damaging the barrier formed by the airway epithelium and stimulating the release of mediators that orchestrate local immune responses and inflammatory process from HBEpC.     

Identification of the major allergenic components in Blomia tropicalis and the relevance of the specific IgE in asthmatic patients.

BACKGROUND: Blomia tropicalis has been reported to be a clinically important allergen in house dust. High prevalence of sensitization to B. tropicalis has been noted in asthmatic patients in Taiwan; however, the allergenic components and its impact on asthmatic patients remain to be clarified. OBJECTIVE: To analyze the prevalence of IgE against B. tropicalis and each allergenic component in asthmatic patients. METHODS: A series of recombinant allergenic components were used for skin tests. The B. tropicalis specific IgE in the serum were measured using the Pharmacia CAP System and immunoblot analysis. RESULTS: A total of 131 patients were included in this study: 44% of these 131 patients were allergic to B. tropicalis, 43% of the 80 B. tropicalis-sensitive patients were allergic to Blo t 5, and 75% of the 65 Blo t 5-sensitive patients were allergic to Blo t 5 fragment 3 (Blo t 5 70-117). The sera IgE binding activity to B. tropicalis was repeatedly tested after Dermatophagoides pteronyssinus absorption, and results showed that most patients were concurrently sensitized to D. pteronyssinus and B. tropicalis. In addition, in 2 (18%) of 11 patients, the B. tropicalis sensitization was caused by the cross-reactivity of D. pteronyssinus. CONCLUSION: A high prevalence of B. tropicalis sensitization was detected in our asthmatic patients, and most of them were concurrently sensitized to D. pteronyssinus and B. tropicalis. The major allergenic component and its IgE binding fragments in Blo t 5 have been identified. These allergenic components can be used for the allergenic determination in B. tropicalis and for further immunotherapy.     

Chronic rhinosinusitis: an enhanced immune response to ubiquitous airborne fungi

BACKGROUND: Chronic rhinosinusitis (CRS) is one of the most common long-term illnesses in the United States. The etiology of CRS is unknown, and no effective treatment has been established. OBJECTIVE: We investigated the hypothesis that abnormal immunologic responses to ubiquitous airborne fungi contribute to the pathogenesis of this disease. METHODS: The proliferative and cytokine responses of PBMCs to extracts from 4 common airborne fungi-including Alternaria , Aspergillus , Cladosporium , and Penicillium -were examined by in vitro culture. Serum specimens were tested for specific IgE and IgG to these fungi. RESULTS: PBMCs from approximately 90% of the patients with CRS, but not those from normal individuals, produced both IL-5 and IL-13 when exposed to Alternaria. Furthermore, PBMCs from patients with CRS produced significantly more IFN-gamma than PBMCs from normal individuals in response to Alternaria (median, 553 pg/mL vs 98 pg/mL; P < .01). Levels of serum IgG antibodies to Alternaria and Cladosporium were clearly increased in patients with CRS compared with normal individuals ( P < .01). Less than 30% of the patients with CRS had specific IgE antibodies to Alternaria or Cladosporium. The increased humoral (serum IgG) response strongly correlated with the increased cellular (IL-5 production) response to Alternaria ( r = 0.619; P < .01). CONCLUSION: Patients with CRS show exaggerated humoral and cellular responses, both T(H)1 and T(H)2 types, to common airborne fungi, particularly Alternaria. The anomalous immune and inflammatory responses to ubiquitous fungi may explain the chronicity of airway inflammation in CRS.     

cDNA cloning and immunologic characterization of a novel EF-1beta allergen from Penicillium citrinum

BACKGROUND: We have identified previously that Penicillium citrinum is the most prevalent Penicillium species in the Taipei area. It is important to delineate the whole spectrum of allergenic components of this prevalent airborne fungus. The purpose of this study was to identify novel P. citrinum allergens through molecular cloning of allergen genes using a cDNA library of P. citrinum and sera from patients with bronchial asthma. METHODS: A lambda-Uni-ZAP XR-based cDNA library of P. citrinum was screened with sera from asthmatic patients. An IgE-binding cDNA clone was isolated and heterologously expressed in Escherichia coli. The frequency of IgE-binding to the expressed protein and the IgE reactivity to allergen subunits were analyzed by immunoblotting. RESULTS: An IgE-reactive cDNA clone (clone B) was isolated by plaque immunoassay. The cDNA insert is 876-bp long and encodes a 228-amino acid polypeptide with a calculated molecular mass of 25 035 Da. Protein database search with the deduced clone B sequence revealed that 121 (53%) and 82 (36%) of the 228 amino acids were identical to those of the elongation factor 1-beta (EF-1beta) proteins from the yeast Saccharomyces cerevisiae and the parasite Echinococcus granulosus, respectively. His-tagged recombinant clone B proteins were constructed and expressed in E. coli. Seven (8%) of the 92 serum samples from patients with bronchial asthma showed IgE-binding to the recombinant clone B protein. Among these seven positive sera, five demonstrated IgE-binding to the C-terminal fragment (aa 119-228) while the other two sera showed IgE reactivity to the N-terminal fragment (aa 1-118) of this newly identified EF-1betaPenicillium allergen. CONCLUSIONS: A novel P. citrinum allergen (Pen c 24) was identified and characterized in the present study. Results obtained provide more information about allergens of prevalent airborne fungi and a basis to understand more about the IgE responses in human atopic disorders and in parasitic infections.     

The role of fungal allergy in bronchial asthma]

Fungus is known to be one of the causative allergens inducing bronchial asthma as are housedustmites, pollen and pet dander. Outdoor airborne fungi such as Cladosporium, Alternaria, Penicillium and Aspergillus are important inducing IgE antibody formation. In addition to these common fungi, the indoor fungi Aspergillus restrictus, Neurospora and Eurotium are important allergenic fungi which have recently been identified. The yeast Candida albicans, is a common commensal of the human oral and vaginal mucosae and gastrointestinal tract and part of the normal flora, is known as one of the main allergens causing bronchial asthma. We examined the allergenicity of mannan (Mn) as a cell-wall constituent and acid protease (CAAP) as a secreted enzyme of C. albicans. We previously reported cases of atopic asthma caused by CAAP and stressed the role of CAAP as an important allergen in mucosal allergy to C. albicans 9). The levels of the antibodies to these antigens in the sera of asthmatic patients who showed positive immediate intradermal response to crude C. albicans (n=86) were measured. Anti-Mn IgE and IgG antibody levels were measured by liquid-phase assay (AlaSTAT). Anti-CAAP and anti-crude C. albicans IgE and IgG antibody levels were measured by RAST and AlaSTAT. Anti-Mn A and anti-Mn B IgE antibody titers were strongly correlated (r=0.87), while anti-Mn A and anti-CAAP IgE titers were not correlated. However, all of the anti-Mn A IgE positive sera and all of the anti-CAAP IgE positive sera were positive for IgE to crude-C. albicans. This indicates that both Mn and CAAP are C. albicans-related allergens. Titers of IgG antibodies to Mn A and crude C. albicans were highly correlated (r=0. 90). Results of inhibition assays performed using other fungal antigens as inhibitors showed that Mn is a cross reactive allergen among different fungi and that CAAP is a C. albicans specific allergen causing human mucosal allergic reaction.     

Development of allergy and IgE antibodies during the first five years of life in Estonian children

BACKGROUND: Epidemiological studies have demonstrated a low prevalence of allergic diseases and atopic sensitization among schoolchildren and young adults in the formerly socialist countries of Central and Eastern Europe as compared to Western Europe. OBJECTIVE: The aim of our study was to prospectively investigate IgE responses to food and inhalant allergens and the development of allergy during early childhood in a population with a low prevalence of atopic disorders. METHODS: In a population-based prospective study, 273 children were followed from birth through the first 5 years of life, recording manifestations of allergy by questionnaires and clinical examinations at 0.5, 1, 2 and 5 years (n = 213). Skin prick tests (SPT) were performed using natural foods (cow's milk, egg white) and commercial extracts of inhaled allergens (cat, dog, D. pteronyssinus, birch, timothy). In addition, serum IgE levels and circulating IgE antibodies against the seven allergens were determined. RESULTS: The prevalence of allergic diseases at 5 years of life was 19%. Atopic dermatitis was the most common allergic disease at all ages. The point prevalence of positive skin prick tests was 7% at 0.5, 1 and 2 years of age, and 3% at 5 years. Circulating IgE antibodies against food allergens were common at all ages, i.e. 13, 23, 36 and 36%, respectively, at 0.5, 1, 2 and 5 years. The prevalence of circulating IgE antibodies to inhalant allergens increased from 1.5% at 0.5 years to 11% at 1, 19% at 2 and 47% at 5 years. The antibody levels were generally low, however. The value of positive SPT and the presence of IgE antibodies in the diagnosis of clinical allergy were low. CONCLUSION: The results of this prospective study carried out in a previously socialist country with a low allergy prevalence among schoolchildren and young adults indicate that transient sensitization in early childhood is followed by a down-regulation of skin reactivity.     

Sensitization to fungi: epidemiology, comparative skin tests, and IgE reactivity of fungal extracts

BACKGROUND: Several fungal species are known to cause severe respiratory and cutaneous allergic diseases. Extracts from several allergenic fungi are used for in vivo and in vitro tests, as standard preparations are still not available. OBJECTIVE: The aims are to define the pattern of in vivo and in vitro IgE reactivity to fungal species in an allergic population with respiratory symptoms; to determine the influence of different extract preparations on diagnostic results; and to evaluate whether there exists a relationship between the diagnostic pattern of reactivity and the pattern of specific IgE reactivity in immunoblots. METHODS: Skin prick tests were applied to a cohort of 4962 respiratory subjects, aged 3-80 years. Fungal extracts from Alternaria, Aspergillus, Candida, Cladosporium, Penicillium, Saccharomyces, and Trichophyton were used, along with extracts from pollens, mites, and animal dander. Demographical and diagnostic data were recorded. IgE detection was carried out with the same allergenic extracts plus Malassezia. Comparative skin tests and IgE detection were carried out using extracts from three commercial suppliers. IgE immunoblots were carried out with the same panel of commercial fungal extracts and were compared with in-house extracts. Data analysis was carried out by grouping the population on the basis of their reactivity to a single, to two or to more than two, mould species. RESULTS: Nineteen percent of the allergic population reacted to at least one fungal extract by means of the skin test. Alternaria and Candida accounted for the largest number of positive tests, and along with Trichophyton they were the main sensitizers in the subset of patients with an isolated sensitization. The prevalence of skin test reactivity increased for these three fungi in the subsets with two associated reactivities and, furthermore, in the subset showing reactivity to more than two mould species. In the latter group, a steady increase of the skin test reactivity was recorded for all the other fungal sources, suggesting a clustered reactivity. Comparative skin and IgE testing with different groups of subjects with a simple pattern of skin reactivity resulted in sensitivity differences between in vivo and in vitro tests, whereas discrepant results were recorded in the subsets of patients with multiple fungi sensitization. Although hampered by the limited reliability of fungal extracts, IgE immunoblots revealed differing patterns of reactivity when sera from the three subsets were used. This suggests a link between the diagnostic reactivity pattern and the IgE sensitization to extracts' components. Age and gender distribution differed among the Alternaria-, Candida-, and Trichophyton-sensitized subjects, but not in the subset with more than two fungi sensitizations. CONCLUSIONS: The preliminary assessment of a new classification of the mould-sensitized population has been reached. The limiting quality of fungal extracts requires future studies using an allergenic molecule-based approach. The diagnostic process and the definition of the reactivity pattern would thus be easy, and it could lead to a novel specific immunotherapy approach.     

Latex allergy: low prevalence of immunoglobulin E to highly purified proteins Hev b 2 and Hev b 13

BACKGROUND: Hevea brasiliensis (Hev b) 2 and Hev b 13 have recently been identified as major latex allergens by detecting specific IgE antibodies in >50% of sera from Hev b latex-allergic individuals. OBJECTIVE: We assessed the prevalence rates for sensitization to extensively purified latex allergens in patients from three diverse geographical areas. METHODS: Native Hev b 2, Hev b 5, Hev b 6.01 and Hev b 13 were purified by non-denaturating chromatography and were used in ELISAs to assess sera from 215 latex-allergic patients and 172 atopic non-sensitized controls from Finland, Spain and the United States to detect allergen-specific IgE antibodies. RESULTS: Unexpectedly, even highly purified Hev b 13 contained epitope(s) to which Hev b 6-specific human IgE antibodies bound effectively. Further purification, however, reduced the prevalence of IgE antibody reactivity to low levels: 15%, 5% and 11% for Hev b 2, and 18%, 30% and 27% for Hev b 13 among latex-allergic Finnish, Spanish and American patients, respectively. Interestingly, Finnish patients had a lower prevalence of Hev b 5-specific IgE antibody (28%) as compared with Spanish (49%) and American (71%) patients. The prevalence of Hev b 6.01-specific IgE reactivity was uniformly >50% in all three populations. CONCLUSION: Neither Hev b 2 nor Hev b 13 appear to be major latex allergens when evaluated in serological assays using highly purified allergens. The reason(s) for the observed differences in published sensitization rates in various geographic regions requires further study. The purity of the allergen preparations has a marked impact on the accuracy of latex-specific IgE antibody detection in epidemiological studies and in the serological diagnosis of latex allergy.