Hari M. Vijay

Fungi and allergic lower respiratory tract diseases

Asthma is a common disorder that in 2009 afflicted 8.2% of adults and children, 24.6 million persons, in the United States. In patients with moderate and severe persistent asthma, there is significantly increased morbidity, use of health care support, and health care costs. Epidemiologic studies in the United States and Europe have associated mold sensitivity, particularly to Alternaria alternata and Cladosporium herbarum, with the development, persistence, and severity of asthma. In addition, sensitivity to Aspergillus fumigatus has been associated with severe persistent asthma in adults. Allergic bronchopulmonary aspergillosis (ABPA) is caused by A fumigatus and is characterized by exacerbations of asthma, recurrent transient chest radiographic infiltrates, coughing up thick mucus plugs, peripheral and pulmonary eosinophilia, and increased total serum IgE and fungus-specific IgE levels, especially during exacerbation. The airways appear to be chronically or intermittently colonized by A fumigatus in patients with ABPA. ABPA is the most common form of allergic bronchopulmonary mycosis (ABPM); other fungi, including Candida, Penicillium, and Curvularia species, are implicated. The characteristics of ABPM include severe asthma, eosinophilia, markedly increased total IgE and specific IgE levels, bronchiectasis, and mold colonization of the airways. The term severe asthma associated with fungal sensitization (SAFS) has been coined to illustrate the high rate of fungal sensitivity in patients with persistent severe asthma and improvement with antifungal treatment. The immunopathology of ABPA, ABPM, and SAFS is incompletely understood. Genetic risks identified in patients with ABPA include HLA association and certain T(H)2-prominent and cystic fibrosis variants, but these have not been studied in patients with ABPM and SAFS. Oral corticosteroid and antifungal therapies appear to be partially successful in patients with ABPA. However, the role of antifungal and immunomodulating therapies in patients with ABPA, ABPM, and SAFS requires additional larger studies.

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.

Isolation and Expression of a cDNA Clone Encoding an Alternaria alternata Alt a 1 Subunit

Alternaria alternata is recognized as an important source of fungal aeroallergens. Alt a 1, the major allergen of this mold, is a dimer of disulfide-linked subunits that migrate in SDS-PAGE under reducing conditions at apparent M(r)s of 14,500 and 16,000. IgE antibodies to this protein are present in the sera of >90% of A. alternata-sensitive individuals. Previous studies from this laboratory showed that the N-termini twenty amino acids of the purified subunits are nearly identical. We now report the isolation of clones from an A. alternata (strain 34-016) cDNA library constructed in lambda(gt)11, using rabbit IgG antiserum against partially purified Alt a 1. One of nineteen clones selected from screens totalling 305,000 pfu (rb51) was sequenced, and determined to harbor an insert of 660 bp. An in-frame open reading frame within the cloned insert encodes a peptide of M(r) 16,960 that bears no significant homology to known allergens or proteins. The size of the rb51 transcript was determined to be approximately 0.7 kb by Northern analysis of A. alternata total RNA. The largely hydrophobic N-terminal region of the peptide contains an alpha-helical domain and other features characteristic of membrane targeting or secretory signals. The peptide sequence downstream of this region matches previously sequenced Alt a 1 N-terminal from two independent sources at 17 of 20, and 24 of 26 positions. Recombinant Alt a 1 expressed as a secreted protein in Pichia pastoris exists as a dimer in conditioned medium, as shown by immunoblotting under nonreducing conditions. Recombinant Alt a 1, like the natural allergen in A. alternata extracts, is also reactive with serum IgE from A. alternata-sensitive individuals.

Allergenic components of isolates of cladosporium herbarum

Abstract Six isolates of Cladosporium herbarum, IMI 49630, IMI 46932, IMI 96220, CBS 121.47, CBS 180.50 and ATCC 38810 were grown on synthetic revised tobacco medium for 28 days. Extracts of the mycelia were prepared and their biochemical and immunological properties were examined. Two isolates, IMI 49632 and CBS 180.50 did not give a significant yield of mycelial mat, and the mycelial extract of ATCC 38810 was only weakly immunoreactive. In IgE immunoblots of sodium dodecylsulphate polyacrylamide electrophoresis gels, the remaining three extracts showed strongly reactive bands at 14, 17, 25, 35, 41,47 and 97 kDa MW and IMI 96220 had two additional bands at 53 and 71 kDa. These extracts had 8 to 10 bands in isoelectric focusing with pl values between 3.75 to 5.2, the two predominant bands being at 4.5 and 5.2. In crossed — radioimmunoelectrophoresis using human atopic sera, IMI 96220 showed one dominant and three minor allergens; IMI 49630 and CBS 121.47 had one dominant and one minor allergen. In direct ...

Molecular Cloning of IgE–Binding Fragments of Alternaria alternata Allergens

Exposure to the hyphomycete Alternaria alternata is recognized as an important risk factor for asthma. IgE immunoblotting has been used to catalogue the number and Mr of allergens in A. alternata extracts, with estimates ranging from 10 to 30, although few are present in nearly all extracts studied. Several A. alternata allergens have been cloned, including a subunit of the major allergen Alt a 1, ribosomal P2 phosphoprotein, aldehyde dehydrogenase and a yeast YCP4 homolog. We have cloned two sequences encoding IgE–binding fragments of allergens from an A. alternata λgt11 cDNA library using pooled atopic sera from A. alternata–sensitive individuals. One is homologous to a region near the C–terminus of hsp70 from Cladosporium herbarum; a near–complete isoallergen variant of A. alternata ribosomal P2 protein was also cloned. Their lacZ fusion proteins had reactivities of 5 and 14%, respectively, with individual atopic sera, indicating that the corresponding allergens are both minor. This study describes one new A. alternata allergen candidate and implicates ribosomal P2 protein as an allergen thtat is stable between independently isolated clones.

An allergenic polypeptide representing a variable region of hsp 70 cloned from a cDNA library of Cladosporium herbarum

Background Extracts of Clado.sporium herharum, a major source of fungal aeroaller‐gens. exhibit a complex profile of IgE‐binding proteins. Yields of conventionally purified allergens from this mold have been insufficient to permit further molecular analyses.

Mold‐sensitivity in children with moderate‐severe asthma is associated with HLA‐DR and HLA‐DQ

To cite this article: Knutsen AP, Vijay HM, Kumar V, Kariuki B, Santiago LA, Graff R, Wofford JD, Shah MR. Mold‐sensitivity in children with moderate‐severe asthma is associated with HLA‐DR and HLA‐DQ. Allergy 2010; 65: 1367–1375.

N-terminus of a major allergen, Alt a I, of Alternaria alternata defined to be an epitope.

A 20mer peptide representing the N-terminus of a major allergen, Alt a I, of Alternaria alternata was synthesized and examined for its antibody binding and antibody induction activities. Alt a I peptide-BSA conjugate reacted with both human IgE and rabbit anti-Alt a I IgG in ELISA, albeit the binding of the peptide to IgE was relatively weak. Control conjugate showed no antibody binding. These results indicated that the N-terminus of Alt a I is an antibody binding site. Moreover, peptide-KLH conjugate and nonconjugated peptide induced both IgG and IgE antibodies in Balb/c mice that recognized both native Alt a I allergen and peptide-BSA conjugate. Since the free peptide was able to induce antibodies in vivo, the peptide may also possess a T cell epitope.

IgE binding synthetic peptides of Alt a 1, a major allergen of Alternaria alternata.

Alternaria alternata protein, Alt a 1 is a major allergen associated with allergy in atopic patients. Although the molecule binds strongly to IgE antibody from patients, the epitopes involved have not been identified or defined. In the present study, we synthesized overlapping peptides spanning the whole sequence and evaluated their IgE binding with sera from patients with Alternaria-induced allergy. The results identified four IgE binding linear regions. Two of these regions K41-P50 and Y54-K63 showed consistent reactivity with all four patients studied. The specific epitopes involved in the immune response may be of value in the immunodiagnosis and probably also in specific immunotherapy.

Purification and Characterization of Alt a-29 from Alternaria alternata

A major protein component reactive with pooled human atopic sera was isolated from a lyophilized broth extract of Alternaria alternata 34-016. By successive chromatography on Whatman DE-52, Sephadex G-100 and Mono Q HR5/5, a low molecular weight antigen was obtained. Comparison with standard proteins on Sephadex G-100 indicated its molecular weight was 31 kD. Non-reduced samples run on SDS-PAGE showed a band at 29.2 kD which reacted strongly with human IgE. After reduction, it produced a doublet pattern on SDS-PAGE with MW 14.5 and 16.0 kD. The doublet pattern was confirmed by Western blotting with pooled human atopic sera. IEF of the protein showed a major component with a PI of 4.15 and two minor components at 4.25 and 4.40. Immunoblots of the IEF bands showed all three were reactive with human IgE. Ion exchange chromatography of the protein on Mono Q HR5/5 resulted in three resolved components, all of which are immunoreactive. Together with the IEF data, this suggests that there are several conformational or structural isoforms of this protein.