House Dust Mite Species Research Articles

Multiplexed transcriptional profiling of Dermatophagoides house dust mites allergens in human epithelium cells.

Allergic asthma, a chronic disease characterized by airway inflammation, poses a significant public health concern. It is well-established that house dust mites (HDMs) are common inducers of allergic responses in individuals, particularly children. In central Taiwan, our research team observed that over 80% of allergic children exhibited sensitization to various HDMs species. This investigation aims to bridge the gap between these observations and a better understanding of the early fundamental mechanisms for preventing allergic diseases. Specifically, our study delves into the impact of crude extracts of HDMs on human epithelial BEAS-2B cells. Our findings, based on RNA sequencing (RNA-seq) analysis, shed light on how three major Dermatophagoides HDMs allergens activate a common Toll-like receptor signaling pathway in human epithelial cells within a 4-h treatment. During this process, the nuclear transcription factor NF-κB translocated into the cell nucleus within 30 min of allergen stimulation, triggering the expression of pro-inflammatory genes such as IL-6 and IL-8 over 4 h. Additionally, when the cells were treated with specific Dermatophagoides microceras (Der m) allergens, it resulted in the upregulation of genes that regulate type 1 diabetes mellitus (T1DM) signaling pathways. This led to the mediation of IL-12A inflammation. Furthermore, there was an increase in gene sets associated with cilia function and the microtubule cytoskeleton in human epithelial cells after treatment with a combination of Der m allergens and Dexamethasone. Additionally, OMICs analysis was conducted to examine the effects of HDMs allergenic stimulation on human epidermal cells. We aimed to improve our understanding of the molecular mechanisms within cells and identify potential targets and natural products in the treatment of asthma caused by HDMs allergens.

Dermatophagoides farinae microRNAs released to external environments via exosomes regulate inflammation-related gene expression in human bronchial epithelial cells.

Dermatophagoides farinae (DFA) is an important species of house dust mites (HDMs) that causes allergic diseases. Previous studies have focused on allergens with protein components to explain the allergic effect of HDMs; however, there is little knowledge on the role of microRNAs (miRNAs) in the allergic effect of HDMs. This study aimed to unravel the new mechanism of dust mite sensitization from the perspective of cross-species transport of extracellular vesicles-encapsulated miRNAs from HDMs. Small RNA (sRNA) sequencing was performed to detect miRNAs expression profiles from DFA, DFA-derived exosomes and DFA culture supernatants. A quantitative fluorescent real-time PCR (qPCR) assay was used to detect miRNAs expression in dust specimens. BEAS-2B cells endocytosed exosomes were modeled in vitro to detect miRNAs from DFA and the expression of related inflammatory factors. Representative dfa-miR-276-3p and dfa-novel-miR2 were transfected into BEAS-2B cells, and then differentially expressed genes (DEGs) were analyzed by RNA sequencing. Protein-protein interaction (PPI) network analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway and Gene Ontology (GO) terms enrichment analyses were performed on the first 300 nodes of DEGs. sRNA sequencing identified 42 conserved miRNAs and 66 novel miRNAs in DFA, DFA-derived exosomes, and DFA culture supernatants. A homology analysis was performed on the top 18 conserved miRNAs with high expression levels. The presence of dust mites and miRNAs from HDMs in living environment were also validated. Following uptake of DFA-derived exosomes by BEAS-2B cells, exosomes transported miRNAs from DFA to target cells and produced pro-inflammatory effects in corresponding cells. RNA sequencing identified DEGs in dfa-miR-276-3p and dfa-novel-miR2 transfected BEAS-2B cells. GO and KEGG enrichment analyses revealed the role of exosomes with cross-species transporting of DFA miRNAs in inflammatory signaling pathways, such as JAK-STAT signaling pathway, PI3K/AKT signaling pathway and IL-6-mediated signaling pathway. Our findings demonstrate the miRNAs expression profiles in DFA for the first time. The DFA miRNAs are delivered into living environments via exosomes, and engulfed by human bronchial epithelial cells, and cross-species regulation may contribute to inflammation-related processes.

Exosomes Derived from Dermatophagoides farinae Induce Allergic Airway Inflammation.

House dust mites (HDMs) are a major source of indoor allergens that cause airway allergic disease. Dermatophagoides farinae, a predominant species of HDMs in China, has demonstrated pathogenic role in allergic disorders. Exosomes derived from human bronchoalveolar lavage fluid have been strongly associated with allergic respiratory diseases progression. However, the pathogenic role of D. farinae-derived exosomes in allergic airway inflammation has remained unclear until now. Here, D. farinae was stirred overnight in phosphate-buffered saline, and the supernatant was used to extract exosomes by ultracentrifugation. Then, shotgun liquid chromatography-tandem mass spectrometry and small RNA sequencing were performed to identify proteins and microRNAs contained in D. farinae exosomes. Immunoblotting, Western blotting, and enzyme-linked immunosorbent assay demonstrated the specific immunoreactivity of D. farinae-specific serum IgE antibody against D. farinae exosomes, and D. farinae exosomes were found to induce allergic airway inflammation in a mouse model. In addition, D. farinae exosomes invaded 16-HBE bronchial epithelial cells and NR8383 alveolar macrophages to release the inflammation-related cytokines interleukin-33 (IL-33), thymic stromal lymphopoietin, tumor necrosis factor alpha, and IL-6, and comparative transcriptomic analysis of 16-HBE and NR8383 cells revealed that immune pathways and immune cytokines/chemokines were involved in the sensitization of D. farinae exosomes. Taken together, our data demonstrate that D. farinae exosomes are immunogenic and may induce allergic airway inflammation via bronchial epithelial cells and alveolar macrophages. IMPORTANCE Dermatophagoides farinae, a predominant species of house dust mites in China, has displayed pathogenic role in allergic disorders, and exosomes derived from human bronchoalveolar lavage fluid have been strongly associated with allergic respiratory diseases progression. However, the pathogenic role of D. farinae-derived exosomes in allergic airway inflammation has remained unclear until now. This study, for the first time, extracted exosomes from D. farinae, and sequenced their protein cargo and microRNAs using shotgun liquid chromatography-tandem mass spectrometry and small RNA sequencing. D. farinae-derived exosomes trigger allergen-specific immune responses and present satisfactory immunogenicity, as revealed by immunoblotting, Western blotting, and enzyme-linked immunosorbent assay and may induce allergic airway inflammation via bronchial epithelial cells and alveolar macrophages. Our data provide insights into the mechanisms of allergic airway inflammation caused with D. farinae-derived exosomes and the treatment of house dust mite-induced allergic airway inflammation.

Cloning and bioinformatics analysis of aquaporin proteins in the European house dust mite, Dermatophagoides pteronyssinus

Aquaporins are highly conserved water channel proteins involved in the transport of solutes across membranes. The few aquaporins identified to date play major roles in excretion in insects. Excretion is a major source of indoor allergens from the house dust mite species, but, to date, aquaporins have not been well studied in these species. We used RNA sequencing to study the transcriptome of the house dust mite Dermatophagoides pteronyssinus to identify putative aquaporin family members. RNA was isolated from more than 2000 mites for 5΄-RACE and 3΄-RACE. Putative aquaporins were identified through BLAST alignments, and primers were designed to amplify the five identified sequences. These cDNA fragments were then inserted into bacterial plasmids for molecular cloning and sequencing of DerpAQP1, DerpAQP2, DerpAQP3, DerpAQP4, and DerpAQP5 (GenBank accessions nos. KY305293, KY305294, KY305295, KY305296, and KY305298, respectively). Phylogenetic analysis of cloned sequences revealed the clustering of these putative aquaporins into two groups with human aquaporins (hAQPs): DerpAQP3 was clustered with hAQP8, hAQP11, and hAQP12; and DerpAQP1, DerpAQP2, DerpAQP4, DerpAQP5 were clustered with hAQP3, hAQP7 hAQP9, and hAQP10. This study provides a foundation for future studies of the importance of these proteins to dust mite functions and allergenicity.

The Allergenic Activity of Blo t 2, a Blomia tropicalis IgE-Binding Molecule

Only few allergens derived from house dust mite (HDM) species have been evaluated in terms of their potential to induce allergic inflammation. In this study, we aimed to evaluate different aspects of the allergenicity and allergenic activity of Blo t 2, a Blomia tropicalis allergen. Blo t 2 was produced as a recombinant protein in Escherichia coli. Its allergenic activity was tested in humans by skin prick test and basophil activation assays, and in mice, by passive cutaneous anaphylaxis and a model of allergic airway inflammation. Sensitization rate to Blo t 2 (54.3%) was similar to that found to Blo t 21 (57.2%) and higher than to Der p 2 (37.5%). Most Blo t 2-sensitized patients showed a low intensity response (99.5%). Blo t 2 elicited CD203c upregulation and allergen induced skin inflammation. Additionally, immunized animals produced anti-Blo t 2 IgE antibodies and passive transfer of their serum to non-immunized animals induced skin inflammation after allergen exposure. Immunized animals developed bronchial hyperreactivity and a strong inflammatory lung reaction (eosinophils and neutrophils). These results confirm the allergenic activity of Blo t 2 and supports its clinical relevance.

Current advances in house dust mite allergen immunotherapy (AIT): Routes of administration, biomarkers and molecular allergen profiling.

Allergy to house dust mites (HDM) is a perennial respiratory disease that affect more than half a billion people worldwide. Dermatophagoides pteronyssinus and D. farinae, two HDM species, are major sources of indoor allergens triggering allergic inflammation. Although symptomatic drugs are widely used to block the allergic reaction, allergen immunotherapy is the only curative treatment of IgE-mediated type I respiratory allergies. In this article, we review recent advances in various routes of allergen immunotherapy. We particularly focus on subcutaneous (SCIT) and sublingual (SLIT) immunotherapy, used as a reference therapy since they have transformed allergic treatments by improving symptoms (asthma and rhinitis) as well as the quality of life of patients. We also highlight recent data in more exploratory routes (i.e., oral, intralymphatic, epicutaneous and intradermal) and discuss respective advantages of various route, as well as their foreseen modes of action. Finally, we provide an update on biomarkers as well as on the relevance of the molecular profiling of allergic individuals related to treatment efficacy or asthma prediction.

Population growth and respiration in the dust mite Dermatophagoides farinae under different temperature and humidity regimes.

Dermatophagoides farinae is an important house dust mite species that causes allergies in humans worldwide. In houses, these mites are commonly found in actively used mattresses and pillows, which provide food (i.e. sloughed skin and microorganisms), moisture, and increased temperature for faster mite development. In mattresses, feeding mites prefer the upper sector, as close as possible to the resting human (temperature 32-36°C, humidity between 55 and 59%). However, mites that are not actively feeding prefer staying at deeper zones of the mattress. Here, we analyzed mite responses to different temperatures (15-35°C) and relative humidity (62-94% RH) in terms of their population size growth and respiration (CO2 production) using lab mite cultures. The intrinsic rate of population increase had a single maximum at approximately 28°C and 85% RH. At 30°C, there were two respiration peaks at RH 90% (smaller peak) and 65% (larger peak). Therefore, there is a mismatch between the optimal temperature/humidity for the population size increase vs. respiration. We propose preliminary hypotheses explaining the two respiration peaks and suggest that future research should be done to elucidate the nature of these peaks.

Recovery of Dermatophagoides pteronyssinus (Acariformes: Astigmata: Pyroglyphidae) is influenced by vacuuming protocol, fabric type and the viability of dust mites

Dermatophagoides pteronyssinus (Trouessart, 1897) (Acariformes: Astigmata: Pyroglyphidae) is amongst the most common house dust mites (HDMs) species in Europe. Vacuuming is commonly employed as a method for the recovery for the subsequent quantification of mite densities. The vacuuming protocols employed and their efficiencies are frequently unreported. In this study, a constant-flow vacuum sampler was used to evaluate the recovery of laboratory-reared D. pteronyssinus from cotton, denim or fleece. Two vacuuming protocols were employed. Protocol 1 (P1) entailed a single unidirectional pass of the vacuum head across the fabrics for 60, 90 or 120 seconds. For Protocol 2 (P2), fabrics were vacuumed twice (laterally followed by diagonally) for the same time periods. Vacuuming was carried out on fabrics seeded with live mites (LM), dead mites (DM), and live mites, which were applied to fabric surfaces for 10 minutes and then euthanized by freezing prior to vacuum sampling (EM). There was a significant difference (P<0.001) in mean HDMs recovery with regard to mite viability (LM, DM, EM), fabric type (cotton, denim, fleece), duration of sampling (60, 90, 120s) and vacuuming protocols employed (P1, P2). The numbers of mites recovered from fleece (compared to cotton and denim) were lower for each treatment type where LM and EM were deployed. Overall, the use of protocol 2 resulted in the greatest mean percentage recovery (>80%) of dust mites for all fabrics combined. Recovery rates were also influenced by entrapment of mites (or active attachment) in the fabric microstructure within the weave. The results have implications for the standardisation of mite recovery from fabrics by means of vacuuming.

Profile of aeroallergen sensitizations in allergic patients living in southern Vietnam.

Climatic and geographical characteristics may alter the plant distribution and thereby the patterns of allergens. To evaluate the profile of allergen sensitization in patients in southern Vietnam and its association with allergic diseases. We collected data of 423 patients who visited the Unit of Allergy and Clinical Immunology, University Medical Center, Vietnam from 2014 to 2021, from their medical records. Patients underwent skin prick tests to the 12 most common aeroallergens. Clinical evaluation and diagnosis of allergic diseases was done in consert with their allergen sensitization status. Mites and cockroach were the most prevalent sensitizing allergens, with the sensitization prevalences as followed: Dermatophagoides farinae (Df) (59.8%), Dermatophagoides pteronyssinus (Dp) (50.4%), Blomica tropicalis (Bt) (49.6%), storage mites mix (10.4%), and cockroach (10.2%). Sensitization to Df was more predominant in males than in females (66% vs 54.1%). Dp-sensitized patients were younger than non-sensitized patients (29.01 ± 13.60 vs. 32.17 ± 14.89) whereas storage mites-sensitized patients were older than the non-sensitized groups (36.25 ± 13.53 vs. 28.76 ± 13.39) (p < 0.05 for all). A considerable proportion of patients with urticaria, allergic rhinitis, and atopic dermatitis were sensitized to mites. Polysensitization to different species of house dust mites (Dp, Df) and storage mites (Bt) was prevalent among patients sensitized to any kind of mites. Among people living in southern Vietnam, HDM mites, and cockroach were the predominant allergens. Further studies on the factors regulating the association between allergen sensitization with allergic diseases and polysensitization are crucial.

Screening of B-cell epitopes of Der-p1 and Der-p2 major aeroallergens by computational approach for designing immunotherapeutics

Introduction and Aim: Allergic diseases are IgE-mediated hypersensitivity reactions affecting approximately 30% of the general population globally. Dermatophagoides pteronyssinus (Der-p) is the most prevalent house dust mite (HDM) species consisting of 23 mite allergen groups. Among these, group 1 and 2 are major allergenic proteins, which causes allergic asthma in 80% of sensitized individuals, with elevated IgE titres in the serum. This study involves in silico analysis of potential B-cell epitopes of group 1 and group 2 of Der-p, which can be utilized in designing immunotherapeutic vaccines. Materials and Methods: Allergen sequences obtained from the database- International Union of Immunological Societies (IUIS), for predicting of B-cell epitopes. The physiochemical properties and secondary structures of the obtained sequence were evaluated. The sequences were further subjected to determining antigenicity, surface accessibility, and prediction of linear and discontinuous B-cell epitope by utilizing IEDB tools. Results: The linear and discontinuous B-cell epitopes of Der-p1 and Der-p2 aeroallergen were predicted. Further, Der-p1 and Der-p2 showed 6 linear epitopes each respectively. Conformational epitopes predicted were 123 of Der-p1 and 72 of Der-p2 respectively, by the ElliPro tool. Based on the structure, antigenicity, and surface accessibility, only 10% of Der-p1 and Der-p2 which binds to B-cell epitopes are linear and the majority are discontinuous. Conclusion: The linear and conformational epitopes of Der-p1 and Der-p2 are predicted using in silico tools. These identified epitopes might be useful for developing epitope-based immunotherapeutics for HDM allergy.

Early Atopic Sensitization to House-Dust Mite in Children with Recurrent Wheeze-A Cross-Sectional Study.

To determine sensitization to house-dust mite (HDM) antigen in under-five children with recurrent wheeze, compare it with nonwheezers, and assess atopic comorbidities in them. A cross-sectional study was done in the Pediatric department of a teaching hospital in North India, in 190 children aged 1-5 y. Out of these, 127 had recurrent wheeze (RW), and 63 had no wheeze (NW). Sensitivity was done by skin prick test (SPT) for two dust mites antigens: Dermatophagoide farinae and Dermatophagoide pteronyssinus antigens. In addition, atopic comorbidities like atopic dermatitis and allergic rhinitis were assessed. Mean age of the study population was 34.52 ± 20.50 mo. SPT positivity for either of the dust mites was 97 (76.4%) in RW and 13 (20.6%) in NW which was significant (p < 0.001, aOR = 12.27). HDM species sensitization for D. pteronyssinus was 55.1% vs. 15.9% (p < 0.001 aOR = 7.81) and D. farinae was 39.4% vs. 9.5% (p < 0.001, aOR = 5.45) in groups, respectively. Mean wheal size in RW Group was also significantly higher than NW group for D. pteronyssinus (2.39 ± 1.44 vs. 0.52 ± 1.19mm, median (IQR) 3 (1-3), p < 0.001), D. farinae (1.80 ± 1.39 vs. 0.32 ± 1.00mm, median (IQR) 2 (0-3), p < 0.001). Allergic rhinitis was present in 55 (43.3%) vs. 7 (11.1%) (p < 0.001), atopic dermatitis in 28 (22%) vs. 2 (3.2%) (p = 0.001) in group 1 and 2, respectively. All children with allergic rhinitis had HDM sensitization in both groups. This study showed early sensitization to HDM in children with recurrent wheeze. Atopic comorbidities were also present in them.

Integrated OMICs Approach for the Group 1 Protease Mite-Allergen of House Dust Mite Dermatophagoides microceras.

House dust mites (HDMs) are one of the most important allergy-causing agents of asthma. In central Taiwan, the prevalence of sensitization to Dermatophagoides microceras (Der m), a particular mite species of HDMs, is approximately 80% and is related to the IgE crossing reactivity of Dermatophagoides pteronyssinus (Der p) and Dermatophagoides farinae (Der f). Integrated OMICs examination was used to identify and characterize the specific group 1 mite-allergic component (Der m 1). De novo draft genomic assembly and comparative genome analysis predicted that the full-length Der m 1 allergen gene is 321 amino acids in silico. Proteomics verified this result, and its recombinant protein production implicated the cysteine protease and α chain of fibrinogen proteolytic activity. In the sensitized mice, pathophysiological features and increased neutrophils accumulation were evident in the lung tissues and BALF with the combination of Der m 1 and 2 inhalation, respectively. Principal component analysis (PCA) of mice cytokines revealed that the cytokine profiles of the allergen-sensitized mice model with combined Der m 1 and 2 were similar to those with Der m 2 alone but differed from those with Der m 1 alone. Regarding the possible sensitizing roles of Der m 1 in the cells, the fibrinogen cleavage products (FCPs) derived from combined Der m 1 and Der m 2 induced the expression of pro-inflammatory cytokines IL-6 and IL-8 in human bronchial epithelium cells. Der m 1 biologically functions as a cysteine protease and contributes to the α chain of fibrinogen digestion in vitro. The combination of Der m 1 and 2 could induce similar cytokines expression patterns to Der m 2 in mice, and the FCPs derived from Der m 1 has a synergistic effect with Der m 2 to induce the expression of pro-inflammatory cytokines in human bronchial epithelium cells.

Contribution of cysteine and serine proteases to proteolytic digestion in an allergy-eliciting house dust mite

The digestive physiology of house dust mites (HDM) is of interest to understand their allergenicity towards humans since many of their allergens are digestive enzymes and/or are excreted into airborne fecal pellets. The aim of this study is to provide insight on the biochemical basis of proteolytic digestion in Dermatophagoides pteronyssinus, the most widespread HDM species. First, assays using non-specific protein substrates on purified fecal and body extracts determined that body-associated activity is almost exclusively dependent on cysteine proteases, and specifically on major allergen Der p 1. By contrast, cysteine and serine proteases contributed similarly to the activity estimated on fecal extracts. Second, the screening of group-specific peptide-based protease inhibitors followed by ingestion bioassays revealed that the human skin-derived cysteine protease inhibitor cystatin A produces a significant reduction in mite feeding (i.e. excreted guanine), and triggers the overproduction of Der p 1 (3-fold increase by ELISA). Noteworthy, the inhibition of cysteine proteases by cystatin A also resulted in a reduction in three non-target serine protease activities. Further incubation of these extracts with exogenous Der p 1, but not with other commercial cysteine proteases, restored trypsin (Der p 3) and chymotrypsin (Der p 6) activities, indicating that Der p 1 is responsible for their activation in vivo. Finally, the role of serine proteases on the mite's digestive physiology is discussed based on their remarkable activity in fecal extracts and the autocoprophagic behavior reported in mites in this study.

The Influence of Indoor Environmental Factors on the Prevalence of House Dust Mites as Aeroallergens in Bandar Abbas Student Dormitories, South of Iran, 2019.

This study was conducted to determine the influence of environmental factors on the prevalence of house dust mites in student dormitories of Bandar Abbas city. In this study, 64 dust samples were collected from seven randomly selected dormitories located in various areas of the Bandar Abbas. The collected mites were isolated and mounted in Hoyer's medium and identified using a morphological key. The associations between the environmental factors and the density of house dust mites were investigated. In total, 1,093 adult mites were collected and identified. They consisted of four species including Dermatophagoides pteronyssinus Trouessart (57.6%), Dermatophagoides farinae Hughes (24.3%) and Dermatophagoides evansi Fain (14.9%) (Acari: Pyroglyphidae), and Cheyletus malaccensis Oudemans (3.2%) (Acari: Cheyletidae). All of the dormitories were contaminated by more than one house dust mites species and the mean density of house dust mites in dormitories was 8.3 ± 0.2 mites/g of dust. There was a significant relationship between average house dust mites density and some of environmental factors such as relative humidity, temperature, floor covering type, and number of occupants (P < 0.05). Results of this study revealed that two major allergenic dust mites, D. pteronyssinus and D. farinae, were the most prevalent and collected from all of dormitories and some of indoor environmental factors found to influence mites' population.

Development of a new multiplex PCR to detect prevalent species of house dust mites in house dust

ABSTRACT Dermatophagoides pteronyssinus and Dermatophagoides farinae are the most common House Dust Mite (HDM) species in home environments worldwide and responsible for HDM allergy. Since the prevalence of HDM-related clinical conditions is linked to exposure to the mite itself, the detection of HDM in the human households gains importance. We aimed to develop a fast and accessible multiplex PCR to detect and distinguish two relevant HDM species in house dust. New primers were designed, and sensitivity analysis was performed. Sequencing of PCR products was also performed to confirm the method’s specificity. The limit of detection of the multiplex PCR for both species was as low as 30 pg µL−1. The application of the multiplex PCR to dust samples also resulted in the identification of both species with high sensitivity. The protocol required small amount of template, reagents and a short reaction time thus presenting an alternative to classically used techniques for HDM identification.

The Density of Dermatophagoides sp. in Households and its Correlation with the Score for Allergic Rhinitis in Jember, East Java

The prevalence of allergic rhinitis (AR) in Indonesia is an estimated 24.3% and increasing each year. The majority cause of AR is house dust mites (HDMs) allergens, especially Dermatophagoides sp. The screening tool to estimate the prevalence and diagnose AF in a population setting is the Score for Allergic Rhinitis (SFAR) questionnaire. This study aimed to investigate the correlation between the density of Dermatophagoides sp. in households and the SFAR in Jember, East Java. The study enrolled 30 housewives as respondents, which were selected by stratified random sampling. Respondents were interviewed using the SFAR questionnaire. The dust sample from each house of the respondent was collected, the HDMs either Dermatophagoides sp. or others were identified and the density of Dermatophagoides sp. was calculated. Data were analyzed using the Pearson Test. Dermatophagoides sp. were found in 21 out of 30 samples and 11 out of 30 respondents had an SFAR of ≥ 7. The Pearson analysis showed the value of p = 0.555 and r = 0.136. Dermatophagoides sp. was the majority of species of HDMs, but there was no significant correlation between the density of Dermatophagoides sp. in households and the SFAR among housewives in Jember.

&lt;p class="Body"&gt;&lt;strong&gt;Sensitivity in allergic asthmatic subjects towards house dust mite allergens&lt;/strong&gt;&lt;/p&gt;

Asthma is a common problem that affects about 20 million peoples in India and can be often under-diagnosed or misdiagnosed. It can be allergic or non-allergic though the former type is more common and prevalent. Allergic asthma can be triggered by many allergens and house dust mites (HDM) are one of the common indoor allergens. The present study emphasizes the significance of house dust mites in allergic asthmatic subjects which is based on 115 asthmatic subjects in Punjab, India. For the quantification and the estimation of total serum Immunoglobulin E and HDM specific IgE, a mixture of 14 allergens and a mixture of two mite allergens viz. Dermatophagoides pteronyssinus and D. farinae were used respectively. Total and specific IgE levels were detected on ImmunoCAP Phadia 100. A statistically significant correlation between total and HDM specific IgE levels of 115 asthmatic subjects was found as compared to control group of 30 non-allergic individuals. The specific IgE levels of 54.78% subjects against the allergen of two mite species were found to be positive. Dust samples were taken from various localities of the houses to identify the diversity of house dust mites which were responsible for allergic asthma. Five common house dust mite species viz. D. pteronyssinus Trouessart, D. farinae Hughes, D. microceras Griffiths and Cunnington, D. aureliani Fain and Euroglyphus maynei Cooreman were identified from the dust. The present study observed that total IgE levels were higher with higher specific IgE levels against the mixture of two mite allergens viz. D. pteronyssinus and D. farinae in the blood serum. D. pteronyssinus was the most abundant and prevalent mite species followed by D. farinae. Therefore, present study concluded that HDM specific IgE levels against the mite allergen of D. pteronyssinus and D. farinae in the serum of allergic asthmatic subjects were found to be higher because of the higher prevalence of these two mites (D. pteronyssinus i.e. 69.80% and D. farinae i.e. 20.72%) in the house of allergic asthmatic subjects as compared to other identified mites.

Expression in Escherichia coli and Purification of Folded rDer p 20, the Arginine Kinase From Dermatophagoides pteronyssinus: A Possible Biomarker for Allergic Asthma.

Arginine kinase (AK) was first identified as an allergen in the Indian-meal moth and subsequently shown to occur as allergen in various invertebrates and shellfish. The cDNA coding for AK from the house dust mite (HDM) species Dermatophagoides pteronyssinus, Der p 20, has been isolated, but no recombinant Der p 20 (rDer p 20) allergen has been produced and characterized so far. We report the expression of Der p 20 as recombinant protein in Escherichia coli. rDer p 20 was purified and shown to be a monomeric, folded protein by size exclusion chromatography and circular dichroism spectroscopy, respectively. Using AK-specific antibodies, Der p 20 was found to occur mainly in HDM bodies, but not in fecal particles. Thirty percent of clinically well-characterized HDM allergic patients (n = 98) whose immunoglobulin E (IgE) reactivity profiles had been determined with an extensive panel of purified HDM allergens (Der f 1, 2; Der p 1, 2, 4, 5, 7, 10, 11, 14, 15, 18, 21, 23 and 37) showed IgE reactivity to Der p 20. IgE reactivity to Der p 20 was more frequently associated with lung symptoms. AKs were detected in several invertebrates with specific antibodies and Der p 20 showed IgE cross-reactivity with AK from shrimp (Litopenaeus vannamei). Thus, Der p 20 is a cross-reactive HDM allergen and may serve as a diagnostic marker for HDM-induced lung symptoms such as asthma.

Identification of continuous immunoglobulin G epitopes of Dermatophagoides farinae allergens by peptide microarray immunoassay.

Dermatophagoides farinae, as a common house dust mite species, is one of the main sources of allergens in the world. At present, Dermatophagoides farinae is found to contain more than 30 groups of allergens. These allergens are used for allergen-specific immunotherapy (AIT) of allergic diseases. During the AIT process, immunoglobulin G (IgG) antibodies can block immunoglobulin E (IgE) antibody-induced allergic reactions in the human body. One of the mechanisms may be that IgG and IgE competitively bind to the same allergic protein, so it is necessary to explore the binding sites (epitopes) of IgG antibodies to allergens. In this study, peptide arrays were constructed to react with the serums from patients with allergic asthma to find the IgG epitopes of several allergens including major allergens (Der f 1, 2) and mid-tier allergens (Der f 4, 5, and 7), and then verified by enzyme-linked immunosorbent assay (ELISA) test. Relevant epitopic sequences were located on the tertiary structure of individual allergens, as reconstructed by homology modeling. One IgG epitope of Der f 1 (90-106aa, NVPSELDLRSLRTVTPI), five IgG epitopes of Der f 4 (61-77aa, ERYQPVSYDIHTRSGDE; 193-209aa, FRSDASTHQWPDDLRSI; 226-242aa, HPFIYHETIYYGGNGIN; 271-287aa, LRWLRNFGTEWGLVPSG; 352-368aa, NDWVGPPTDQHGNILSV), and one IgG epitope of Der f 5 (84-101aa, RYNVEIALKSNEILERDL) were identified. IgG epitopes of Der f 2, 7 were not found. There are overlaps between the IgG and IgE epitopes of Der f 1, 4, and 5. These findings not only reflect the practicality of peptide array and ELISA test in the allergen IgG epitope identification, but also provide more information for further understanding of the human immunological changes during AIT and the molecular mechanisms of IgG blocking IgE activity.

Identification and Characterization of Immunogenic RNA Species in HDM Allergens that Modulate Eosinophilic Lung Inflammation.

Environmental allergens such as house dust mites (HDM) are often in complex forms containing both allergic proteins that drive aberrant type 2 responses and microbial substances that induce innate immune responses. These allergen-associated microbial components play an important role in regulating the development of type 2 inflammatory conditions such as allergic asthma. However, the underlying mechanisms remain largely undefined. The protocol presented here determines the structural characteristics and in vivo activity of allergen-associated immunostimulatory RNA. Specifically, common allergens are examined for the presence of double-stranded RNA (dsRNA) species that can stimulate IFN responses in lungs and restrainthe development of severe lung eosinophilia in a mouse model of HDM-induced allergic asthma. Here, we have included the following three assays: Dot blot to show the dsRNA structures in total RNA isolated from allergens including HDM species, RT-qPCR to measure the activities of HDM RNA in interferon stimulating genes (ISGs) expression in mouse lungs and FACS analysis to determine the effects of HDM RNA on the number of eosinophils in BAL and lung, respectively.