[An epidemiologic study on prevalence of allergen-specific IgE antibodies in school children living in rural area of Saitama Prefecture, Japan].

BACKGROUND: In order to estimate the sensitization to inhalant allergen in childhood, prevalence of allergen-specific IgE antibodies was retrospectively surveyed on sera from school children aged from 9 to 15 living in rural area of Saitama prefecture, Japan, in 2001 and five years ago from 2001 (1996). METHODS: Allergen-specific IgE antibodies against Japanese cedar pollen (JCP), ragweed, sweet vernal grass, Dermatophagoides pteronyssinus (Dp), house dust (HD) and cat dander were examined on sera from 79 school children aged from 9 to 11 (group A) and 119 school children aged from 12 to 15 (group B) collected in 2001, and sera from 117 school children aged from 9 to 11 and 56 school children aged from 12 to 15 collected in 1996. Furthermore, a questionnaire survey about diagnosis of any allergic diseases and symptoms associated with the diseases was conducted on the same subjects in 2001. RESULTS: On the survey from school children, group A and B, collected in 2001, percentage of positive cases for allergen-specific IgE antibodies against any of 6 allergens were 49 in both of the groups for JCP, 10 and 12 for ragweed, 18 and 19 for sweet vernal grass, 39 in both of the groups for Dp, 42 in both of the groups for HD, 23 and 14 for cat dander, respectively. On the survey from school children collected in 1996, there is no significant difference from the percentage of positive cases for allergen-specific IgE antibodies against each allergen in 2001. Among the subjects followed-up on the survey in both of the years, the percentage of positive cases for allergen-specific IgE antibodies against each of six allergens on the survey in 2001 increased as compared with those in 1996. As a result of questionnaire survey in 2001, percentage (70: group A, 89: group B) of positive cases for allergen-specific IgE antibodies against any 6 allergens in the subjects with any symptoms associated with allergic diseases, but not diagnosed, was significantly higher than the percentage (34: group A, 48: group B) of that in the subjects without any symptoms associated with the diseases. CONCLUSION: Further prospective study is required to clarify a cause of allergic diseases in childhood, and it should be taken precautionary measures during childhood against sensitization with inhalant allergen to prevent allergic diseases in surveyed area.

Dilution method to refold bacterially expressed recombinant Der f 2 and Der p 2 to exhibit the secondary structure and histamine-releasing activity of natural allergens.

BACKGROUND: Structurally refolded recombinant forms of major house dust mite group 2 allergens, Der f 2 and Der p 2, expressed in Escherichia coli, were prepared by solubilizing the insoluble products with urea and subsequently dialyzing against buffer. In this study, we determined conditions for refolding the urea-denatured recombinant Der f 2 and Der p 2 by one-step dilution as an alternative to dialysis, which requires several steps of handling and much time and cost. METHODS: The insoluble bacterial product containing recombinant Der f 2 was solubilized with a buffer containing 8 M urea, and the solution was diluted to various urea concentrations. The refolding efficiency in each dilution was estimated from the height of the peak corresponding to the folded recombinant Der f 2 and that containing the aggregated form on anion exchange chromatography. The structure and allergenicity of the purified recombinant Der f 2 and Der p 2 refolded using the dilution method were analyzed based on circular dichroism and a basophil histamine-releasing assay, respectively. RESULTS: Although the refolding efficiency decreased as the urea concentration in the dilution increased, experimental conditions whereby the protein and urea concentrations in the dilution were less than 0.5 mg/ml and 0.8 M, respectively, achieved maximum refolding efficiency. The recombinant allergens prepared by the dilution method exhibited the secondary structure and histamine-releasing activity of natural allergens purified from mite culture. CONCLUSIONS: The dilution method established in this study is more convenient in terms of handling, time, and cost than the dialysis method and will be useful for large-scale production and for the preparation of numbers of mutants to analyze IgE epitopes.

Maturation of the activities of recombinant mite allergens Der p 1 and Der f 1, and its implication in the blockade of proteolytic activity.

Recombinant pro-Der p 1 expressed in yeast Pichia pastoris was convertible into the prosequence-removed mature Der p 1 with full activities of cysteine protease and IgE-binding with or without N-glycosylation of the mature sequence as well as pro-Der f 1. The active recombinant variants will be the basis for various future studies. The major N-terminus of pro-Der p 1 with low proteolytic activity was the putative signal-cleavage site, while that of pro-Der f 1 contained not only the equivalent site but also 21 residues downstream, and pro-Der f 1 retained significant activity. Contribution of the N-terminal region of the Der p 1 prosequence including an N-glycosylation motif on effective inhibition of proteolytic activity of pro-Der p 1 was suggested.