Cloning,bioinformatics analysis,and expression of the dust mite allergen Der f 5 of Dermatophagoides farinae

Crude extracts of house dust mites are used clinically for diagnosis and immunotherapy of allergic diseases, including bronchial asthma, perennial rhinitis, and atopic dermatitis. However, crude extracts are complexes with non-allergenic antigens and lack effective concentrations of important allergens, resulting in several side effects. Dermatophagoides farinae (Hughes; Acari: Pyroglyphidae) is one of the predominant sources of dust mite allergens, which has more than 30 groups of allergen. The cDNA coding for the group 5 allergen of D. farinae from China was cloned, sequenced and expressed. According to alignment using the VECTOR NTI 9.0 software, there were eight mismatched nucleotides in five cDNA clones resulting in seven incompatible amino acid residues, suggesting that the Der f 5 allergen might have sequence polymorphism. Bioinformatics analysis revealed that the matured Der f 5 allergen has a molecular mass of 13604.03 Da, a theoretical pI of 5.43 and is probably hydrophobic and cytoplasmic. Similarities in amino acid sequences between Der f 5 and allergens of other domestic mite species, viz. Der p 5, Blo t 5, Sui m 5, and Lep d 5, were 79, 48, 53, and 37%, respectively. Phylogenetic analysis indicated that Der f 5 and Der p 5 clustered together. Blo t 5 and Ale o 5 also clustered together, although Blomia tropicalis and Aleuroglyphus ovatus belong to different mite families, viz. Echimyopodidae and Acaridae, respectively.     

粉尘螨主要变应原Derf1基因的改造与可溶性表达

目的在大肠杆菌中提高粉尘螨1类变应原（Derf1）的可溶性表达。方法用RT-PCR方法扩增得到Derf1的全长序列与成熟肽序列（mDerf1）；以已知DerP5基因的前导序列替换Derf1前导序列和原酶序列，重新构建出rDerf1基因；将上述3个基因分别克隆入原核表达载体pGEX-4T-1中表达，经Western-blot对三者的表达产物进行分析鉴定。结果Western-blot表明，pGEX-Derf1与pGEX-mDerf1的表达产物分别为分子量约63000与51000的重组蛋白质，重组蛋白质主要存在于细胞裂解液的沉淀中。pGEX-rDerf1大量表达了可溶性目的蛋白质，分子量约53000，并被成功地分离纯化。以上三种表达产物均可被鼠抗GST抗体与螨过敏患者血清特异地识别。结论表达了含Derf1，mDerf1与rDerf1的三种GST融合蛋白质，它们均具免疫反应性，纯化获得了GST-rDerf1融合蛋白质，为后期的诊断及治疗奠定了基础。     

Activated charcoal suppresses breeding of the house dust mite, Dermatophagoides pteronyssinus, in culture

House dust mite sensitized asthmatics are advised to practice allergen avoidance. Charcoal pillows are used in Korea with unsubstantiated claims regarding their efficacy in alleviating asthma symptoms. We tested the effects of activated charcoal on breeding of house dust mites in culture. Twenty live adult house dust mites (Dermatophagoides pteronyssinus) were inoculated, 10 replicates, on culture media containing 0%, 1%, 3%, 5%, 10%, and 20% activated charcoal and incubated at 25 degrees C and a relative humidity of 75%. After four weeks, the mean numbers of live house dust mites were 286, 176, 46, 16, 7, and 0 for the 0%, 1%, 3%, 5%, 10%, and 20% charcoal-containing culture media, respectively. Thus, activated charcoal suppresses breeding of house dust mites and offers a new promising method for house dust mite control.     

Sensitization to aeroallergens in Korean children: a population-based study in 2010

We performed this study to assess the prevalence of sensitization to aeroallergens and to analyze the difference between prevalence rates according to children's ages and residential areas. In this nationwide cross-sectional study, first grade students of 45 elementary schools and 40 middle schools were randomly selected, and skin prick tests were performed for 18 inhalant allergens between October and November 2010. Of 7,829 analyzed subjects, 3,753 (47.9%) were sensitized to at least one aeroallergen. Sensitization to Dermatophagoides farinae was found to be the most prevalent in elementary schoolchildren (32.4%), followed by Dermatophagoides pteronyssinus, Tyrophagus putrescentiae, Japanese hop, and oak. In middle schoolchildren, D. pteronyssinus yielded the highest prevalence (42.7%), followed by D. farinae, T. putrescentiae, Japanese hop, and cat. In middle schoolchildren, the sensitization rate to aeroallergens in metropolitan, urban, and rural areas was 57.2%, 54.3%, and 49.8%, respectively (P = 0.019). In this age group, the sensitization rate in low, middle, high, and very high income groups was 53.8%, 51.8%, 59.0%, and 59.6%, respectively (P = 0.002). In conclusion, the sensitization rate is 47.9% and house dust mite is the most prevalent allergen in the pediatric population in Korea. The rate is higher in metropolitan areas and the highest income group than in rural areas and low income groups.     

New sensitization to house dust mites in cefteram-induced occupational asthma: a case report

Occupational asthma (OA) can improve after cessation of exposure; however, some patients suffer from persistence or aggravation of their asthmatic symptoms. Here we report a case of a new sensitization to house dust mites during the follow-up period in a 37-year-old female patient with OA induced by cefteram pivoxil powder (cefteram powder). She was previously diagnosed with OA caused by inhalation of cefteram powder. Consequently, she left her job and had been well for 9 subsequent years. She began to experience aggravation of her rhinitis and asthmatic symptoms again several months prior to presentation. Her skin-prick test results had converted to strongly positive responses to two types of house dust mites. The serum levels of eosinophil cationic protein (ECP) and the total and specific immunoglobulin (Ig)E levels against the two types of house dust mites were elevated. An inhalation challenge test with Dermatophagoides farinae was performed, and significant bronchoconstriction (21.1% reduction in the forced expiratory volume in the first second) with asthma symptoms was observed at 10 minutes. To our knowledge, this is the first case demonstrating a new sensitization to house dust mites in a patient with OA caused by cefteram powder. Regular monitoring, including skin-prick tests and measurement of specific serum IgE/ECP levels, may help to screen potential cases.     

Inflammatory and remodeling events in asthma with chronic exposure to house dust mites: a murine model

Although animal models with ovalbumin have been used to study chronic asthma, there are difficulties in inducing recurrence as well as in maintaining chronic inflammation in this system. Using a murine model of house dust mite (HDM)-induced bronchial asthma, we examined the airway remodeling process in response to the chronic exposure to HDM. During the seventh and twelfth weeks of study, HDM were inhaled through the nose for three consecutive days and airway responsiveness was measured. Twenty-four hours later, bronchoalveolar lavage and histological examination were performed. The degree of overproduction of mucus, subepithelial fibrosis, and the thickness of the peribronchial smooth muscle in the experimental group was clearly increased compared to the control group. In addition, HDM-exposed mice demonstrated severe airway hyperreactivity to methacholine. In the bronchoalveolar lavage fluid, the number of total cells and eosinophils was increased; during the twelfth week, the number of neutrophils increased in the experimental group. With regard to changes in cytokines, the concentrations of IL-4, IL- 13, and transforming growth factor-beta (TGF-beta) were increased in the experimental group. The data suggest that eosinophils, IL-4, IL-13, and TGF-beta might play an important role in the airway remodeling process and that neutrophils may be involved with increased exposure time.     

Human T cell responses to beta-galactosidase.

The peripheral blood of most normal individuals has been shown to contain T cells that respond to beta-galactosidase (beta-Gal), presumably as a result of natural priming. Three T cell clones (clones 1,2,4) specific for beta-Gal were isolated from peripheral blood mononuclear cells (PBMC) after pretreatment with leucine methyl ester (LeuOMe); a fourth clone from the same individual was isolated from untreated cells. All four clones were CD4+ CD8- alpha beta TcR+ and clone 1 was additionally shown to be cytotoxic. Epstein-Barr virus (EBV) transformed B cell lines were derived from LeuOMe-treated or untreated PBMC and used to study the efficiency of presentation of beta-Gal to one of the clones. The results indicated that B cells transformed after LeuOMe treatment presented beta-Gal at lower concentrations than untreated controls. beta-Gal would therefore appear to be a highly suitable model antigen for studies of immunoregulation in humans.     

Association Between Serum IgE Levels and the CTLA4 +49A/G and FCER1B -654C/T Polymorphisms in Korean Children With Asthma

Purpose

T cells play a central role in cell-mediated immunity, atopic disease, and asthma. The balance of CD28/cytotoxic T-lymphocyte antigen 4 (CTLA4)-derived signal transduction plays an important role in the activation of T cells and an increased immunoglobulin E (IgE) response. The aim of the current study was to investigate the association between polymorphisms in the genes encoding both CTLA4 and the high-affinity IgE receptor 1B (FCER1B) and serum IgE levels in Korean children with asthma.

Methods

We enrolled 238 controls and 742 children with asthma. The CTLA4 +49A/G and FCER1B -654C/T polymorphisms were genotyped by PCR-restriction fragment length polymorphism analysis.

Results

We observed no difference in the distribution of CTLA4 +49A/G among controls, children with asthma, and those with atopic asthma. In contrast, the GA genotype of CTLA4 +49A/G in children with atopic asthma was significantly higher compared to that in those with non-atopic asthma. Moreover, significantly higher log Dp/Df-specific IgE levels were found in children with asthma and those with atopic asthma carrying one or two copies of the CTLA4 +49A versus those homozygous for +49G. Gene-gene interactions between CTLA4 and FCER1B with the heterozygote and homozygote of variant genotypes were associated with the log Dp/Df-specific IgE levels, but not asthma development. In addition, children with Dp/Df (+) asthma carried an elevated combined genotype of risk allele compared to those with Dp/Df (-) asthma.

Conclusions

The CTLA4 +49A/G polymorphism may contribute to the production of IgE in Korean children with asthma, especially in Dp/Df-specific IgE levels, but not in the direct development of asthma. In addition, Dp/Df-specific IgE levels with a FCER1B -654C/T polymorphism may involve additive effects.     

T cell responses to a mixture of Mycobacterium tuberculosis peptides with complementary HLA-DR binding profiles

The T cell response to a mixture of eight peptides derived from sequences of the Mycobacterium tuberculosis 16-, 19- and 38-kD antigens (MTBmix-8) has been studied. The peptides were selected on the basis of complementary binding to nine HLA-DR molecules (HLA-DR1 to DR9). MTBmix-8 at 6.25 and 50 μg/ml gave rise to significant stimulation (P< 0.05) of peripheral blood mononuclear cells (PBMC) from healthy tuberculin-positive and both untreated and treated diseased subjects, but not in any of a control group of healthy tuberculin-negative subjects. MTB-mix-8 stimulated proliferation of PBMC from healthy tuberculin-positive individuals at lower concentrations than the individual component peptides. However, the maximal stimulation achieved was only slightly higher than that achieved with individual peptides. MTBmix-8 also stimulated the production of interferon-gamma (IFN-γ) in vitro. Using the mean ± 2 s.d. of the values for IFN-γ production in the tuberculin-negative population as a cut-off, MTBmix-8 at 6.25 μg/ml was able to detect infection with a sensitivity of 100% in untreated patients, 87% in treated patients, and 82% in tuberculin-positive controls. The corresponding figures for the most potent single peptide (16p91–110) were: 66% in untreated patients, 71% in treated patients and only 42% in controls. Thus, using the IFN-γ-based assay, which has the additional advantages of speed and does not require radioactivity, the mixture of peptides is more sensitive than single peptides in diagnosing infection.     

In vivo induction of CD4+ T cell responses by antigens covalently linked to synthetic microspheres does not require adjuvant

Macrophages, dendritic cells or B lymphocytes have been shownto play a major role in the presentation of soluble antigensto CD4⁺ T cells. In contrast, the capacity of these cells topresent particulate antigens such as bacterial or parasiticantigens to T cells remains controversial. To investigate thisquestion, well defined particulate antigens were prepared bycovalent linkage of proteins or peptides to 1 µm in diametersynthetic microspheres. The T cell immunogenicity of such particulateantigens was analyzed in vitro and in vivo. In vitro, a solubleprotein such as hen egg lysozyme (HEL) coupled to beads stimulateda strong proliferative T cell response of lymph node cells fromHEL-primed mice or of specific T cell hybridomas. HEL coupledto beads was presented to the specific T cell hybridomas bysplenocytes or by peritoneal macrophages, but not by lymphomaB cells. Immunization of mice with several different proteinantigens or with a synthetic peptide covalently linked to beadsinduced strong CD4⁺ T cell responses in the absence of adjuvant.The strong in vivo immunogenicity of proteins coupled to beadsdid not result from a non-specific adjuvant effect of beadssince covalent linkage of the antigen to beads was strictlyrequired to induce T cell responses in the absence of adjuvant.In vivo treatment by carrageenan showed that macrophages arerequired for the in vivo stimulation of T cell responses bythese particulate antigens. Thus, these results demonstratedthe role of phagocytic cells, especially macrophages, for invivo presentation of particulate antigens. These particulateantigens represent an interesting approach for the developmentof new vaccines, and for the in vivo analysis of the role ofvarious antigen presenting cells in T cell activation and differentiation.     

Design of a ProDer f 1 vaccine delivered by the MHC class II pathway of antigen presentation and analysis of the effectiveness for specific immunotherapy

Dermatophagoides farinae (Der f 1) is one of leading cause for allergic asthma, and allergen-specific immunotherapy (SIT) is currently recognized as the only etiological therapy to ameliorate asthmatic symptom. The current study was designed on the major histocompatibility complex (MHC) class II pathway, invariant chain (Ii)-segment hybrids as vaccine basis to explore the efficacy of Der f 1 hybrid vaccine by virtue of Ii as carrier in enhancing the protective immune response to asthma. Initially, we engineered a fused molecule, DCP-IhC-ProDer f 1, to deliver ProDer f 1 antigen via specific dendritic cell-targeting peptides to dendritic cells (DCs). Then the DCP-IhC-ProDer f 1 was immunized to the asthmatic models of murine induced by ProDer f 1 allergen. The findings showed that the cytokine repertoire in the murine model was shifted after SIT, including stronger secretion of IFN-γ and IL-10, and a decreased production of IL-4 and IL-17. ELISA determination revealed that the hybrid displayed weak IgE and IgG₁ reactivities, and IgG_2a levels were elevated. Furthermore, DCP-IhC-ProDer f 1 treatment inhibited inflammatory cell infiltration in the lung tissues. Our results suggest that the DCP-Ihc-ProDer f 1 may be used as a candidate SIT against asthma.     

The fraction 1 and V protein antigens of Yersinia pestis activate dendritic cells to induce primary T cell responses

The F1 and V antigens of Yersinia pestis, despite acting as virulence factors secreted by the organism during infection, also combine to produce an effective recombinant vaccine against plague, currently in clinical trial. The protective mechanisms induced by rF1 + rV probably involve interactions with dendritic cells (DC) as antigen uptake, processing and presenting cells. To study such interactions, naive ex vivo DC from bone marrow, spleen and lymph node were cultured with rF1, rV or combined antigens and demonstrated to secrete interleukin (IL)-4 and IL-12 into the culture supernatant. Cytokine production in response to pulsing was dependent on the maturity of the bone marrow-derived DC culture, so that pulsed 8-day-old cultures had accumulated significantly more intracellular IL-4 and IL-12 than unpulsed cells. DC, pulsed with rF1 + rV for 2-24 h, were able to prime naive autologous lymph node T cells to proliferate in an antigen dose-dependent manner, with an order of potency of 3d bone marrow-derived DC (BMDC) > 7d BMDC > splenic DC. Significantly, cell-free supernatants from rF1 + rV-pulsed BMDC and splenic DC were also able to induce specific primary responses effectively in naive T cells, suggesting that these supernatants contained stimulatory factor(s). This study suggests an important role for DC, or factors secreted by them, in the induction of protective immunity to plague by the rF1 and rV antigens.     

T cell responses to orbital antigens in thyroid-associated ophthalmopathy.

Thyroid-associated ophthalmopathy (TAO) is most likely to be a T cell-mediated disease, in which cytokines released in the extraocular muscles activate fibroblasts, increasing glycosaminoglycan production. The nature of the orbital antigen recognized by the infiltrating T cells is unclear, although it is possible that there is cross-reactivity between this and a thyroid autoantigen to explain the close association with thyroid autoimmunity. We have tested the ability of human and porcine eye muscle antigen preparations to stimulate proliferation of circulating T cells from healthy subjects and patients with TAO or Graves' disease without clinical TAO. Occasional responses were seen, particularly after depletion of CD8+ T cells, and two out of 10 TAO patients responded to eye muscle proteins of 25-50 kD after fractionation of antigens on gels and subsequent elution. There was no disease-specific response of T cells to R1, R14, D1 and 1D3, recombinant proteins identified from screening an eye muscle cDNA library with sera from patients with autoimmune thyroid disease. We have also found that interferon-gamma (IFN-gamma) production by T cells from TAO patients was not stimulated by eye muscle membrane antigens or by 1D3. These results suggest that the frequency of circulating T cells responding to eye muscle antigens in TAO is low, and that several candidate orbital antigens, including the 64-kD protein 1D3, are unlikely to be important T cell autoantigens in this condition.     

A mouse model of the atopic eczema/dermatitis syndrome by repeated application of a crude extract of house-dust mite Dermatophagoides farinae

BACKGROUND: We cultured Dermatophagoides farinae (Df), one of the most common mites in house dust and the most important allergen among natural allergens. With this material, we attempted to produce an animal model of the atopic eczema/dermatitis syndrome (AEDS). METHODS: We cultured Df mites in high density and prepared a crude extract of Df (DfE) together with the culture medium. We applied the extract to the back skin of NC/Nga and BALB/c mice three times per week for 8 weeks. RESULTS: In the NC/Nga group, dryness or scaling appeared on the skin, and scratching behavior increased at the second week in the DfE-treated group. Skin erosion and hemorrhage occurred at the fourth week. The epidermis thickened and deepened into the upper dermis, in which mast cells were highly accumulated, corresponding with the skin lesion of AEDS patients. Specific IgE and IgG to DfE and total IgE were elevated in the sera. Mice treated with an extract of mite culture medium did not develop skin lesions. In the BALB/c group, mice developed specific IgE and IgG to DfE, however, no typical skin lesions appeared. Mast cells in the upper dermis did not increase. CONCLUSIONS: Repeated painting of Dermatophagoides extract produced IgE-associated AEDS-like lesions on the skin of NC mice.     

Altered antigenicity of M-177, a 177-kDa allergen from the house dust mite Dermatophagoides farinae, in stored extract

BACKGROUND: A high molecular weight allergen, M-177 (177 kDa) was isolated from Dermatophagoides farinae using a specific antibody raised to an allergenic clone Mag 3, which was obtained by immunoscreening a mite cDNA library. The potent IgE reactivity of M-177 is comparable with that of Der f 2. OBJECTIVE: The aim of this study was to analyse the molecular characteristics and the allergenic activity of M-177 in stored mite extracts. METHODS: Antigens were analysed by immunoblotting and enzyme-linked immunosorbent assay (ELISA; inhibition). Allergenic activity was estimated from IgE reactivity and the results of a histamine release assay. RESULTS: The intact M-177 molecule was present in high concentrations in fresh extract obtained from purified mite bodies, but was only detected in small amounts in stored extracts. Instead of the intact molecule, anti-Mag 3 antibody detected various cross-reactive antigens in the stored preparations. Studies of a stored liquid extract showed that these cross-reactive antigens were produced by the degradation of M-177, and that this change was suppressed by the addition of protease inhibitors. Interestingly, the allergenic activity of the fragmented M-177 (sM-177) isolated from the stored extracts was greater than that of the intact antigen. Specific IgE reacted with sM-177 in 84.2% of 38 sera samples from patients allergic to mites, while 65.8% were positive for M-177-specific IgE. Similarly, the histamine release test showed that sM-177 had greater allergenic activity in vitro. ELISA inhibition indicated that the increased allergenic activity resulted from alteration of the antigenicity with the degradation of M-177. CONCLUSIONS: M-177 is a protease-sensitive allergen. The breakdown products of M-177 provoked higher allergenic activity than the intact allergen.     

T-cell sensitization to epitopes from the house dust mites Dermatophagoides pteronyssinus and Euroglyphus maynei

Background Dermatophagoides pteronyssinus and Euroglyphus maynei frequently occur in house dust but little is known about primary sensitization to the less abundant E. maynei. Objective To determine the occurrence of primary sensitization to E. maynei by T-cell responses and the crossreactivity to D. pteronyssinus. Methods The proliferative response ot peripheral blood cells to overlapping peptides from Derp I and Eur m 1 were measured as well as to peptides from Der f 1, an allergen not found in the study environment. Results The most frequent and strongest responses were to Der p 1 peptides especially in the region 105–133. However, 3/17 responders to mite peptides were stimulated predominantly by Eur m 1 peptides and a further two had their highest response to an Eur m 1 peptide. There was very little crossreactivity between Der p 1 and Eur m 1 peptides and very little response to peptides from Der f 1. Conclusion E. maynei group 1 allergens are a significant source of primary T-cell sensitization and have little T-cell crossreactivity with D. pteronyssinus or D. farinae.     

A monoclonal antibody to a determinant of the rat T cell antigen receptor expressed by a minor subset of T cells

A hybridoma producing the monoclonal antibody HIS42 was isolatedfrom a fusion between spleen cells from a BALB/c mouse immunizedwith rat thymocytes and the fusion partner SP2/0. This antibodyrecognizes a minor subset of T cells in every haplotype of rattested so tar. The subpopulation of HIS42 positive T cells containsboth CD4+ and CD8+ cells, in the same ratio as found in theperipheral T cell population. When bound to Sepharose beads,HIS42 induces T cell proliteration in the presence of Interleukin-2.In contrast to lymph node T cells, a number of thymocytes werefound to express HIS42 only in the cytoplasm or together withmembrane expression. Most bright HIS42 surface labelled thymocyteswere also positive for MRC OX-44, a marker predominantly identifyingmature thymocytes. SDS-PAGE analyses of the membrane moleculeslmmunoprecipitated by HIS42 show two bands on unreduced gels.One of these bands (85 kd) runs as two separate bands at 35and 48 kd on reduction. The other much weaker broad band ({smalltilde}100 kd) is hardly affected by reduced conditions. Takentogether these data suggest that HIS42 is directed against adeterminant on the rat T cell receptor for antigen, which iscommon to a small number of T cells.