Horng-Der Shen

Microarrayed allergen molecules: diagnostic gatekeepers for allergy treatment

Type I allergy is an immunoglobulin E (IgE)‐mediated hypersensitivity disease affecting more than 25% of the population. Currently, diagnosis of allergy is performed by provocation testing and IgE serology using allergen extracts. This process defines allergen‐containing sources but cannot identify the disease‐eliciting allergenic molecules. We have applied microarray technology to develop a miniaturized allergy test containing 94 purified allergen molecules that represent the most common allergen sources. The allergen microarray allows the determination and monitoring of allergic patients’ IgE reactivity profiles to large numbers of disease‐causing allergens by using single measurements and minute amounts of serum. This method may change established practice in allergy diagnosis, prevention, and therapy. In addition, microarrayed antigens may be applied to the diagnosis of autoimmune and infectious diseases.

Respiratory fungal allergy.

Fungal allergy including allergic rhinitis, conjunctivitis, bronchial asthma, and allergic bronchopulmonary mycoses results from exposure to spores. In this review we have dealt with the common allergenic fungi and allergens, immunopathogenesis, diagnostic assays, and the possible control of allergy in the future based on epitope-specific immunotherapy and vaccination.

Immunobiology of Fungal Allergens

In recent years, considerable attention has been paid in obtaining purified relevant allergens from fungi associated with allergy. Using molecular biology techniques, a number of mold allergens have been obtained by cloning the genes encoding the allergens. Currently, about 70 fungal allergens have been approved by the International Allergen Nomenclature Committee. In this review, we have presented major allergens from Aspergillus, Penicillium, Cladosporium, and Alternaria and discussed their immunochemical characteristics and their role in the diagnosis of allergy and possible usefulness in understanding the pathogenesis of the disease. The structure-function properties and the potential role of these recombinant allergens in the immunomodulatory therapy also are presented.

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.

cDNA Cloning and Immunological Characterization of a Newly Identified Enolase Allergen from Penicillium citrinum and Aspergillus fumigatus

Background:Penicillium citrinum and Aspergillus fumigatus are prevalent indoor airborne fungal species that have been implicated in human respiratory allergic disorders. It is important to understand the allergenic profile of these fungal species. The purpose of the present study is to characterize a newly identified enolase allergen from P. citrinum and A. fumigatus. Methods: Fungal proteins were separated by two-dimensional (2D) gel electrophoresis and blotted onto polyvinylidene difluoride membranes. Protein spots that reacted with IgE antibodies in serum samples from asthmatic patients were identified and the N-terminal amino acid sequences were determined by Edman degradation. The peptide sequences obtained were utilized in cloning the cDNA of the allergen genes by reverse transcriptase-polymerase chain reaction and the 5′- and 3′-rapid amplification cDNA end reactions. Results: Our results from 2D immunoblotting identified a 47-kD IgE-reactive component in the extracts of P. citrinum and A. fumigatus. The N-terminal amino acid sequences of the 47-kD proteins are homologous to those of fungal enolases. The corresponding enolase cDNA from P. citrinum contains 1,552 bp and encodes a protein of 438 residues. In A. fumigatus, the isolated enolase cDNA has 1,649 bp and contains a 438-amino acid open reading frame. The deduced amino acid sequences of these two enolases have 94% identity. These enolases from P. citrinum and A. fumigatus were expressed in Escherichia coli as a His-tagged protein and designated as rPen c 22 and rAsp f 22, respectively. Sera from 7 (30%) of the 23 Penicillium-sensitized asthmatic patients showed IgE binding to the 47-kD P. citrinum component (Pen c 22) and rPen c 22. In addition, six of seven Pen c 22-positive serum samples have IgE immunoblot reactivity to the 47-kD A. fumigatus component (Asp f 22) and rAsp f 22. A polyclonal rabbit antiserum generated against the N-terminal peptide of Pen c 22 can react with Pen c 22, rPen c 22, Asp f 22 and rAsp f 22. In addition, the presence of IgE cross-reactivity between rPen c 22 and rAsp f 22 and between enolases from A. fumigatus and Alternaria alternata was also detected by immunoblot inhibition. Conclusions: These results demonstrated that a novel enolase allergen from P. citrinum (Pen c 22) and A. fumigatus (Asp f 22) was identified. In addition, IgE cross-reactivity between enolase allergens from A. fumigatus and P. citrinum and between enolases from A. fumigatus and A. alternata was also detected. Results obtained provide more information on fungal enolase allergens.

Analysis of sequence polymorphism of a major mite allergen, Der p 2

Background The major house dust mite allergen Der p 2 has been regarded as an important allergen involved in the immunopathogenesis of allergic asthma and eczema. Objectives To determine the degree of sequence polymorphism exists in Der p 2 at both the genomic DNA and cDNA levels.

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.

Molecular cloning and immunological characterization of the house dust mite allergen Der f 7

Background The allergen Der p 7 from Dermatophagoides pteronyssinus has been defined by molecular cloning and shown to be an important specificity in 50% of miteallergic patients.

Molecular and immunological characterization of Pen ch 18, the vacuolar serine protease major allergen of Penicillium chrysogenum

Background: We have suggested previously that the 32 and 34 kDa major allergens of Penicillium chrysogenum (also known as P. notatum) are the vacuolar (Pen ch 18) and the alkaline (Pen ch 13) serine proteases, respectively, of P. chrysogenum. The purpose of this study is to characterize the 32 kDa allergen of P. chrysogenum and its immunoglobulin E (IgE)cross‐reactivity with Pen ch 13 allergen.

Characterization of the house dust mite allergen Der p 7 by monoclonal antibodies

The house dust mite allergen Der p 7, which was defined by cDNA cloning, has been shown to react with about 50% of allergic sera and corresponds to or is antigenically related to at least three different sized components in mite extracts. To characterize these entities, monoclonal antibodies (MoAbs) were generated by immunizing BALB/c mice affinity‐purified Der p 7‐GST (glutathione S‐transferase) fusion protein. MoAbs WH9 and WH22 showed positive reactivity to recombinant Der p 7 negative reactivity to GST and the Der p 5‐GST fusion protein in ELISA and immunoblotting. The specificity of both MoAbs was confirmed by inhibition of the ELISA activity by recombinant Der p 7 but not by the recombinant Der p 5. Immunoblot analysis demonstrated that both MoAbs showed reactivities to components with molecular weights (mol. wt.) of 31, 30 and 26kDa reactive to both MoAbs. At least six major forms with different pI or size were indicated by 2‐D gel analysis. In addition to characterization of Der p 7, both MoAbs may also be considered for use in the standardization of Der p 7 in mite extracts.