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OBJECTIVES: Bedding in childcare centers (CCCs) can hold house dust mite (HDM) allergens. This study examined whether HDM allergen levels can be reduced through the distribution of an educational newsletter on bedding control to parents of CCC children in Korea. METHODS: All 38 CCCs were measured for Der 1 (sum of Der f 1 and Der p 1) concentrations on classroom floors and bedding before the intervention. Educational newsletters on children’s bedding control were sent to 21 CCCs by mail, and teachers were asked to distribute the newsletters to the parents of the children (intervention group). The remaining 17 CCCs were not sent newsletters (control group). The measurement of Der 1 concentrations in 38 CCCs was repeated after the intervention. Dust samples were collected with a vacuum cleaner and analyzed using enzyme-linked immunosorbent assay methods. RESULTS: The Der 1 concentrations on the bedding were significantly higher than those on the floors in 38 CCCs at baseline (p<0.05). Although changes of the Der 1 concentrations for the control group (n=17) were not significant, Der 1 concentrations for the intervention group (n=21) decreased significantly from 2077.9 ng/g dust to 963.5 ng/g dust on the floors and from 3683.9 ng/g dust to 610.4 ng/g dust on bedding (p<0.05). CONCLUSIONS: The distribution of educational newsletters on bedding control to parents may be an effective means of controlling HDMs in CCCs.
Subject(s)
Child , Humans , Allergens , Dust , Enzyme-Linked Immunosorbent Assay , Korea , Parents , Periodicals as Topic , Postal Service , Pyroglyphidae , VacuumABSTRACT
OBJECTIVES: Bedding in childcare centers (CCCs) can hold house dust mite (HDM) allergens. This study examined whether HDM allergen levels can be reduced through the distribution of an educational newsletter on bedding control to parents of CCC children in Korea. METHODS: All 38 CCCs were measured for Der 1 (sum of Der f 1 and Der p 1) concentrations on classroom floors and bedding before the intervention. Educational newsletters on children’s bedding control were sent to 21 CCCs by mail, and teachers were asked to distribute the newsletters to the parents of the children (intervention group). The remaining 17 CCCs were not sent newsletters (control group). The measurement of Der 1 concentrations in 38 CCCs was repeated after the intervention. Dust samples were collected with a vacuum cleaner and analyzed using enzyme-linked immunosorbent assay methods. RESULTS: The Der 1 concentrations on the bedding were significantly higher than those on the floors in 38 CCCs at baseline (p<0.05). Although changes of the Der 1 concentrations for the control group (n=17) were not significant, Der 1 concentrations for the intervention group (n=21) decreased significantly from 2077.9 ng/g dust to 963.5 ng/g dust on the floors and from 3683.9 ng/g dust to 610.4 ng/g dust on bedding (p<0.05). CONCLUSIONS: The distribution of educational newsletters on bedding control to parents may be an effective means of controlling HDMs in CCCs.
Subject(s)
Child , Humans , Allergens , Dust , Enzyme-Linked Immunosorbent Assay , Korea , Parents , Periodicals as Topic , Postal Service , Pyroglyphidae , VacuumABSTRACT
Objective To construct and express a chimeric gene with T-/B-cell epitopes of the major allergen group 1 from Dermatophagoides farina(Der f 1). Methods Two chimeric genes,Der f 1A and Der f 1B,were synthesized as B1-T1-B2-T2-B3-T3-B4-T4-B5-T5-B6 and B1-B2-B3-B4-B5-B6-T1-T2-T3-T4-T5 pattens. Two recombinant vectors,pET-28a(+)-Der f 1A and pET-28a(+)-Der f 1B,were constructed for prokaryotic expression. These chimeric genes were induced by 1 mmol/L IPTG (final concentration),digested with restriction enzymes and sequenced. The chimeric proteins were analyzed by SDS-PAGE and Western blotting. Results After digestion by restriction enzymes and sequencing,the recombinant vectors were constructed successfully. The specific bands for chimeric proteins were visible by SDS-PAGE and Western blotting,suggesting that these proteins were purified successfully. Further analyses were performed for IgE-binding properties of Der f 1A and Der f 1B to sera from patients sensitized to house dust mite. Compared with the parental allergens Der f 1,Der f 1A and Der f 1B had reduced IgE-binding capacity(both P0.05). Conclusion Two chimeric proteins are expressed successfully,which contain T-/B-cell epitopes of Der f 1 and provide a basis for specific immunotherapy.
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PURPOSE: The environmental factors human rhinoviruses (HRVs) and house dust mites (HDMs) are the most common causes of acute exacerbations of asthma. The aim of this study was to compare the chemokine production induced by HRVs in airway epithelial cells with that induced by other respiratory viruses, and to investigate synergistic interactions between HRVs and HDMs on the induction of inflammatory chemokines in vitro. METHODS: A549 human airway epithelial cells were infected with either rhinovirus serotype 7, respiratory syncytial virus (RSV)-A2 strain, or adenovirus serotype 3 and analyzed for interleukin (IL)-8 and regulated on activation, normal T-cell expressed and secreted (RANTES) release and mRNA expression. Additionally, activation of nuclear factor (NF)-kappaB and activator protein (AP)-1 were evaluated. The release of IL-8 and RANTES was also measured in cells stimulated simultaneously with a virus and the HDM allergen, Der f1. RESULTS: HRV caused greater IL-8 and RANTES release and mRNA expression compared with either RSV or adenovirus. NF-kappaB and AP-1 were activated in these processes. Cells incubated with a virus and Der f1 showed an increased IL-8 release. However, compared with cells incubated with virus alone as the stimulator, only HRV with Der f1 showed a statistically significant increase. CONCLUSIONS: IL-8 and RANTES were induced to a greater extent by HRV compared with other viruses, and only HRV with Der f1 acted synergistically to induce bronchial epithelial IL-8 release. These findings may correspond with the fact that rhinoviruses are identified more frequently than other viruses in cases of acute exacerbation of asthma.
Subject(s)
Humans , Adenoviridae , Antigens, Dermatophagoides , Arthropod Proteins , Asthma , Chemokine CCL5 , Chemokines , Cysteine Endopeptidases , Epithelial Cells , Interleukin-8 , Interleukins , NF-kappa B , Pyroglyphidae , Respiratory Syncytial Viruses , Rhinovirus , RNA, Messenger , Sprains and Strains , T-Lymphocytes , Transcription Factor AP-1 , VirusesABSTRACT
Background: House dust mite (HDM) allergen quantification in house dust samples before and after the allergen elimination is one means of convincing the target population about the health benefits of allergen removal from their environment. Objective: To produce local reagents for quantification of Der f 1 (major allergen of Dermatophagoides farinae) in dust samples from houses of HDM allergic Thai patients. Methods: Recombinant Der f 1 was used for immunization of a BALB/c mouse for hybridoma production. Polyclonal antibody (PAb) to whole body extract of D. farinae was prepared from an immunized rabbit. A sandwich ELISA (MAb-allergen-PAb) was used, in comparison with the commercialized reagents (Indoor Biotechnology, UK), to quantify Der f 1 in dust samples. Results: Two hybridoma clones, Df1-1 and Df1-2, were established. Their secreted MAbs (MAbDf1-1 and MAbDf1-2, respectively) bound to the homologous antigen as well as native Der f 1 and a crude extract of D. farinae. Epitopes of MAbDf1-1 and MAbDf1-2 were located at amino acid residues 206NSQHYGISNYCQ217 and 283DYW---NSWD-WGDSG298 of Der f 1. MAbDf1-1 had higher affinity to Der f 1 than the MAbDf1-2. A sandwich ELISA (MAbDf1-1-allergen-PAb) and commercialized reagents (MAb1-allergen-MAb2 sandwich ELISA) were used in comparison for quantification of Der f 1 in 42 dust samples collected from bedrooms and living rooms of 21 houses of the HDM allergic patients. All of the 42 dust samples measured by both ELISAs had the Der f 1 levels higher than 2 mg per gram of fine dust which is the HDM allergy sensitizing level. In addition, Der f 1 levels in 41 samples (except 1 sample from a living room) measured by the MAbDf1-1-PAb and MAb1-MAb2 sandwich ELISAs were higher than 10 mg per g of dust which is the morbidity level of HDM allergen. The local sandwich ELISA showed a high coefficient correlation (r = 0.91) in measuring known amounts of recombinant and native Der f 1. The results indicate that the reagents produced in the present study can be used for measuring the environmental levels of HDM Der f 1. The assay can also be used for standardization of the HDM extract for monitoring patient's allergenic status or for immunotherapeutic purpose.
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Background: Different mattress materials may affect the accumulation of allergens. Objective: To compare the amount of group 1 dust mite allergens (Der p1 + Der f1) on mattresses made of different kinds of materials before and after use. Methods: Sixty new mattresses made of kapok, synthetic fiber, coconut fiber and sponge-like polyurethane, were placed in the house officers’ dormitory at Siriraj hospital, Thailand. The dust samples were collected before (0), 1, 2, 3, 6, 9 and 12 months after the mattresses were used. Group 1 dust mite allergens were analyzed using two-site monoclonal antibody ELISA. Results: Der f1 made up 86.7 % of group 1 allergens found in the matress dust. After the 2nd month, only the mean level in sponge-like polyurethane mattress was under 2 µg/g dust (sensitized level). At the 6th month, the mean levels were 13.1 in coconut, 21.7 in kapok and 17.3 µg/g dust in synthetic fiber, all of which were more than 10 µg/g dust (symptomatic level). At the 9th month, the level in sponge-like polyurethane mattress was increased to 11.2 µg/g. At 12th month the level in coconut fiber, sponge-like polyurethane synthetic fiber and kapok mattresses were 20.2, 22.4, 28.9 and 32.2 µg/g dust respectively. Conclusions: The accumulation rate in kapok and synthetic mattresses was significantly higher than coconut and sponge-like polyurethane mattresses. The mean level of group 1 mite allergens exceeded 10 µg/g dust after the 6th month of use in coconut fiber, kapok and synthetic fiber and at the 9th month in sponge-like polyurethane mattress.
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Objective:To construct the plant expression vector of Der f1 allergen of Dermatophagoides pteronyssinu and expression in tobacco lamina.Methods:The Der f1 gene was amplified from the glycerin bacterium which contained pET28a(+)-Der f1 plasmid,cloned into the pMD 19-T plasmid,and then sequenced.The Der f1 gene was digested by ClaⅠand SalⅠ,and cloned into potato virus X (PVX) to construct plant expression vector PVX-Der f1,and then was transformed agrobacterium tumefaciens.The positive one was selected to infect tobacco lamina for expressing target protein.The protein was identified and analysed by SDS-PAGEand Western blot.Results:Digestion and sequence analysis confirmed that the plant expression vector was correct,and the SDS-PAGE and Western blot results showed that the molecular weight of the protein was about 34M_r and it could specific binding with positive serum.Conclusion:The plant expression vector of Der f1 is successfully constructed and the recombinant protein is also produced.
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Our objective was to clone, express and characterize adult Dermatophagoides farinae group 1 (Der f 1) allergens to further produce recombinant allergens for future clinical applications in order to eliminate side reactions from crude extracts of mites. Based on GenBank data, we designed primers and amplified the cDNA fragment coding for Der f 1 by nested-PCR. After purification and recovery, the cDNA fragment was cloned into the pMD19-T vector. The fragment was then sequenced, subcloned into the plasmid pET28a(+), expressed in Escherichia coli BL21 and identified by Western blotting. The cDNA coding for Der f 1 was cloned, sequenced and expressed successfully. Sequence analysis showed the presence of an open reading frame containing 966 bp that encodes a protein of 321 amino acids. Interestingly, homology analysis showed that the Der p 1 shared more than 87 percent identity in amino acid sequence with Eur m 1 but only 80 percent with Der f 1. Furthermore, phylogenetic analyses suggested that D. pteronyssinus was evolutionarily closer to Euroglyphus maynei than to D. farinae, even though D. pteronyssinus and D. farinae belong to the same Dermatophagoides genus. A total of three cysteine peptidase active sites were found in the predicted amino acid sequence, including 127-138 (QGGCGSCWAFSG), 267-277 (NYHAVNIVGYG) and 284-303 (YWIVRNSWDTTWGDSGYGYF). Moreover, secondary structure analysis revealed that Der f 1 contained an a helix (33.96 percent), an extended strand (17.13 percent), a ß turn (5.61 percent), and a random coil (43.30 percent). A simple three-dimensional model of this protein was constructed using a Swiss-model server. The cDNA coding for Der f 1 was cloned, sequenced and expressed successfully. Alignment and phylogenetic analysis suggests that D. pteronyssinus is evolutionarily more similar to E. maynei than to D. farinae.