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Notes: 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-1β) 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-1βPenicillium 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.

Notes: Background:  Dermatophagoides pteronyssinus (Dp) and D. farinae (Df) mites are the most important source of indoor aeroallergens. Most Dp mite allergens identified to date have relatively low molecular weights (MWs). Identification of high-MW mite allergens is a crucial step in characterizing the complete spectrum of mite allergens and to provide appropriate tools for diagnostic and therapeutic application.Methods:  The full-length Der p 11 cDNA clone was isolated using cDNA library immunoscreening, the 5′–3′ rapid amplification of cDNA ends (RACE) system and polymerase chain reactions (PCR). The whole cDNA insert and its PCR-derived DNA fragments (p1 to p4) were generated and expressed in the Escherichia coli expression system. The allergenicity of the recombinant protein and its peptide fragments was examined by IgE immunodot assays. The IgE-binding reactivity of rDer p 11 was analyzed in the serum of 50 asthmatic children with positive reactivity to Dp mite extract. Its recombinant peptide fragments were also examined by immunodot assays in 30 mite-allergic children.Results:  Der p 11 cDNA consists of a 2625-bp open reading frame encoding a 103-kDa protein with 875 amino acids. It exhibits significant homology with the paramyosin of other invertebrates. The protein sequence alignment of this newly identified Dp mite allergen (denominated as Der p 11) revealed over 89% identity with Der f 11 and Blo t 1. Among 50 Dp-sensitive asthmatic children, rDer p 11 showed positive IgE-binding reactivity to 39 patients (78%). Using immunodot assays, multiple human IgE-binding activities were demonstrated in all four fragments of Der p 11. Using immunoblot assays, the dominant IgG-binding epitope for monoclonal antibody (mAb642) was located in fragment p3 only. In immunoblot assays, cross-inhibition between rDer p 11 and rDer f 11 was up to 73–80% at concentrations of 100 μg/ml.Conclusions:  This study confirms that the newly identified recombinant Der p 11 is a novel and important high-MW Dp mite allergen for asthmatic children. Our data also indicates that human IgE-binding major epitopes are scattered over the entire molecule of Der p 11.

Notes: Allergenic components of Candida albicans fractionated by SDS–polyacrylamide gel electrophoresis (PAGE) and transferred to nitrocellulose membranes were identified using sera from 30 asthmatic patients who showed positive skin test and RAST (radio-allergosorbent test) to C. albicans. The IgE-binding yeast components in the complex antigen preparation were then detected by reaction with enzyme-labelled anti-human IgE antibodies. They were confirmed by Coomassie blue R-250 staining of the membrane to visualize all protein bands after reaction with the enzyme substrate. The IgE-binding patterns of the sera tested were heterogeneous, displaying a total of 16 identifiable components with molecular weights ranging from 20 to 94 kD. A 40 kD component showed the highest IgE-binding frequency, being recognized by 23 (77%) of the 30 sera examined. The other 15 allergenic components identified were recognized by less than 25% of the sera tested. Only two of the 30 serum samples contained IgE antibodies reactive with seven to eight allergenic components. Ten of the 30 sera reacted with only one allergenic component, and the remaining serum samples recognized two to five of the 16 identified allergens. Results described in this study are applicable to allergen standardization work and provide a basis for further study on the role of C. albicans in clinical allergy.

Notes: Serum levels of IgG subclass and house dust mite (Dermatophagoides pteronyssinus, Dpt) specific IgG4 were evaluated during immunotherapy in asthmatic children. Asthmatic children undergoing long-term immunotherapy (more than 2 years) posed a mean value of total serum IgG4 or Dpt-specific IgG4 antibodies significantly higher than that of patients prior to receiving immunotherapy, asthmatic (placebo) controls, or patients undergoing short-term immunotherapy (less than 1 year) (P 〈 0.05). The mean levels of serum Dpt-specific IgG4 in all asthmatic groups were also significantly higher than in the non-allergic controls (P 〈 0.01). Moreover, the mean level of Dpt-specific IgG4 tended to increase during immunotherapy. A significant correlation between total serum IgG4 and Dpt-specirk IgG4 antibodies was noted (r= 0.6243; P 〈 0.001). Serial follow-up reveals that Dpt-specific IgG4 levels usually rose significantly with clinical improvement in asthmatic children during immunoiherapy. These results suggest that the anti-mite-specific IgG4 antibody may serve as an indicator for clinical outcome of mite allergy during immunotherapy.

Notes: The prevalance of positive specific IgE antibodies to house dust mites (Dermatophagoides pteronyssinus: D. farinae) was determined by enzyme-linked immunosorbent assay (ELISA) in 5097 (61%) volunteers of 8345 schoolchildren aged between 7 and 14 yr from two government schools, All of them filled out a questionnaire concerning allergic symptoms. Among them, 412 (8·1%) children showed a positive reaction to at least one of the two mite allergens, the range varying between 5·6 and 11·2% according to the child's age. Boys had higher prevalence of positive mite specific IgE than girls (9·8% vs. 6·4%, P 〈 0·01), with the overall male to female ratio 1·5:1. The prevalence of bronchial asthma in boys and girls was 5·3% and 3·3% respectively. The positive mite specific IgE antibody in children with asthma and allergic rhinitis was 52% (103 of 198) and 28·7% (193 of 673) respectively. The mean levels of mite specific IgE were not significantly related to the age of onset and severity of asthmatic symptoms (P 〉 0·1), but were significantly different among subjects with current and past asthma (P 〈 0·001). It is suggested that the mire-specific IgE may play a role in the pathogenesis of bronchial asthma in children.

Notes: Allergens and antigens of Bermuda grass pollen fractionated by SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes were identified using twenty-one sera of Bermuda grass pollen-allergic patients. The IgE- and IgG-binding pollen components transferred to nitrocellulose were detected by reaction with enzyme-labelled anti-human IgE and anti-human IgG, respectively. There was heterogeneity in both IgE- and IgG-binding patterns of the allergic sera tested. Fourteen pollen components, ranging in molecular weight from 16000 to 88000 daltons, bound to IgE antibodies. Only two of the fourteen allergens identified reacted with IgE antibodies of more than 50% of the twenty-one allergic sera. The pollen component with a molecular weight of 32000 daltons showed by far the highest frequency of IgE binding, being recognized by sixteen (76%) of the twenty-one sera examined. Fifteen (71 %) of the twenty-one sera tested had IgE antibodies that reacted with more than one of the fourteen allergenic components identified. Pollen components recognized by IgE antibodies also reacted with IgG antibodies, and there were components only recognized by IgG antibodies. Results obtained from this study should be useful both clinically and in research.