Michiko Haida

Allergens of the house dust mite Dermatophagoides farinae—Immunochemical studies of four allergenic fractions

Analysis of the allergenic components of the house dust mite Dermatophagoides farinae was performed. Four different types of proteins reactive with human anti-mite IgE antibodies were identified as four distinct radioprecipitins in radioimmunoelectrophoresis. These were designated as mite allergen reactive with human IgE antibodies (Me) Me 1, Me 2, Me 3, and Me 4 according to their electrophoretic mobility from the slower to the faster. Me 1 and Me 2 were the first and the second prevailing allergens, respectively, in 133 atopic patients subjected to the study. Me 1, on purification by gel filtration and anion exchange chromatography, elicited a single radioprecipitin by radioimmunoelectrophoresis. Me 1 also elicited a single band in sodium dodecyl sulphate-polyacrylamide gel electrophoresis and a single peak in high-performance liquid chromatography. Both methods elicited identical values of 17,000 daltons for the molecular weight of Me 1. The isoelectric point of Me 1 was suggested to be 8.0. Me 1 elicited higher skin reactions compared to that obtained by the crude mite extract when it was assessed in seven atopic patients by serial titration intradermal skin testing.

Characterization of the proteases in the crude mite extract.

Characterization of the proteases was performed in the crude mite extract fractionated by Sephacryl S-200 gel filtration. Three peaks of protease activities were detected in the fractions. From the results of substrate specificity and susceptibility to the inhibitors, PK.1 protease (about 60 kD) is suggested to be a trypsin-like protease of mites. From the results of susceptibility to various agents, PK.2 (about 30 kD) and PK.3 (about 20 kD) proteases may be cysteine proteases, e.g., papain and cathepsin B. PK.3 protease existed in the precipitate of 60% ammonium sulfate fractionation. The data in the present study suggest the possibility that Dermatophagoides farinae I allergen might be a cysteine protease probably derived from the gastrointestinal tract of the house dust mite.

Isolation of Cysteine Protease in the Crude Mite Extract, Dermatophagoides farinae

In order to study the relationship between cysteine protease and Der f I, which is one of the major allergens in the mite, Dermatophagoides farinae, isolation of cysteine protease was attempted using various column chromatographies. Both the potent cysteine protease activity and the allergenic activity were detected in the same fractions by anion exchange chromatography on a DEAE-Sephacel, gel chromatographies and chelating Sepharose 6B chromatography. In the double immunodiffusion test, the finally isolated fraction and rabbit anti-Der f I sera reacted to give a single precipitation line which fused completely with the precipitation line formed by Der f I and anti-Der f I sera. Sequence analysis for the first 10 N-terminal amino acids from cysteine protease and Der f I were identical. These results strongly suggest that cysteine protease of mites may be Der f I allergen and that measuring cysteine protease activity may possibly become a beneficial method for detecting Der f I allergens.

IgG1 Antibodies to House Dust Mite (Dermatophagoides farinae) and Late Asthmatic Response

Thirteen asthmatic patients sensitive to mite were challenged by inhalation of an extract of mites (Dermatophagoides farinae). Seven showed dual bronchial reactions and 5 showed isolated immediate responses. No patient showed an isolated late reaction. Six of seven patients with dual reaction had higher IgG1 antibodies than the 5 patients with isolated immediate reaction when examined before the challenge. A similar result was obtained in terms of levels of immune complex. IgE, IgG4 and total IgG antibodies were not predictive for late reaction. These results suggest that there is a close correlation of the presence of high IgG1 antibodies with a propensity to develop late asthmatic responses. The meaning of this observation is discussed.

Allergens of the house dust mite Dermatophagoides farinae. II. Immunological characterization of four allergenic molecules.

In a previous study, we detected four major allergens from the house dust mite extract prepared from the body of Dermatophagoides farinae, employing radioimmunoelectrophoresis. The four allergenic proteins were designated as Me1, Me2, Me3 and Me4. The most prevalent allergen was found to be Me1 and its molecular weight was determined to be 17,000 daltons. In this study, the molecular weight of the second prevalent allergen, Me2, was determined to be 27,000 daltons using the immunoblotting method. The molecular weights of the other two major allergens, Me3 and Me4, were also estimated.

Synthesis of Biologically Active Recombinant Der f II

A cDNA library corresponding to mite protein was screened employing anti-Der f II antibody. Two possible clones containing plasmids pFL1 and pFL11 were obtained. The two plasmids had insertions of about 500 basepairs. The DNA sequences of the two insertions were determined, from which the amino acid sequences were deduced. The amino acid sequence of the purified native Der f II protein could be determined to 45 residues from the N terminus. As a result of comparison, we concluded that the cDNAs prepared from live Dermatophagoides farinae mite corresponded to the mite allergen Der f II. The recombinant Der f II was biologically active.

Inhibition of Allergen-Induced Bronchoconstriction in Sensitized Guinea Pigs by Orally Administered Allergen

Crude mite extract (CME) was orally administered to guinea pigs sensitized to CME. It was shown that such treatment reduces the bronchoconstrictive response upon allergen provocation. Isolated tracheae taken from guinea pigs orally administered CME allergen showed less contraction in response to CME as compared to those obtained from sensitized but not orally treated animals. The oral administration of allergens seemed to attenuate the bronchial hyperresponsiveness of sensitized animals to a non-specific chemical stimulus (histamine). IgE antibodies titrated by 8 days passive cutaneous anaphylaxis, and IgG1 and IgG2 antibodies measured by ELISA were comparable in the sera obtained from animals before and after CME treatment.