Susheela Sridhara

Identification of serine protease as a major allergen of Curvularia lunata

Background:  Several proteins from Curvularia lunata have been identified as important fungal allergens. It will be worthwhile to study the functional aspects of these allergens. The present study aimed at purifying a major allergen and determining its biological function.

Allergenicity assessment of transgenic mustard (Brassica juncea) expressing bacterial codA gene

Background:  Assessing the allergenicity and toxicity of genetically modified (GM) crops is essential before they become a regular part of our food supply. The present study aimed to assess the allergenicity of Brassica juncea (mustard) expressing choline oxidase (codA) gene from Arthrobacter globiformis that provides resistance against abiotic stresses.

Sensitization to blackgram in patients with bronchial asthma and rhinitis: clinical evaluation and characterization of allergens.

Background:  Legumes are important causative agents of type I hypersensitivity in south Asia and Europe but such studies are lacking in Indian population. The present study investigates blackgram sensitization in asthma and rhinitis patients and identifies immunoglobulin E (IgE)‐binding proteins.

Allergens of Curvularia lunata during cultivation in different media

BACKGROUND Despite enormous efforts toward the standardization of fungal extracts, only a few extracts have been characterized that are relevant for the diagnosis and immunotherapy of allergy-associated disorders. OBJECTIVE The goal of this study was to determine the optimum growth condition of Curvularia lunata, an important fungal allergen for quality raw material, and to analyze the C lunata extract for IgE- and IgG-binding proteins. METHODS C lunata was grown in synthetic (Czapeck Dox medium), semisynthetic (Sabourauds broth [SB]), and natural media (potato dextrose [PD]) for different periods of time. The extracts were probed for allergenic and antigenic activity, with pooled patient sera and polyclonal antibodies raised against C lunata by means of ELISAs, immunoblots (in vitro), and intradermal tests (in vivo). RESULTS The growth of C lunata was better in semisynthetic media (ie, SB) compared with other types of media. Dry weight and protein content was maximum in the 7-day culture of SB. ELISA with pooled sera from C lunata-sensitive patients exhibited that cultures grown in SB for 11 to 13 days and PD plus 1.0% agar for 5 days were the most potent. Intradermal tests with 11- to 13-day SB culture extract showed maximum skin reactivity in allergy patients. Immunoblots with patient sera showed 10 to 14 IgE-binding proteins in 5- to 15-day SB cultures. Analysis of 5- to 15-day SB extract with rabbit sera showed 10 to 16 IgG-binding proteins. CONCLUSIONS The extract from 11- to 13-day SB cultures were most biologically potent (intradermal tests) and showed high antigenic and allergenic reactivity (ELISA and immunoblot). The addition of yeast extract did not affect the dry weight and protein content of the C lunata extract. Furthermore, addition of agar in PD medium increases the dry weight and protein content of the fungal mat. Synthetic media was not suitable for mass cultivation of C lunata.

Allergenic cross-reactivity of Curvularia lunata with other airborne fungal species

Background: Curvularia lunata is an important fungus for respiratory allergic disorders. Previous studies indicated cross‐reactivity of Curvularia with other fungi. However, the cross‐reactive allergenic component (s) were not identified. The present work was carried out to study the shared allergenic components of C. lunata and others.

Immunobiochemical Analysis of Cross-Reactive Glutathione-S-Transferase Allergen from Different Fungal Sources

Clinical observations suggest the presence of cross-reactive allergens. There is a need to identify these cross-reactive allergens to improve the treatment used for allergic disorders. The present study was aimed to identify and characterize a cross-reactive allergenic protein from fungi. Allergen extracts of various fungi viz. Alternaria alternata, Aspergillus fumigatus, Cladosporium herbarum, Curvularia lunata, and Epicoccum purpurascens showed GST enzymatic activity ranging from 0.765 to 1.004 Δ340 nm/min/µg where as activity of rGST was 1.123 Δ340 nm/min/µg. Immunoblot with GST antibodies showed a band of approximately 26 kDa in all these fungal extracts. Sera of fungal allergy patients showed the presence of IgE antibodies to GST. Rabbit antibodies raised against the fungal extracts reacted with rGST confirming the presence of GST-like protein in these extract. ELISA inhibition using GST antibodies revealed inhibition with C. herbarum, A. alternata, C. lunata, A. fumigatus, and E. purpurascens demonstrating that fungal GST competes for binding to anti-GST. In summary, a GST-like protein was recognized as cross-reactive allergen in these fungal extracts.

Allergens of Epicoccum nigrum grown in different media for quality source material.

Background: The Epicoccum nigrum (EN) extract used in allergy disorders exhibits batch‐to‐batch variations in protein composition and allergenic potency. In this study, the allergens of EN grown in different media were investigated.

Studies on shared antigenic/allergenic components among fungi

Fungal allergens have been found to be one of the most prevalent aeroallergens in India. Knowledge of shared/unique components among different fungi is necessary for proper diagnosis and treatment of patients allergic to fungi. In the present study, crude extracts (CE) of 11 common fungi (Alternaria alternata, Aspergillus flavus, Asp. fumigatus, Asp. niger, Asp. tamarii, Asp. versicolor, Cladosporium herbarum, Curvularia lunata, Mucor hiemalis, Penicillium citrinum, and Fusarium solani) were characterized by isoelectric focusing (IEF), SDS‐PAGE, and immunoblot. On IEF (pI 3–9), the number of protein bands was found to be greatest (46) in M. hiemalis extract. SDS‐PAGE exhibited a varied number of bands, generally 18–40, with mol. mass ranging from 14 to 100 kDa. IgG‐specific immunoprint using rabbit anti‐F. solani CF antibodies demonstrated a mol. mass distribution of shared antigenic proteins of 14–100 kDa in most of the fungi. Shared allergenicity was observed in a number of allergenic proteins in fungal extracts with mol. mass ranging between 14 and 70 kDa on IgE‐specific immunoblot using pooled sera of patients allergic to Fusarium. A 45‐kDa protein was found to be common among these fungi on immunoblot with patients as well as with rabbit antibodies. F. solani CF extract contained more antigenic/allergenic proteins than F. solani CE. It was concluded that F. solani CF shared several antigenic/allergenic components with CE of other common fungi. This fact needs to be taken into account when fungal extracts are used in diagnosis and immunotherapy of allergic patients.

Purification and Characterization of a Major Cross-Reactive Allergen from Epicoccum purpurascens

Background:Epicoccum purpurascens (formerly nigrum) (EP), is a ubiquitous saprophytic mould found both indoors and outdoors. Several studies have reported sensitization to EP in 5–7 % of different populations worldwide. The diagnosis of mould allergy requires a standardized fungal extract that contains all its important allergenic proteins. The crude allergen extract from EP was standardized earlier, however none of its allergens have been purified. Methods: A major allergen from spore-mycelia extract of EP was purified using concanavalin A (Con A) Sepharose chromatography, gel filtration and electro-elution. The allergen isolated was characterized for its IgE-binding ability and cross-reactivity with five well-known allergenic fungi by ELISA and immunoblot. Results: A 33.5-kD glycoprotein allergen of EP, Epi p 1, was purified to homogeneity. All the EP allergic patients’ sera tested recognized this protein. Periodate modification of Epi p 1 showed partial loss in IgE binding while proteinase K treatment caused complete loss in binding to IgE. Dose-dependent inhibition in binding of rabbit anti Epi p 1 antibodies was obtained with Epi p 1, Aspergillus fumigatus, Alternaria alternata, Curvularia lunata, Cladosporium herbarum and Fusarium solani in ELISA. Rabbit antibodies to all the above five fungi recognized Epi p 1 in immunoblot, confirming that Epi p 1 shares common epitopes with the fungi tested. Conclusion: A major glycoprotein allergen of 33.5 kD was purified from EP which cross-reacts with other fungi. Hence this glycoprotein can be exploited to reduce the panel of allergen extracts used for therapy of mould allergy.

Purification and characterization of a cross-reactive 45-kD major allergen of Fusarium solani.

Background:Fusarium solani (FS) is an important source of fungal allergen. A 45-kD major allergen of FS showed reactivity with patients’ sera sensitive to many fungi. Objectives: To purify and characterize a 45-kD common allergenic protein from FS, which may be useful for the diagnosis of and therapy for fungal allergy. Methods: FS culture filtrate extract was separated on SDS-PAGE; 45-kD protein was electroeluted and purified on C18 column using reverse-phase high-pressure liquid chromatography (rpHPLC). The purified protein was functionally and biochemically characterized by in vitro and in vivo methods. Results: The 45-kD protein showed a single peak on rpHPLC. The N-terminal amino acid sequence of this protein did not show homology to enolase or known fungal proteins. It showed cross-reactivity with Epicoccum nigrum, Curvularia lunata, Cladosporium herbarum and Alternaria alternata by ELISA and ELISA inhibition using rabbit antibodies raised against these fungi. IgE ELISA inhibition with patients’ sera positive to different fungi demonstrated allergenic cross-reactivity of the 45-kD protein with other fungal extracts. This 45-kD protein released a significant amount of histamine in FS-allergic patients. Conclusion: A cross-reactive 45-kD allergenic/antigenic protein was purified to homogeneity and characterized. It has prospects for use in allergen therapy.