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Studies on mold allergens.

AIlergen extracts have been used for the diagnosis and treatment of allergy for over 70 years, millions of patients being involved each year. Despite this, little is known concerning the nature of these materials, either in terms of allergen and antigen content or in terms of their ability to desensitize patients. Moreover, allergen extracts prepared by different procedures and marketed by different companies vary considerably in composition and content of allergen materials; some may contain irritants and extraneous materials. Obviously such poorly standardized extracts will influence diagnostic results as well as proposed immunotherapy Pollen has been widely studied as an aeroallergen for many years, but far less is known about fungal spores, even though these are present in the air in concentrations in excess of those of pollen grains. A recent survey in North American homes revealed that Cladosporium is one of the species most often encountered both indoors and outdoors. Other common molds isolated from homes are Alternaria, Penicillium, Helminthosporium and Aspergillus. It has long been established that respiratory allergic symptoms can be caused by inhalation of air-borne mold spores. The present therapy attempts to desensitize patients by injections of crude extracts of the appropriate molds.

A programme was eshtablished under the leadership of Hari M. Vijay, in the Drug Toxicology Division, on mold allergens. This is providing the basic scientific knowledge and reference materials that will eventually permit regulations to be drawn up governing commercial preparations being sold for desensitization therapy.

Purification of A. alternata crude extract by gel-filtration on Sephadex G-100 led to the discovery that A.alternata produced, in addition to the major allergen protein mixture (94, 77, 66 and 59 Kda MW by immunoblotting) a related cross-reacting protein (16 and 18 Kda MW), antigenic and immunogenic for the production of IgG antibodies but with little or no allergenic activity (hypoallergen). Cross-reactivity between the major and hypoallergen components was observed in immunoblotting and crossed-immunoelectrophoresis and fused-rocket immunoelectrophoresis using rabbit antisera to the purified major and hypoallergens, suggesting that the hypoallergen carries a dominant epitope of the major allergen. From the point of view of immuno-therapy, it is evident the antigenicity of the hypoallergen component, its cross-reactivity with the major allergen component, its low allergenicity and its ease of purification are all excellent characteristics for a therapeutic agent capable of desensitizing patients with minimal risk of anaphylaxis. Moreover, the hypoallergen could also be used for in vitro (by radio-allergosorbent assay) diagnosis of mold allergenic patients.

A better understanding of the structure of the allergen and its gene would allow design of non-allergenic fragments carrying the antigenic determinant similar to the hypoallergen. These should be better therapeutic agents than the crude extracts in use at present or the purified and modified allergens now being developed. Currently, mRNA is being isolated from the culture of A. alternata, and production of monoclonal antibodies of the IgE and IgG classes to the major and hypoallergen is in process. Cloning of the allergens will be attempted using the sequence information and the specific antisera (monoclonal antibodies to the major and hypoallergen).

In 1981, the Allergen Standardization Subcommittee of the International Union of Immunological Societies undertook the production of serveral allergen extracts that would meet WHO specifications as International Reference Preparations. Since 1983, an International Collaborative Study has been carried out on candidate Alternaria extracts. Major difficulties were encountered in source materials. For example, extracts were prepared from strains which could not be verified as being Alternaria, some strains were identified as Alternaria sp. but not specifically Alternaria alternata. This is important as this species is a cause of bronchospasm in the significant number of patients. It is essential therefore that the A. alternata isolates included in a reference preparation be properly characterized for their purity, allergenic potency and presence of major allergenic components. In view of the problems with the source materials, it was initially concluded that an International Reference Preparation of Alternaria sp. could be obtained but not specifically one of Alternaria alternata. This obviously would present problems for allergen standardization of this organism. The group therefore undertook further investigation of four well characterized isolates of A. alternata, and have reported that laboratory cultures of these isolates, grown on synthetic revised tobacco medium, were found suitable for a reference preparation. The batch-to-batch reproducibility of one of the isolates was investigated by biochemical and immunological techniques. It was found that a reference preparation of A. alternata, suitable for standardization purposes, can be obtained from any batch of the stable isolate of this organism (isolate 34-016), against which the potency of commercial extracts can be adequately measured.
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Author:Vijay, Hari M.
Publication:Canadian Chemical News
Date:Nov 1, 1989
Words:764
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