PubMed Health⌕ Search

Biomedical subjects

M D Chapman

Publications and source records attributed to M D Chapman.

169 records · Page 10Linked to original sources
PubMedDate not supplied

Identification of a novel cat allergen--cystatin.

BACKGROUND: Cat allergen is an important cause of sensitization among children with asthma in Japan. Although there is good evidence that cats produce other allergens, only one major allergen, Fel d 1, has been studied in detail. AIMS: To identify and define the molecular structure of the other potential cat allergens. METHODS: A cat skin cDNA library was screened using IgE antibodies to cat dander and selected clones were sequenced and expressed. RESULTS: One cDNA clone contained an open reading frame encoding a 98-amino acid residue protein. Sequence homology searches revealed a high degree of identity with bovine and human cystatin A, 79 and 75%, respectively. This cat cystatin clone contained the conserved cysteine protease motif and two of three lipocalin motifs. By plaque immunoassay, 60-90% of cat allergic sera had IgE Ab to cat cystatin. This cysteine protease inhibitor motif was partially conserved in dog allergens, Can f 1 and Can f 2, which are lipocalins. Recombinant cystatin was produced in Escherichia coli cells and purified as an 11-kD protein, corresponding to the predicted MW of cystatin. The structure of cat cystatin was modeled on human cystatin B using the SWISS-MODEL. CONCLUSION: A newly identified allergen, cystatin, has been cloned from cat skin and is a member of the cysteine protease inhibitor family.

Allergens↗
PubMedDate not supplied

Sequence polymorphisms and antibody binding to the group 2 dust mite allergens.

BACKGROUND: The group 2 allergens Der p 2, Der f 2 and Eur m 2 are 14-kD proteins with > 80% sequence identity. Isoforms within each genus have been identified which differ by 3-4 amino acids. The aim of this study was to investigate the importance of these substitutions to antibody binding. METHODS: Recombinant allergens were expressed and purified from Escherichia coli. ELISA and skin testing were used to evaluate antibody binding. Molecular modeling of the tertiary structure was preformed to examine the location of substitutions. RESULTS: The three Der f 2 isoforms and two of three of the Der p 2 isoforms reacted with all monoclonal antibodies (mAb). Der p 2.0101, the isoform with aspartate at position 114, bound all mAb except 1D8. Substitution of asparagine for aspartate restored binding of rDer p 2.0101 to mAb 1D8 and increased the correlation coefficient for IgE binding from 0.72 to 0.77. The three Der p 2 isoforms showed comparable skin test reactivity to nDer p 2 and commercial extract. rEur m 2.0101 bound to all mAb except 7A1 and when compared with rDer p 2 for IgE binding, r(2) = of 0.58 (n = 72). Lep d 2 did not react with mAb or with Dermatophagoides spp. allergic sera. Modeling revealed that Eur m 2, Lep d 2 and Tyr p 2 retain the tertiary fold of Der p 2 and the substitutions are on the surface. CONCLUSIONS: mAb could distinguish isoform substitutions. IgE binding showed a good correlation among all isoforms, thus the recombinant allergens are useful for diagnosis.

Allergens↗
PubMedDate not supplied

Molecular cloning of German cockroach (Blattella germanica) allergens.

Allergens produced by cockroaches (CRs) are an important cause of IgE antibody responses and asthma. Using molecular cloning and nucleic acid hybridization techniques, we have identified and sequenced several important allergens produced by the German CR (Blattella germanica) and studied their expression in the American CR (Periplaneta americana). Principal allergens include Bla g 2 (36-kD protein) and Bla g 4 (21-kD protein), to which 60-70% of CR-allergic patients make IgE antibodies. Bla g 2 is only expressed by B. germanica, whereas DNA encoding Bla g 4 is present in P. americana, but is not transcribed into mRNA. Sequence homology searches have identified Bla g 2 as an aspartic protease and Bla g 4 as a calycin. Other CR allergens that have been cloned include a glutathione transferase and a troponin. These studies will enable recombinant allergens to be expressed and used to investigate the role of CR allergens in asthma.

Allergens↗
PubMedDate not supplied

Defined epitopes: in vivo and in vitro studies using recombinant allergens.

BACKGROUND: The aim of the studies was to investigate the biologic activity of recombinant cockroach and mite allergens and their potential for use in diagnosis and treatment of allergic disease. METHODS: Cockroach allergens Bla g 2, Bla g 4 and Bla g 5 and mite group 5 allergens were produced in bacterial expression vectors and used for immediate skin and serum IgE antibody tests. RESULTS: The cockroach allergens showed very good skin test reactivity in allergic patients, giving positive reactions at 10(-2)-10(-5) microg/ml; controls were negative at 10(0) microg/ml. These reactions correlated with serum IgE antibody results. The prevalence of reactivity to group 5 mite allergens varied with exposure. There was a high prevalence (70%) of sensitization to Blomia tropicalis allergen, Blo t 5, among patients from Brazil and Singapore, whereas < 20% of patients from Charlottesville, US and Manchester, UK gave positive skin tests to Blo t 5 (p < 0.001). CONCLUSIONS: The results show that recombinant allergens retain biologic activity and suggest that cocktails of two to four recombinant allergens could be used for diagnostic or therapeutic purposes. The phased introduction of recombinant allergens should improve the management of allergic disease.

Allergens↗