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As a method of evaluating the effect of inactivators on allergens while suppressing the effect of inactivator on the assay, we developed new dot-blot method that combines immunostaining and protein detection methods. This method is useful for evaluating whether the inactivator can inactivate allergens rather than removing them from the assay., (© The Author(s) 2021. Published by Oxford University Press on behalf of Japan Society for Bioscience, Biotechnology, and Agrochemistry.)

Der f 1, a major house dust mite allergen and member of the papain-like cysteine protease family, can provoke immune responses with its proteolytic activity. To understand the role of Der f 1 in inflammatory immune responses, we studied the mechanism of the regulation of interleukin (IL)-8 expressions in human basophilic cell KU812 by proteolytically active recombinant Der f 1. Not only production of IL-8 mRNA was induced but also the DNA binding activity of activator protein-1 (AP-1) and phosphorylation of NF-κB p65 were increased in Der f 1-treated KU812. Furthermore, Der f 1 induction of IL-8 expression was sensitive to pharmacological inhibition of ERK and p38 mitogen activated protein kinase (MAPK) pathways. Der f 1 also activated ERK and p38 MAPK phosphorylation and rapidly induced reactive oxygen species (ROS) production. The antioxidant N-acetyl-cysteine (NAC) inhibited phosphorylation of ERK, but not p38, suggesting that secretion of IL-8 in KU812 cells treated with Der f 1 is dependent on ROS, ERK MAPK and p38 MAPK. We describe the mechanism of Der f 1-induced IL-8 secretion from human basophilic cells, which are thought to be important for allergic inflammation independent of IgE antibodies. These findings improve our understanding of the inflammatory immune response in human basophils to protease allergens. [ABSTRACT FROM AUTHOR]

In test systems for diagnostics of type I allergy, recombinant allergens in native conformation are used, because immunoglobulins E (IgE), responsible for the development of this type of allergy, recognize conformational epitopes of protein allergens. To obtain recombinant allergens of the Dermatophagoides farinae house dust mites (HDM), Der f 1 and Der f 2, two systems of heterological expression were used: Escherichia coli and Nicotiana benthamiana plants. IgE from sera of children allergic to HDM were shown to recognize the recombinant Der f 2 protein synthesized in both E. coli and N. benthamiana plants, as well as the recombinant Der f 1 protein produced in N. benthamiana plants, while mature form of Der f 1 produced in E. coli did not interact with IgE. [ABSTRACT FROM AUTHOR]

Amino acid sequence variations have possible influences on the allergenicity of allergens and may be important factors in allergen standardization. This study was undertaken to investigate the sequence polymorphisms of group 1 and 2 allergens from Korean isolates of the house dust mites Dermatophagoides farinae and D. pteronyssinus. cDNA sequences encoding group 1 and 2 allergens were amplified by RT-PCR and compared the deduced amino acid sequences. Der f 1.0101, which appeared in 64.0 % of the 50 sequences analyzed, was found to be predominant. Among the Der p 1 sequences, Der p 1.0102 and 1.0105 were predominant (58 %). Among the Der f 2 sequences, Der f 2.0102 (40.7 %) and a new variant with Gly at position 42 (27.8 %) were predominant. The deduced amino acid sequences of 60 Der p 2 clones were examined, and 28 variants with 1-5 amino acid substitutions were found. Interestingly, all of the Der p 2 sequences had Thr instead of Lys at position 49. Two variants (Leu40, Thr49, and Asn114 (26.6 %); Val40, Thr49, and Asn114 (20.0 %)) were found to be the most predominant forms of Der p 2. Der p 1 has a high rate of sporadic substitutions and the group 2 allergens show a more regular pattern with orderly associations of amino acid substitutions. Der f 1 and Der p 2 from Korean mite isolates have unique amino acid sequence polymorphisms. These findings provide important data for house dust mite allergen standardization. [ABSTRACT FROM AUTHOR]

Abstract: Brown-Norway rats (n =113) sensitized and challenged with nDer f 1 allergen were used to examine the contribution of lung sensory nerves to ozone (O3) exacerbation of asthma. Prior to their third challenge rats inhaled 1.0ppm O3 for 8h. There were three groups: (1) control; (2) vagus perineural capsaicin treatment (PCT) with or without hexamethonium; and (3) vagotomy. O3 inhalation resulted in a significant increase in lung resistance (R L) and an exaggerated response to subsequent allergen challenge. PCT abolished the O3-induced increase in R L and significantly reduced the increase in R L induced by a subsequent allergen challenge, while hexamethonium treatment reestablished bronchoconstriction induced by allergen challenge. Vagotomy resulted in a significant increase in the bronchoconstriction induced by O3 inhalation and subsequent challenge with allergen. In this model of O3 exacerbation of asthma, vagal C-fibers initiate reflex bronchoconstriction, vagal myelinated fibers initiate reflex bronchodilation, and mediators released within the airway initiate bronchoconstriction. [Copyright &y& Elsevier]

Dust mites, cockroaches, and pets (cats, dogs) are common in homes worldwide, and many species are the source of potent allergens which cause allergic diseases. These diseases are influenced by genetic predisposition and environmental exposure. Generally, the levels of house dust mite (Der p 1 and Der f 1) and cockroach (Bla g 1, Bla g 2) allergens are used as markers of indoor exposure to arthropods. This article reviews the findings of allergens Der p 1, Der f 1, and Bla g 1 in randomly selected urban households in Zagreb (Croatia) measured from 2006 to 2010 and compares them with exposure to arthropod allergens in other countries. In short, house dust mite allergen levels in Croatian homes are low, but exposure is common; Der p 1 was found in 73 % and Der f 1 in 83 % of the households. By contrast, exposure to cockroach allergen Bla g 1 was both low and uncommon (13 %). Exposure to multiple allergens associated with sensitisation and asthma was not frequent in urban homes in Croatia. However, further studies should include monitoring of both arthropod and pet allergens in high-risk populations in inland and coastal Croatia. They should also investigate a complex dose-response relationship between exposure and sensitisation/asthma development, especially in early childhood. [ABSTRACT FROM AUTHOR]

Pyroglyphid mites are primarily associated with allergen exposure at home; hence the name house dust mites. However, we have found numerous studies reporting pyroglyhid mite levels in public and occupational settings. This review presents the findings of house dust mite allergens (family Pyroglyphidae, species Dermatophagoides) as potential work-related risk factors and proposes occupations at risk of house dust mite-related diseases. Pyroglyphid mites or their allergens are found in various workplaces, but clinically relevant exposures have been observed in hotels, cinemas, schools, day-care centres, libraries, public transportation (buses, trains, taxies, and airplanes), fishing-boats, submarines, poultry farms, and churches. Here we propose a classification of occupational risk as low (occasional exposure to mite allergen levels up to 2 μg g-1), moderate (exposure between 2 μg g-1 and 10 μg g-1), and high (exposure >10 μg g-1). The classification of risk should include factors relevant for indoor mite population (climate, building characteristics, and cleaning schedule). To avoid development or aggravation of allergies associated with exposure to house dust mites at work, occupational physicians should assess exposure risk at work, propose proper protection, provide vocational guidance to persons at risk and conduct pre-employment and periodic examinations to diagnose new allergy cases. Protection at work should aim to control dust mite levels at work. Measures may include proper interior design and regular cleaning and building maintenance. [ABSTRACT FROM AUTHOR]

Immunoassay methods are generally used for measuring of allergenic substances. However, they need special facilities, skilled handling, and time-consuming procedure. In this work, a fiber-optic immunoassay system which could measure allergen by fluorescent intensities of immune complexes formed by allergens and fluorescently labeled antibodies was established. Immune complexes absorbed on the optical fiber probe surface, and excitation light was injected into the probe, then evanescent field is created in the proximity of the probe. The fluorophores were excited by the evanescent light, and fluorescence was detected by a photo diode. The target allergen detected by our system was Der f1 derived from Dermatophagoides farinae that is one of the house dust mite and major source of inhaled allergens. The fluorophore used labeling on detecting antibody was cyanine 5. The system enabled to detect and quantitatively determine of Der f1. The measurement range was from 0.24 to 250 ng/ml, and the result competes with ELISA. The measurement time was 16 min/sample. The immunoassay system was applied to measurement of Der f1 from actual dust samples. Calculated values of Der f1 showed good correlations between the fiber-optic fluoroimmunoassay and ELISA. [ABSTRACT FROM AUTHOR]

Anionic surfaces can interact with a variety of biological molecules and when used on bedding material may have the potential of binding with and sequestering protein allergens such as those produced by dust mites. However, no method exists to evaluate the efficacy of this claim. The purpose of this study was to develop a method using enzyme-linked immunosorbent assay (ELISA) to evaluate the capability of anionic treated cotton bedding sheet material to retain medically important dust mite allergens in direct comparison with nonanionic cotton bedding sheet material from a single manufacturer. Anionic, and nonanionic cotton bedding sheets were challenged singularly with water suspended pulverized dust mite Dermatophagoides farinae, water suspended pulverized dust mite D. pteronyssinus, or water alone as a negative control. All dried inoculums were subjected to four sequential extraction series using water in the first two separate extractions and water with surfactant in the last two separate extractions. All four extractions were assessed by ELISA using monoclonal antibodies specific for medically important protein allergens Der f 1 (D. farinae) or Der p 1 (D. pteronyssinus), respectively. There were a total of 25 replicates for each assessment. Our findings demonstrate that nearly all of the Der f 1 and Der p 1 was released from the nonanionic fabric after the first water extraction. Conversely, most of the Der f 1 and Der p 1 allergens were retained by the anionic fabric after both water extractions and was released only after surfactant was used. Our data indicated that >80% of Der f 1 and Der p 1 was released from the nonanionic fabric after the initial two water extractions while 80% of Der f 1 and Der p 1 was retained by anionic fabric after the same extractions. The method proved to be robust and data was reproducible. Protein extraction using less stringent to high stringent conditions demonstrated the effectiveness of anionic fabric in retaining dust mite proteins that cause allergy. This method can be used to assess other materials while allergists should consider the use of anionic bedding as it effectively retained dust mite allergens Der f 1 and Der p 1 until laundering can remove them from the household. [ABSTRACT FROM PUBLISHER]

Abstract: House dust mite (HDM) allergens are the most common allergens involved in the induction of IgE-mediated hypersensitivity. Recently, epicutaneous sensitization with HDM allergens has been emphasized in the development of atopic dermatitis (AD); however, direct stimulation of canine keratinocytes by mite allergens has not been well investigated. In the present study, we investigated the effects of Der f 1, a major allergen of Dermatophagoides farinae, on cytokine and chemokine gene expression in a canine keratinocyte cell line, CPEK. CPEK constitutively expressed mRNA for TNF-α, IL-12p35, IL-18, GM-CSF, TGF-β, IL-8/CXCL8, TARC/CCL17, CTACK/CCL27 and MEC/CCL28. Of all the cytokines and chemokines investigated in CPEK, transcription levels of GM-CSF, IL-8/CXCL8 and TNF-α mRNA were significantly enhanced by stimulation with Der f 1. The present results suggest that Der f 1 can directly augment inflammatory cytokine and chemokine production from keratinocytes, and may initiate allergic inflammation independently of Type-I hypersensitivity. [Copyright &y& Elsevier]

Abstract: A major house dust mite allergen Der f 1 belongs to the papain-like cysteine protease family. This study investigated whether Der f 1 can cleave the latency-associated peptide (LAP) of transforming growth factor (TGF)-β via its proteolytic activity and activate latent TGF-β. We found that Der f 1 can cleave LAP and induce the activation of latent TGF-β, leading to functional Smad signaling. Importantly, these actions of Der f 1 were inhibited by cysteine protease inhibitor E64 or inactivation of the protease activity by heat. Thus, latent TGF-β may be a direct target of Der f 1 protease activity. [Copyright &y& Elsevier]

Abstract: The group 1 mite allergens Der f 1 and Der p 1 are potent allergens excreted by Dermatophagoides farinae and Dermatophagoides pteronyssinus, respectively. The human immunoglobulin E antibody responses to the group 1 allergens show more cross-reactivity than the murine immunoglobulin G antibody responses, which are largely species specific. Here, we report the crystal structure of the mature form of Der f 1, which was isolated from its natural source, and a new high-resolution structure of mature recombinant Der p 1. Unlike Der p 1, Der f 1 is monomeric both in the crystalline state and in solution. Moreover, no metal binding is observed in the structure of Der f 1 despite the fact that all amino acids involved in Ca2+ binding in Der p 1 are completely conserved in Der f 1. Although Der p 1 and Der f 1 share an extensive sequence identity, comparison of the crystal structures of both allergens revealed structural features that could explain the differences in murine IgG and human IgE antibody responses to these allergens. There are structural differences between Der f 1 and Der p 1 that are unevenly distributed on the allergens'' surfaces. This uneven spatial arrangement of conserved versus altered residues could explain both the specificity and cross-reactivity of antibodies against Der f 1 and Der p 1. [Copyright &y& Elsevier]

Abstract: Endotoxins and allergens represent the major relevant contents of the atmospheric bioaerosol with regard to the triggering and exacerbation of allergic diseases. In this study, mattress concentrations of endotoxin and indoor allergens were measured in three hospitals in the alpine climate of Bavaria and in adjacent homes. Dust was collected from each of 10 mattresses according to a standardized protocol, and endotoxin was analyzed with the Limulus Amebocyte Lysate (LAL) test, indoor allergens Der p 1, Der f 1 and Fel d 1 were analyzed by ELISAs. The concentration of endotoxin in the mattresses did not differ significantly between different cities. The percentiles of endotoxin were significantly higher in hospitals than in homes. The concentrations of mite allergens (Der p 1 and Der f 1) in the dust were significantly lower in all hospitals than in homes. There was no significant difference of mite allergens between different time points. The concentrations of Fel d 1 were significantly higher in the autumn than in the summer (median: 1376 vs. 478ng/g). No significant differences of Fel d 1 were found between hospitals and homes or between different hospitals. As Fel d 1 concentrations reached levels at which cat allergic patients can experience symptoms, efforts had to be made to reduce the concentrations of Fel d 1 especially in hospitals. In contrast, mite allergens were low in hospitals, which can be clinically beneficial for patients with mite allergies. [Copyright &y& Elsevier]

Background: Although exposure to mite allergens is an important risk factor for the production of IgE and is associated with various allergic diseases, there has been uncertainty as to the route of exposure by which sensitization occurs. Cystatin A is a skin-derived dominant inhibitor against proteolytic activity of major mite allergens, Der f 1 and Der p 1, and blocks the upregulation of IL-8 release from human keratinocytes stimulated with the allergens. We analyzed whether the stimulation of keratinocytes with the allergens upregulates the release of granulocyte-macrophage colony-stimulating factor (GM-CSF), which has many actions relevant to allergic diseases including atopic dermatitis, and if so, whether cystatin A can block this process. Methods: Normal human keratinocytes and the human keratinocyte cell line HaCaT were stimulated with recombinant group 1 allergens in the absence or presence of cystatin A. Results: Stimulation with the recombinant allergens upregulated the release of GM-CSF from normal human keratinocytes in a culture with high calcium concentration and HaCaT cells, which could be inhibited by the addition of cystatin A. The allergens exhibiting proteolytic activity did not digest cystatin A. Proteolytic activity of recombinant Der f 1 was partially regenerated after incubation with keratinocytes even without preactivation by L-cysteine. Conclusion: Proteolytic activity of recombinant Der f 1 and Der p 1 upregulates GM-CSF and IL-8 release from keratinocytes in vitro, suggesting possible contributions to sensitization through the skin and the perpetuation of atopic dermatitis, as well as a homeostatic role for cystatin A against inflammation of the skin. Copyright © 2007 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

The aim of this study was to determine the level of two major mite allergens Dermatophagoides pteronyssinus (Der p 1) and Dermatophagoides farinae (Der f 1) in 30 urban homes in Zagreb, Croatia, using the enzyme immunoassay with two monoclonal antibodies which has been established as the reference method for indoor allergen analysis. Dust samples were taken by vacuuming a carpeted area and collected on cellulose filters. The ranges of Der p 1 and Der f 1 were (0.1-12.5) µg g-1 (median 0.32 µg g-1) and (0.1-31.2) µg g-1 (median 0.35 µg g-1), respectively. Der p 1 and Der f 1 (>2 µg g-1) associated with increased risk of sensitization to mite allergens were found in approximately 16 % homes for each allergen. The sum of allergen (Der p 1 + Der f 1) exceeded the lower threshold in 27 % of homes. Analytical evaluation of the ELISA assay showed satisfactory results for precision (intra-assay CV <6.9 %, inter-assay CV<13.3 %), accuracy (91 % to 93 %), and sensitivity (2 ng mL-1). The ELISA assay for the measurement of dust mite allergens demonstrated very good analytical characteristics for routine laboratory use, and will provide the essential basis for our future studies of various indoor allergens. [ABSTRACT FROM AUTHOR]

Background: Weak signals for allergen-specific IgE are a problem in murine models for the study of allergies. It has been reported that the removal of IgG from murine sera enhances signal intensity. Very recently, buffer solutions designed to enhance signals in immunoassays have been developed and made commercially available. Methods: Sera from mice immunized either with a recombinant form of one of the major mite allergens Der p 1, Der f 1 and Der f 2, or with ovalbumin adsorbed to alum were used for the assays. Total IgE was measured by a sandwich enzyme-linked immunosorbent assay (ELISA). Allergen-specific IgE was assayed using plates coated with the allergens after the removal of IgG from sera with protein G-coupled sepharose beads in wells of other plates or with the use of commercially available enhancer solutions without the removal of IgG. IgE binding was detected with horseradish peroxidase-conjugated anti-murine IgE monoclonal antibody as the secondary antibody. Results: Significant levels of total IgE were produced after the immunizations. The in-well pretreatment of diluted sera (1/10 dilution) with protein G-coupled beads enhanced the signals for allergen-specific IgE. The use of the enhancer solutions for dilution of the sera and secondary antibody and prolonged incubation remarkably enhanced the signals at a more extensive dilution of sera (1/200 or less) without the removal of IgG. Conclusions: An ELISA simply modified with the use of immunoreaction enhancer solutions has advantages in terms of signal intensity and ease of handling for the detection of allergen-specific murine IgE and would be useful for the study of allergies with murine models. Copyright © 2006 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Background: Although allergen avoidance is known to be important for treating atopic diseases, there is a very limited amount of time for clinical education of patients on this topic. Methods: We compared the effect of the thorough home visit counseling (>60 minutes per visit) for avoiding house dust mites (HDMs) with that of regular guidance in our clinics (10 minutes per patient). We enrolled 36 children with asthma (7 years of age or younger; mean, 3.8) in this study under an informed consent. After enrolling the 24 patients for the home visit, 12 families were enrolled as controls for the regular clinical guidance. Between June 1995 and June 1996, we visited the homes of 24 children with asthma enrolled in this study every month and performed a thorough HDM-avoidance counseling of more than 60 minutes (home visit counsel) at each visit. We compared the effects of this counseling with those of the regular clinical guidance given (10 minutes per patient) to the remaining 12 children with asthma. We also evaluated the effect of home visit counseling on children of two subgroups, i.e., an atopic (with positive IgE antibody against HDM) and a nonatopic (without detectable IgE antibodies against 8 common allergens) subgroup. Results: Home visit counseling markedly reduced the frequencies of asthma attacks (p < 0.000001), the required theophylline dosages (p < 0.0005), and the levels of HDM allergens (p < 0.0005) in the atopic subgroups, whereas the effect of regular counseling on these 3 items was relatively less (p < 0.05 or not significant). Surprisingly, home visit counseling also markedly reduced the asthma attacks (p < 0.00001) and theophylline dosages (p < 0.00001) of children with non-atopic asthma. Conclusions: These results suggest that thorough allergen avoidance counseling is effective for children with non-atopic asthma as well as atopic asthma. [ABSTRACT FROM AUTHOR]

Background Polymorphic sequence substitutions in the major mite allergens can markedly affect immunoglobulin E binding and T cell responses, but there are few studies on environmental isolates from Dermatophagoides pteronyssinus and none for D. farinae. Objective To determine the sequence variation of the group 1 and 2 allergens from environmental D. pteronyssinus and D. farinae. Methods RNA from each species was isolated from homes in Bangkok and the sequence of Der p 1, Der p 2, Der f 1, and Der f 2 determined from cDNA produced by high fidelity polymerase chain reactions. Results The enlarged data set revealed preferred amino acid substitutions in residues 19, 81, and 215 of Der p 1 as well as sporadic changes. Der p 2 showed frequent variations with clusters of amino acid substitutions, but the canonical Der p 2.0101 was not found in any of 17 sequences. Der f 2 showed variants with clusters of substitutions similar to Der p 2 but in different amino acid positions and without any structural concordance. Der f 1 in contrast to the other allergens had few amino acid sequence substitutions. Conclusions The sequence information on variants provides data important for the optimal design of allergen formulations and useful for the genetic engineering and structure–function analyses of the major allergens. [ABSTRACT FROM AUTHOR]

Background: The major house dust mite group 1 allergens Der p 1 and Der f 1 are the most potent indoor allergens. Der p 1 cleaves human cell surface molecules, the low-affinity IgE receptor (CD23/FcεRII), the α-subunit of the IL-2 receptor (CD25), and a protease inhibitor α1-antitrypsin, and in vitro and in vivo studies suggested the importance of its proteolytic activity in the pathogenesis of allergy. Recently, we established an efficient system to prepare correctly folded active recombinant Der p 1 and Der f 1 (Der p 1-N52Q and Der f 1-N53Q) with similar molecular sizes, secondary structures and allergenicities as their natural types. To evaluate whether Der p 1-N52Q and Der f 1-N53Q are suitable for use in future in vitro and in vivo studies as alternatives to the natural types, we investigate their proteolytic activity to cleave the human proteins and IgE-eliciting activity in mice. Methods: Proteolytic activities of Der p 1-N52Q and Der f 1-N53Q against a short peptide substrate, a collagen substrate Azocoll, human CD23 and CD25 expressed on the cells and human α1-antitrypsin were analyzed by kinetic assays for proteolysis of the fluorogenic or colorimetric substrates, flow cytometry and sodium dodecyl sulphate-polyacrylamide gel electrophoresis, respectively. Mice were intraperitoneally immunized with Der p 1-N52Q and Der f 1-N53Q adsorbed on Alum, and the serum IgE levels were measured by sandwich ELISA. Results: Der p 1-N52Q and Der f 1-N53Q showed proteolytic specificities against the short peptide substrate, Azocoll, human cell surface CD23 and CD25 and human α1-antitrypsin, and elicited significant serum IgE levels in immunized mice. Conclusion: The recombinant forms, Der p 1-N52Q and Der f 1-N53Q, will be useful tools as alternatives to the natural Der p 1 and Der f 1 for various in vitro and in vivo analyses. Copyright © 2005 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Background: With 63% of US children under 5 years of age in regular child care, day-care facilities could be an important source of exposure to indoor allergens. Objective: This study examined levels of 7 indoor allergens in 89 day-care facilities in 2 North Carolina counties. Methods: At each facility, a questionnaire was administered, observations were made, and vacuumed dust samples were collected from carpeted and noncarpeted areas of one room. Allergen concentrations were measured with antibody-based ELISAs. Results: Each allergen was detected in a majority of facilities (52% to 100%). Geometric mean concentrations were 5.19 μg/g for Alternaria alternata, 2.06 μg/g for Can f 1, 1.43 μg/g for Fel d 1, 0.21 U/g for Bla g 1, 0.20 μg/g for Der p 1, 0.10 μg/g for Der f 1, and 0.01 μg/g for Mus m 1. Concentrations for 5 of the 7 allergens were not statistically different from concentrations found in southern US homes sampled in the National Survey of Lead and Allergens in Housing. In rooms with carpet and hard-surfaced flooring, levels of A alternata, Can f 1, Der f 1, Der p 1, and Fel d 1 were statistically higher on carpet. Conclusions: In this survey of day-care facilities in North Carolina, detectable levels of indoor allergens were commonly found. For many young children and day-care staff, day-care facilities might be a source of clinically relevant exposures to indoor allergens. [Copyright &y& Elsevier]

Abstract: Although mite major group 1 allergens, Der p 1 and Der f 1, were first isolated as cysteine proteases, some studies reported that natural Der p 1 exhibits mixed cysteine and serine protease activity. Clarifying whether the serine protease activity originates from Der p 1 or is due to contamination is important for distinguishing between the pathogenic proteolytic activities of group 1 allergens and mite-derived serine proteases. Recombinant mite group 1 allergens would be useful tool for addressing this issue, because they are completely free from contamination by mite serine proteases. Recombinant Der p 1 and Der f 1, and highly purified natural forms exhibited only cysteine protease activity. However, commercially available natural forms exhibited both activities, but the two activities were eluted into different fractions in size-exclusion column chromatography. The substrate specificity associated with the serine protease activity was similar to that of Der f 3. These results indicate that the serine protease activity does not originate from group 1 allergens. [Copyright &y& Elsevier]

Background: The major house dust mite group 1 allergens Der p 1 and Der f 1, which belong to the papain-like cysteine protease family, are the most potent of indoor allergens. However, little information is available on the location of IgE epitopes. Objective: We investigated the allergenicities of recombinant proforms and mature forms of Der p 1 and Der f 1 to compare them with natural Der p 1 and Der f 1 and to obtain information on the conformational IgE-binding epitopes. Methods: Secreted pro-Der p 1 and pro-Der f 1 and their mutants without hyperglycosylation expressed in yeast were converted to mature forms. We purified the proforms and mature forms and analyzed their apparent molecular sizes and secondary structures by means of gel-filtration and circular dichroism analysis and their allergenicities by means of assays for IgE binding, IgE-binding inhibition, and basophil histamine release. The tertiary structure of pro-Der f 1 was predicted by molecular modeling. Results: The recombinant mature forms exhibited similar molecular sizes, secondary structures, and allergenicities as their natural types. On the other hand, their proforms exhibited different secondary structures and less allergenicities than the mature forms in all sera and volunteers tested. Molecular modeling revealed that the prosegment is anchored at the prosegment-binding loop and the substrate-binding cleft on the surface of the mature portion. Conclusions: Our studies indicate that the prodomains of Der p 1 and Der f 1 reduce allergenicity and that the major conformational IgE epitopes commonly found in a broad population of patients exist within the 2 regions blocked by the prosegments. Recombinant Der p 1 and Der f 1 and the findings in the present study will be the basis for allergen standardization and the design of safer and more effective allergen vaccines. [ABSTRACT FROM AUTHOR]

Transformation of a model legume Lotus japonicus accession Miyakojima MG-20 was examined using Agrobacterium tumefaciens with a binary expression vector. Using the improved transformation method, we introduced a major allergen gene from a house dust mite, Der f 1, into MG-20. Analyses by Southern hybridization, reverse transcription (RT)-PCR, and Western blotting showed that the Der f 1 gene was integrated into the genome of L. japonicus, expressing the gene product in the T1 lines. Our results imply future application of oral allergen-specific immunotherapy using legume plants. [Copyright &y& Elsevier]

Exposure to house dust mite (HDM) allergens can lead to the development of allergic complaints. Mattress covers seem to be an obvious option for lowering allergen exposure in sensitized individuals. Previous studies have shown that Dermatophagoides pteronissinus was the most prevalent HDM species in the Netherlands. In the present study, we investigated the effect of mattress covers on Der p 1 and Der f 1 concentrations in dust samples in three areas in the Netherlands; Groningen, Utrecht and Rotterdam. Dust was obtained from mattresses of 277 patients at the beginning of the study and after 12 months of the placebo-controlled intervention. It was analysed for allergen content by immunoassay. The differential effect of the intervention on Der p 1 vs. Der f 1 was analysed in a subgroup with Der p 1+Der f 1>1 μg/g dust ( N=161). It was tested whether the intervention caused a significant change in the Der f 1/Der p 1 ratio. At t=0 we found very similar levels of the group 1 allergens of both species. The relatively high prevalence of D. farinae in our study was geographically restricted: the median Der f 1/Der p 1 ratio was 11.1 in the Rotterdam area compared with 1.32 in the Utrecht area and 0.33 in the Groningen area. Analysis of our data showed that the favourable intervention effect found for the combined allergen data (reduction factor=2.9, P<0.001) is essentially due to a favourable effect of the intervention on the Der f 1 levels only (reduction factor=3.6, P<0.001). The effect on the Der p 1 level was remarkably small (reduction factor: 1.2, P=0.48). In the intervention group, the Der f 1/Der p 1 ratio decreased after 12 months by a factor 2.0, whereas in the placebo group it increased (probability of the intervention effect: P<0.005). Mite-impermeable covers are more effective in reducing the level of Der f 1 than that of Der p 1. [ABSTRACT FROM AUTHOR]

Background: Exposure to indoor allergens has already been shown to occur in many public places, including workplaces, in several countries. Aim of this study was to measure the levels of house dust mites, cat and cockroach allergens in indoor workplaces (offices and archives) in Italy and to evaluate the possible relationships between allergen levels and building characteristics, type of ventilation, indoor relative humidity and temperature. Methods: Der f 1 and Der p 1, Fel d 1, Bla g 2 were measured by ELISA in dust samples collected from floors or upholstered seats of 160 workplaces. Results: Detectable Der p 1 levels were found in 86 (54%) workplaces Der f 1 in 87 (55%), Fel d 1 in 86 (54%) and Bla g 2 in 3 (1.9%). Der p 1 allergen concentrations expressed per weight were higher than the proposed sensitization thresholds in 7 samples, Der f 1 in 5, Fel d 1 in 6 and Bla g 2 in 3. The highest allergen levels were detected in samples from upholstered seats. A significant correlation was found between Der f 1 level on floors, expressed per surface, and indoor temperature (r=0.39; P<0.01). Conclusions: In our study we found that upholstered seats in workplaces in Italy may constitute a significant reservoir both of house dust mites and cat allergens. Exposure to these allergens in workplaces may represent a risk factor for elicitation of symptoms and/or induction/maintenance of inflammation in allergic individuals and might also constitute a risk factor for sensitization. [Copyright &y& Elsevier]

Background: Information about the influence of housing and occupant characteristics on mite allergen concentrations is crucial to determine which methods could be used to decrease exposure of susceptible subjects. Objectives: To identify housing and occupant characteristics that are associated with mite allergen concentrations in house dust collected from living rooms and mattresses. Methods: We collected dust samples from 750 homes in the northeastern US. The influence of various characteristics on concentrations of mite allergens (Der p 1 and Der f 1) was studied using multiple linear regression analysis. Results: Some characteristics, like absence of air conditioners, the presence of mold or mildew, and a lower temperature were consistently associated with higher concentrations of both mite allergens in dust from all sampling locations. However, none of these factors changed Der p 1 or Der f 1 concentrations by more than a factor of 2. People of white ethnic background had roughly two times higher mite allergen concentrations, while family income, family size, and education level only marginally influenced mite allergen concentrations. Conclusions: Various housing characteristics have some influence on mite allergen concentrations, and could possibly be used to decrease exposure of susceptible subjects. However, only a limited percentage of the variation in mite allergen concentrations was explained by these characteristics. [ABSTRACT FROM AUTHOR]

House dust mites are common sources of indoor allergens. In Reykjavik, Iceland, 9% of the young adult population had serum-specific IgE to Dermatophagoides pteronyssinus. Sensitization to mites is usually assumed to be due to exposure to house dust mites in the indoor environment. This investigation was carried out to measure the concentrations of house dust mite allergens and to investigate which species of mites were present in beds in Iceland. A total of 197 randomly selected adults were visited at home using the European Community Respiratory Health Survey (ECRHS) II Indoor protocol. Dust samples were collected from mattresses for measurement of house dust mite allergen concentrations and to estimate the number and type of house dust mites. Additional samples from mattresses and floors were collected from the homes of 10 patients with positive skin prick tests (SPT) to D. pteronyssinus. House dust mite allergen concentrations were measured using ELISA and examination of mite species was carried out using microscopy. Climatic parameters were assessed using psychrometer readings in the bedrooms and outdoors. We found two single mite specimens, both D. pteronyssinus, in two dust samples. Mite allergen analyses indicated that two other dust samples had Der f 1 results close to the cut-off of 0.1 μg/g of dust. No samples were positive for Der p 1. In an additional collection of dust from the homes of 10 SPT-positive patients no Dermatophagoides spp. were found. Reykjavik citizens are exposed to extremely low amounts of house dust mite allergens in their homes. Possible alternative sources for sensitization are discussed, such as bird nests, exposure from travelling abroad, or other mites or invertebrates that cross-react with house dust mite allergens. Our findings suggest that exposures other than to house dust mites indoors are possible sources of mite allergen exposure. [ABSTRACT FROM AUTHOR]

The level of the Dermatophagoides mite group 1 (Der 1) allergens in reservoir dust has been used as an index of exposure in most studies. However, the mite allergen level in reservoir dust cannot directly reflect the personal exposure level. We sought to develop a new method for quantifying the Der 1 allergens on bedding and human skin surfaces as an index of exposure to mite allergens. Samples were obtained with a small adhesive tape from the forearm skin of 30 healthy volunteers and from their regularly used mattresses. The level of Der 1 allergens collected onto the adhesive tape was measured by a newly developed sensitive fluorometric ELISA for Der p 1 and Der f 1. The Der 1 allergens could be detected in all the samples from bedding surfaces and in 28 of the 30 samples from skin surfaces. The Der 1 levels by adhesive tape sampling from the mattresses correlated with those by reservoir dust sampling. The sampling of the skin and bedding surface with adhesive tape correlated, but skin sampling did not correlate with reservoir sampling. The Der 1 allergens on bedding surfaces and on human skin surfaces could be quantified with a very simple sampling technique. The system developed in this study will provide a new tool for the assessment of mite allergen exposure in daily life. [ABSTRACT FROM AUTHOR]

We studied the effect of a spray containing 1% benzyl benzoate, an acaricide, and 1% tannic acid (‘Lowal’; a protein-denaturing substance), on concentrations of major allergens from house dust mite (HDM) species Dermatophagoides pteronyssinus and D. farinae (Der p 1 and Der f 1, respectively) in carpets. In a double-blind, placebo-controlled study with crossover design, 30 homes of children with HDM sensitization and asthma were included. All houses showed ≥ 400 ng/g of Der p 1 + Der f 1 in carpet dust. The first treatment was performed on day 0 (group 1 active treatment, n = 15; group 2 placebo treatment, n = 15). After 2 and 8 weeks, dust samples were collected for quantification of mite allergens. After a 2-week washout period, the second treatment was performed (group 1 placebo treatment; group 2 active treatment). Again, carpet dust was collected after 2 and 8 weeks. Twenty-two of 30 families completed the trial: 14/15 in group 1 and eight of 15 in group 2. On day 0, there was no significant difference in mite allergen exposure between group 1 and group 2 (1,498 vs. 2,239 ng/g of Der p 1 + Der f 1, respectively). In group 1, the geometric mean for the difference of mite allergen concentration comparing day 0 and week 6 was 196 ng/g (95% CI: -7,161 and 8,401) for the first treatment (active) and 15 ng/g (95% CI: -1,079 and 1,292) for the second treatment (control). In group 2, the difference was 66 ng/g (95% CI: -398 and 1,515) for the first treatment (control) and 609 ng/g (95% CI: 186 and 9,264) for the second treatment (active). Comparing placebo and active treatment in total, there was a significant decrease following placebo treatment after 14 days (p = 0.026). After 8 weeks, active treatment was superior to placebo treatment (p = 0.049), but the allergen reduction achieved was < 20% (median 1,500 ng/g on day 0 vs. 1,250 ng/g after 8 weeks). We conclude that the slight mite allergen reduction on carpets... [ABSTRACT FROM AUTHOR]

Background: HDM distribution varies between geographic areas and may be affected by housing characteristics. We quantified Der p 1 and Der f 1 and assessed the relationships between their levels and housing characteristics in homes of nonallergic subjects. Methods: Der f 1 and Der p 1 were measured by ELISA in dust samples from living-room floors and mattresses of 44 homes of nonallergic subjects in Pavia. Information about household characteristics was obtained by questionnaire. Results: Der p 1 and Der f 1 concentrations (μg/g dust, median) were 0.34 and 7.8 on mattresses, and 0.15 and 0.83 on living-room floors. Higher Der f 1 levels on mattresses were associated with synthetic pillows (P<0.05), and (only when expressed as μg/m2) with bedding washing temperature of ≤60°C (P<0.05). Der f 1 levels were higher on the living-room floors of homes located on lower (≤first) floors (P<0.05). Good correlations were found between Der p 1 and Der f 1 expressed as μg/g dust and μg/m2 on both mattresses and living-room floors. Conclusions: In homes of nonallergic subjects in northern Italy, Der f 1 exceeded the threshold for sensitization in a high proportion of mattress samples. [ABSTRACT FROM AUTHOR]

Background: The role of mite allergen exposure in sensitization and development of asthma has been widely recognized. Previous studies have shown that Dermatophagoides pteronyssinus and Blomia tropicalis were the most prevalent house dust mites in Brazil, while D. farinae was rarely found. The aim of this study was to measure Der f 1 and Der p 1 allergen levels in Brazilian asthmatics’ and controls’ homes. Methods: Sixty-four houses (32 asthmatic, 32 control) were visited for dust sampling from five sites. Der f 1 and Der p 1 levels were measured by two-site monoclonal-antibody-based ELISAs. Results: The highest levels of Der f 1 and Der p 1 allergens were found in bedding samples from both asthmatics’ and controls’ homes. However, the geometric mean of Der f 1 levels (15.8 μg/g of dust) was significantly higher than for Der p 1 (8.2 μg/g of dust) in these samples. In addition, allergen levels ≥10 μg/g of dust were found in 60–80% of the samples for Der f 1 and about 50% for Der p 1. Conclusions: These results indicate that high levels of Der f 1 allergen are present in both asthmatics’ and controls’ homes, in contrast to previously reported data. Therefore, studies on exposure to mites should be performed in different cities, seasons and times, since the mite fauna might be subject to variations. Knowledge of the mite fauna will certainly improve the means of investigating the association between allergen exposure and sensitization, allowing to establish the inclusion of new mite extracts in inhalant skin test sets, and even to detect monosensitized patients with respiratory allergy.Copyright © 2000 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Der f 1 is a major house dust mite allergen belonging to the cysteine protease family. Because of the great demand for clinical and research use of this allergen, much effort to establish an efficient method of preparing purified Der f 1 has been made. We constructed an isopropyl-β-D-thiogalactopyranoside-inducible expression plasmid to produce the pro-form of Der f 1 in Escherichia coli. The recombinant product was accumulated as insoluble inclusion bodies in cells. The solubilized inclusion bodies were found to be successfully renatured by two-step gel filtration chromatography. About 70 mg of pro Der f 1 were properly refolded by this method from 1 liter of culture. Acid treatment of the renatured pro Der f 1 resulted in the autocatalytic removal of the pro-sequence. The obtained mature form of Der f 1 bound IgE in patient sera and induced the release of histamine from peripheral blood leukocytes equally to native Der f 1. Furthermore, mature Der f 1 obtained by this method had identical protease activity with native Der f 1. We also discussed the contribution of the pro-sequence and the sugar chain of Der f 1 to its antigenic and enzymatic activity. This is the first report to produce an active mature form of recombinant Der f 1 in E. coli.Copyright © 2000 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Background: It has been hypothesized that changes in heating systems and insulation of homes in developed countries have generated an indoor climate favorable to organisms that excrete allergens inducing sensitization and allergic disease. The purpose of this study was to determine the influence of the installation of highly insulated windows and central heating systems on indoor climate, and mite‐allergen (Der f 1) and mold spore concentrations. Methods: The bedrooms of 98 apartments were examined before and 7 months (mean) after installation of insulated windows and central heating systems. The air‐exchange rate, temperature, and humidity were measured. In settled dust on carpets and mattresses, the number of colony‐forming mold spores and the Der f 1 concentration were determined. The inhabitants completed a questionnaire about their lifestyles and housing conditions. Results: The air‐exchange rate decreased from geometric mean 0.73 to 0.52 per hour (P=0.029). Temperature (mean 13.4 vs 17.5°C, P<0.001), and absolute humidity (mean 4.6 g vs 6.2 g H2O/kg air, P<0.001) increased. Relative humidity remained nearly unchanged (mean 47.6 vs 49.1%). Der f 1 concentrations on carpets (geometric mean 0.65 vs 1.28 μg/g dust, P<0.001) and mattresses (geometric mean 1.56 vs 2.40 μg/g, P=0.002) increased. Among the fungi that were analyzed, only the thermotolerant species Aspergillus fumigatus increased (geometric mean 20 vs 60 colony‐forming units/g carpet dust, P=0.02). Conclusions: The findings of this study suggest that the installation of insulated windows and central heating systems is associated with an increase of Der f 1 concentrations in carpet and mattress dust and A. fumigatus in carpet dust in apartment bedrooms. [ABSTRACT FROM AUTHOR]

Background Chemical agents such as tannic acid and detergents have been shown to introduce non-random bias in allergen measurement. Objective We investigated how several substances that are commonly found in floor dust (carpet fresheners, powdered pesticides, and table salt) affected immunoassays of purified standard allergens. Methods Three sets of experiments were conducted to: (1) screen for interference with allergen enzyme-linked immunosorbent assay (ELISA); (2) test for concentration-response; and (3) assess the site-of-action of a given dust additive (i.e. the effect on allergen binding to primary or secondary antibody). The ELISAs are commercially available two-site monoclonal antibody assays for Der p 1, Der f 1, and Fel d 1, and a monoclonal/polyclonal assay for Bla g 1. Outcomes are reported in terms of reaction rate (colour change per unit time), which is directly proportional to the amount of bound allergen. Results In the initial screening experiments, carpet fresheners tended to decrease Der p 1 assay reaction rates, increase Der f 1 assay rates, and produce little change in Fel d 1 assay rates. Three carpet fresheners decreased Der p 1 assay rate responses in a concentration-dependent manner. Two carpet fresheners noticeably increased Der f 1 assay reaction rates in both the screening and the concentration-response tests. Powdered pesticides increased reaction rates in the Bla g 1 assays and increased the slope of the dilution curve compared with that of the purified allergen. Salt decreased the reaction rates of Bla g 1 assays at allergen concentrations greater than 0.01 U/mL. For each of the four allergens, the largest effects of dust additives occurred when secondary antibody binding was altered. Conclusions Some common household dust components can introduce systematic error into immunoassays for arthropod allergens. [ABSTRACT FROM AUTHOR]

Several studies have demonstrated the role of domestic exposure to mite in mite allergic patients. However, only one limited study have measured mite exposure at work in offices. The aim of our study was to compare group 1 mite allergen levels in carpets of offices and bedrooms. Thirty bedrooms and 27 offices were randomly selected. Dust samples were performed according to the international recommendations. Mite allergens were measured using ELISA based assay. A dramatic difference was found in group 1 mite allergen levels between bedrooms (mean: 18.4 μg/g) and offices (mean: 0.32 μg/g). We conclude that mite exposure in offices does not seem to be at risk for atopic and non atopic workers. [Copyright &y& Elsevier]