Rapid visual detection of the allergen Dermatophagoides farinae in house dust by loop-mediated isothermal amplification.

Dermatophagoides farinae is considered to be an important factor causing some allergic diseases, such as urticaria, allergic rhinitis, asthma, and other interrelated diseases. Avoiding exposure to allergens is the most effective way to reduce allergic reactions. In this study, we successfully established a loop-mediated isothermal amplification (LAMP) method for the detection of D. farinae DNA target internal transcribed spacer (ITS) and D. farinae 1 allergen (Der f 1) genes. The turbidity-monitoring system and visual fluorescent reagents were used to verify the test results of LAMP assay. Following optimization of the primers and reaction temperatures, the amplification sensitivity, specificity, and efficiency of the method for detecting D. farinae were assessed. There was no cross-reaction with other arthropod species that are commonly found in indoor environmental dust, such as Dermatophagoides pteronyssinus, Alophagoides ovatus, Periplaneta americana, Anopheles sinensis, and Musca domestica. Furthermore, the sensitivity of LAMP assay for detecting D. farinae DNA was 10 times greater than that of conventional PCR. The positive detection rate by the LAMP method was greater than the conventional PCR for both single D. farinae mites and D. farinae mites in indoor dust. A new type of LAMP method for D. farinae based on the Der f 1 and ITS genes was, therefore, successfully established. This study is the first time to detect the D. farinae allergen using LAMP assay. This assay could be useful as a model for the rapid detection of allergens produced by other house dust mites in the future.

Allergen detection and logistic multifactor analysis of allergic rhinitis.

OBJECTIVE: This study aims to investigate the allergens in children with allergic rhinitis (AR) and AR-related influencing factors. PATIENTS AND METHODS: The clinical data of 230 children with AR admitted to our hospital from June 2020 to June 2021 were retrospectively analyzed and included in the observation group. The clinical data of 230 healthy children during the same time period were included as the control group. All children had been tested for allergens using serum allergens, and the clinical data were collected by telephone questionnaires. Univariate and multivariate logistic regression were used to analyze the risk factors affecting AR. RESULTS: A total of 230 children with AR was included in this study, and some of them had two or more allergens. The proportion of house dust mite was the highest among the inhaled allergens, about 75.22%. Shrimp accounted for the highest proportion of food allergens, about 40.87%. Compared with the control group, the proportion of floating population, home heating, allergy history, asthma and other general information in the observation group was higher. At the same time, the proportion of environmental factors such as second-hand smoke, number of residents (≤ 3), daily ventilation and cleaning (no), domestic animals, domestic plants, decoration within 2 years, and living environment (rural) in the observation group was higher. In addition, the proportion of family factors such as delivery mode (cesarean section), family history of allergic rhinitis, parents' education level (middle school and above) in the observation group was higher (p < 0.05). Univariate logistic regression analysis showed that allergic history, asthma, second-hand smoke, floating population, number of residents, domestic animals, decoration within 2 years, delivery mode, and family history of allergic rhinitis were the risk factors affecting the incidence of AR in children (p < 0.05), and daily window ventilation and cleaning were the protective factors (p < 0.05). The multivariate logistic regression analysis showed that asthma, second-hand smoke, floating population, decoration within 2 years, family history of allergic rhinitis and domestic animals were independent risk factors for the occurrence of AR (p < 0.05), and daily ventilation and cleaning were protective factors for the occurrence of AR in children (p < 0.05). CONCLUSIONS: The proportion of house dust mite in inhalation allergens and shrimp in food allergens were the highest in AR children. The incidence of AR was closely related to asthma, second-hand smoke, floating population, decoration within 2 years, family history of AR and domestic animals, etc. Targeted measures could effectively prevent the occurrence and recurrence of AR. At the same time, daily ventilation and cleaning were the protective factors which could reduce the incidence and occurrence of AR in children.

Detection and assessment of dust mite allergens in an indoor environment in Anhui, China.

Dust in the home environment is thought to be a potential trigger for increasing allergic diseases, such as allergic rash, rhinitis, asthma, and other conditions, associated with dust mites. To verify the status of dust mite prevalence in indoor surroundings, we collected 189 dust samples from the air conditioner filters (n = 75) and floors (n = 114) of households, schools, and hotels in the Anhui area, China. All samples were measured for dust mite breeding rate and breeding density under light microscopy and analyzed for dust mite species Dermatophagoides farinae 1 (Der f 1) and Dermatophagoides pteronyssinus 1 (Der p 1) allergen using enzyme-linked immunosorbent assay (ELISA). The dust mite breeding rates were 34.67% (26/75) and 20.18% (23/114), respectively, in the dust samples from the floor and air conditioning filters. The breeding density was the highest in households (10/g), followed by schools (9/g) and hotels (4/g). ELISA indicated that the allergen threshold (2.0 µg/g dust) of Der f 1 was exceeded in only two samples and Der p 1 in one sample. Additionally, a questionnaire was used to investigate the health knowledge on allergic diseases involved in indoor facilities, finding that most allergy sufferers were aware that indoor dust might be responsible for their conditions. The findings suggest that regular maintenance of indoor hygiene and cleaning of air-conditioning filters should reduce the risks of exposure to indoor allergens.

House dust mite allergens and nitrated products: Identification and risk assessment in indoor dust.

Air pollutants can potentially lead to nitration of allergic proteins, thus promoting sensitization of these allergens. However, little is currently known about the nitration status of house dust mite (HDM) allergens. We identified the occurrence of nitrated products of two major HDM allergens Der f 1 and Der p 1 in dust samples collected from college dormitories in eastern China and assessed their associated health risk. The results showed that both non-nitrated and nitrated forms of the two allergens were detected in the dust in the range of non-detected (ND)-10.6, 1.44-15.4, ND-22.4, ND-7.28 µg/g for non-nitrated Der f 1, nitrated Der f 1, non-nitrated Der p 1 and nitrated Der p 1, respectively. The median rates of nitration were determined as 74.0% for Der f 1 and 20.4% for Der p 1 at consideration of one nitration site. Further analysis reveals that the levels of HDM allergens and their nitrated products were found to be generally higher during winter, in dormitories of lower altitude and with female occupants. Furthermore, the calculated risk indexes were at considerably high levels. Our findings suggest that nitrated HDM allergens have already accumulated in the environment at such significant levels and their associated health risk calls for our immediate attention.

Simultaneous assessment of organophosphate flame retardants, plasticizers, trace metals, and house dust mite allergens in settled house dust.

Settled house dust (SHD) is a reservoir for various contaminants, including endocrine-disrupting chemicals (EDCs), trace metals, and house dust mite allergens. This study aimed to characterize various chemical and biological contaminants in SHD and identify determinants governing the indoor contaminants. In total, 106 SHD samples were collected from 106 houses in Seoul and Gyeonggi Province, Korea, in 2021. Bedding dust samples were collected from 30 of these 106 houses. All participants completed a questionnaire comprised of housing and lifestyle-related factors. The samples were analyzed for 18 organophosphate flame retardants (OPFRs), 16 phthalates, five alternative plasticizers (APs), seven trace metals, and two house dust mite allergens (Dermatophagoides farinae type 1 [Der f1] and Dermatophagoides pteronyssinus type 1 [Der p1]). A multiple regression analysis was conducted to identify the determinants governing the concentrations and profiles of various contaminants. OPFRs, phthalates, APs, and trace metals were detected in all SHD samples, indicating ubiquitous contamination in indoor environments. Among the three EDC groups, APs were detected at the highest concentrations (geometric mean [GM] (geometric standard deviation, [GSD]): 1452 (1.6) µg/g in total), followed by phthalates (GM (GSD): 676 (1.4) µg/g in total) and OPFRs (GM (GSD): 10 (1.4) µg/g in total). Der f1 was detected in all bedding dust samples with significantly higher levels than Der p1 (GM (GSD): 0.1 (1.8) µg/g vs. 1.4 × 10-3 (2.3) µg/g). The concentrations of OPFRs, plasticizers, and trace metals in SHD were significantly associated with the type and number of electronic appliances and combustion activities. Der f1 was significantly associated with the number of occupants and water penetration. Ventilation, vacuum cleaning, and wet cleaning or dry mopping significantly reduced the levels of most contaminants in SHD. As residents are persistently exposed to a wide array of pollutants, comprehensive and adequate measures are required to prevent potential exposures.

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.

Vishniacozyma victoriae (syn. Cryptococcus victoriae) in the homes of asthmatic and non-asthmatic children in New York City.

BACKGROUND: Indoor environments contain a broad diversity of non-pathogenic Basidiomycota yeasts, but their role in exacerbating adverse health effects has remained unclear. OBJECTIVE: To understand the role of Vishniacozyma victoriae exposure and its impact on human health. METHODS: A qPCR assay was developed to detect and quantify an abundant indoor yeast species, Vishniacozyma victoriae (syn. Cryptococcus victoriae), from homes participating in the New York City Neighborhood Asthma and Allergy Study (NAAS). We evaluated the associations between V. victoriae, housing characteristics, and asthma relevant health endpoints. RESULTS: V. victoriae was quantified in 236 of the 256 bedroom floor dust samples ranging from less than 300-45,918 cell equivalents/mg of dust. Higher concentrations of V. victoriae were significantly associated with carpeted bedroom floors (P = 0.044), mean specific humidity (P = 0.004), winter (P < 0.0001) and spring (P = 0.001) seasons, and the presence of dog (P = 0.010) and dog allergen Can f 1 (P = 0.027). V. victoriae concentrations were lower in homes of children with asthma vs. without asthma (P = 0.027), an association observed only among the non-seroatopic children.

Occupants' Health and Their Living Conditions of Remote Indigenous Communities in New Zealand.

The New Zealand Ministry of Health reported that respiratory disease affects 700,000 people, annually costs New Zealand NZ$7.05 billion, and is the third-highest cause of death. The hospitalisation rate for asthma of Maori communities is 2.0 higher than that of other ethnic groups, and hospitalisation rates for deprived homes are 2.3 times higher than those of the least deprived homes. Based on physical data and evidence, which were drawn from a mixed methodology that includes field studies of the indoor microclimate, dust-mite allergens, mould growth, and occupants' Respiratory Health Survey of a number of sample houses of Maori communities in Minginui, Te Whaiti, Murupara, and Rotorua of New Zealand, the study identifies unhealthy indoor thermal conditions, thresholds or ranges of indoor micro-climate related to different levels of dust-mite allergen and mould growth, the most common type of indoor mould, and correlations between dust-mite and mould and correlations. The study not only identified that the poor health of occupants is closely related to their inadequate living conditions, but also identifies the threshold of indoor micro-climates to maintain indoor allergens at the acceptable level, which can be used as a guideline to maintain or improve indoor health conditions for future housing development or retrofitted old housing.

Describing fluctuating indoor aerosol dust measurements with application to house dust mite allergens.

Measuring house dust mite aeroallergen concentrations is essential in understanding mite allergen exposure. Physically, the aerolized house dust mite faeces are part of indoor particulate matter. We studied the statistical ways of summarizing measurements of fluctuating mite aeroallergen exposure inside homes through indoor particulate matter. To study emissions from beddings, we measured the time-related airborne dust concentration after shaking a duvet. Analysis was performed both by a method based on the estimated mean and by a non-linear model. Twenty-eight studies reported a sum of concentrations; only one also reported the peak. In our four experiments on shaking a duvet (245 to 275 measurements each), the peak value was two to four times higher than the mean. The mean-based and non-linear models both predicted the sum of concentrations exactly. A 1% upper prediction bound and the non-linear model predicted the peak emission rate moderately well (64 to 92%, and 63 to 93%, respectively). Mean levels of indoor particulate matter measurements differ substantially from peak concentrations. The use of the mean is only sufficient to predict the sum of concentrations. We suggest that, mite aeroallergen measurements should include information on the peak as well as the mean.

Indoor allergen exposure in primary school classrooms in New Zealand.

AIM: Indoor allergens exposure is a risk factor for respiratory symptoms in sensitised children. There is limited data on indoor allergen exposures in New Zealand schools. METHODS: Vacuumed floor dust samples were collected from 136 classrooms in 12 primary schools and analysed for allergens from the house dust mite Dermatophagoides pteronyssinus (Der p 1), cat dander (Fel d 1), cockroach (Bla g 2), cow dander (Bos d 2), horse dander (Equ c 4) and peanut (Ara h 2) by ELISA. RESULTS: House dust mite, cat, cockroach, cow and horse allergens were detected in 96.4%, 100%, 2.2%, 27.0% and 59.9% respectively in the classrooms dust samples. Thirty-one (22.6%) classrooms had Fel d 1 levels of >8.0µg/g while none had Der p 1 levels of >10.0µg/g. Only one classroom had detectable levels of peanut allergen. CONCLUSIONS: House dust mite allergen levels were low in New Zealand classrooms while about a quarter of classrooms had cat allergen levels that may be associated with respiratory symptoms.

Comparison of the Allergenic Potency of House Dust Extract and House Dust Mite Allergen Extract for Subcutaneous Allergen Immunotherapy.

Subcutaneous allergen immunotherapy (SCIT) with non-standardized house dust (HD) extracts has been used in Japan since 1963 for house dust mite (HDM)-allergic patients. Since the potencies of HD extracts are unknown, the allergenic potency of HD extracts was examined by comparing with a standardized HDM allergen extracts. The major allergen content of HDM in the extracts was measured using a sandwich enzyme-linked immunosorbent assay (ELISA). The immunoglobulin E (IgE) inhibitory activities of the extracts were measured by a competitive ELISA. The extract concentrations giving 50% inhibition of IgE binding (log10 IC50) were determined from dose-response curves and defined as inhibitory activities. A linear regression line was constructed from the log10 IC50 values of the standardized HDM extract to interpolate the relative potency of the HD extract with strength of 1 : 10 w/v (HD 1 : 10). The amounts of major allergens (Der f 1, Der p 1 and Der 2) were 116.3 µg/mL in the HDM allergen extract (100000 Japanese Allergy Units [JAU]/mL) and 0.77 µg/mL in the HD 1 : 10. The inhibitory activity (log10 IC50 values) of HD 1 : 10 was 2.389 ± 0.078, indicating the allergenic potency was between 200 and 2000 JAU/mL. Based on regression analysis (R2 >0.99), the allergenic potency of HD 1 : 10 was estimated to be 842 ± 128 JAU/mL. The present study determined the major allergen content of HD extract, which contributes to its allergenic potency. The allergenic potency of HD 1 : 10 was ca. 100-fold less than that of HDM allergen extract.

Is there an association between indoor allergens and the severity of atopic dermatitis?

BACKGROUND: Atopic dermatitis (AD) is a multifactorial inflammatory skin disease with frequent hypersensitivity to allergens. However, the role of exposure to indoor allergens on AD severity is unclear. METHODS: Children aged 0-17 years with active AD from central Chile were recruited; disease severity was evaluated with SCORAD and POEM scores. A home environment survey was applied to parents. Bedroom dust samples were collected for all subjects and analyzed by multiplex assay to quantify dust mite (Der p1, Der f1), dog (Can f1), cat (Fel d1), and alternaria alternata (Alt a1) allergens. RESULTS: Twenty-five children aged 3.9 ± 3.8 years were included. Fifty-two percent were female. Mean SCORAD was 29 ± 14 (range 11-61), and mean POEM was 10.7 ± 6.2. No direct association was found between tobacco exposure, pet ownership, aerosol use, visible dust, or home carpets/rugs with SCORAD (all P > 0.05). Dust samples from all homes had Can f1 and Fel d1 allergens, regardless of pet ownership. Homes that had indoor dogs or cats had significantly higher amounts of these allergens (P < 0.001). Forty percent of homes had dust mite allergens, and none had alternaria alternata. Children with AD living in homes with elevated dust mite and animal dander allergen concentrations had higher SCORAD than those from homes with low allergen concentrations (40 ± 13 vs. 26 ± 13, P = 0.025). CONCLUSIONS: High concentrations of indoor allergens may influence AD severity in children. Further studies assessing indoor allergens and allergen sensitization are warranted to fully evaluate the role of indoor allergens on AD.

[Domestic exposure to moulds and mite allergens in Parisian patients]. / Exposition domestique aux moisissures et aux allergènes d'acariens de patients parisiens.

INTRODUCTION: Moulds and mite allergens present in indoor environments are well known for their effects on respiratory health. METHODS: From 2011 to 2015, the Paris Service for Environmental Health (SPSE) conducted investigations in 293 dwellings following medical referral. These audits included fungal analysis of air (in 12% of dwellings), in mattress surface and floor dust (24%), and mite allergen quantifications in mattresses and carpets (18%). RESULTS: Indoor air fungal concentrations are not significantly different from those in outdoor air. When there is no ventilation or when the system is malfunctioning, an increase in indoor/outdoor air ratios is observed, indicating mould enrichment in the dwelling's indoor air. With regard to house dust samples, fungal spore concentrations vary according to the media from which samples were collected. Mattress fungal contamination is higher in dwellings where observed surface moulds exceed 1 per square meter. In the same way Der p1 mite allergens levels are greater in mattress dust in dwellings where mould contamination is visible. CONCLUSIONS: This study describes the levels of contamination in the dwellings of Parisian patients.

How clean is your house? A study of house dust mites, allergens and other contents of dust samples collected from households.

Household dust contains an array of constituents, including house dust mites (HDM) and the HDM allergen, Der p 1, which can cause sensitivities such as asthma and eczema. Vacuuming can help alleviate symptoms, yet little is understood about cleaning behaviour in different households. This pilot study investigated the contents of dust from four household types (students; over 65 s; and families with and without pets). This was then related to cleaning behaviours and perceptions of cleanliness. Our investigation found that HDMs and Der p 1 were present in all households and sampling locations, including participants' cars. The median Der p 1 was greatest in the living room, though results varied. Demographic group was a determinant for the number of human and pet hairs present in dust. Surprisingly, vacuuming was the most disliked task overall. This information requires consideration when developing cleaning products and advising individuals with dust-related health issues.

[House Dust and Its Adverse Health Effects].

In this review, we examine house dust and its effect on inhabitants' health. Residential house dust includes components from plants, pollens, microorganisms, insects, skin flakes, hairs and fibers. It also includes materials contaminated with chemicals from combustion, furniture, interior materials, electronics, cleaning agents, personal care products. Nowadays, most people spend their time indoors. Thus, dust is an important medium of exposure to pollutions. According to United States Environmental Protection Agency Exposure Factors Handbook, the estimated amount of dust ingestion is 30 mg/day for adults, and 60 mg/day for children over 1 year of age. Since 2003, we have been conducting epidemiological studies to find the association between the indoor environment and the inhabitants' health. The levels of mite allergens, endotoxins, and ß-1,3-d-glucan in house dust were measured as biological factors. Semi volatile organic compounds (SVOC) such as phthalates and phosphate flame retardants (PFRs) in dust were also analyzed. As a result, we found that the ORs (95%CI) of nasal and optical symptoms of sick building syndrome (SBS) were 1.45 (1.01-2.10) and 1.47 (1.14-1.88), respectively, when there was a 10-fold increase in the levels of mite allergens. There was no association of mite allergens with allergies. Endotoxins and ß-1,3-d-glucan did not show any association with SBS. Regarding SVOC, increased levels of phthalates and PFR increased the risk of allergies. The association between phthalates and increased risk of allergies was clearer among children than adults. There were no gold standards of dust sampling and pretreatment methods. Thus, caution is needed when comparing findings of various studies. Methods should accurately reflect exposure levels.

Efficacy of air purifier therapy in allergic rhiniti.

BACKGROUND: With the rising prevalence of allergic rhinitis, the utility of indoor environmental management deserves increasing scrutiny. This research aims at evaluating the ability of air purifiers to be a therapy of allergic rhinitis. METHODS: 32 subjects (25±13.5 years old) diagnosed with allergic rhinitis were selected and HEPA air purifiers placed in their bedrooms for 4 months. Before the intervention and each month, dust samples were collected with a vacuum cleaner and the dust collector assessed for allergen content. Additionally, static dust collectors were left in place all month to collect dust by sedimentation. Particulate matter (PM) was assessed in terms of PMindoor/outdoor ratios. The Rhinitis Quality of Life Questionnaire (RQLQ) was used to assess symptoms. RESULTS: Der p 1 (78 (30,82) ng/g) was the dominant dust mite allergen in air samples of patients' bedroom as well as static collections. Der f1 (444 (345,667) ng/g) was the dominant allergen in bedding. Der f1 levels in both air and bed sampling significantly decreased after initiation of HEPA air purifiers (P<0.05). PM1.0indoor/outdoor, PM2.5indoor/outdoor, PM10indoor/outdoor all decreased (P<0.001) with the HEPA filtration intervention. According to RQLQ data, HEPA filtration was associated with improvements in activity limitation, non-nasal-eye symptoms, practical problems, and nasal symptoms (P<0.001). CONCLUSION: HEPA air purifiers can effectively reduce PM and HDM allergen concentration in the indoor air, and thereby improve clinical manifestations of patients with AR.

Prevalence of house dust mite allergens in low-income homes with evaporative coolers in a semiarid climate.

House dust mites are typically absent in homes in arid and semiarid climates due to low humidity. Evaporative "swamp" cooling significantly increases indoor humidity in dry climates and is suspected of promoting dust mite survival in these regions. We investigated the prevalence and concentration of mite allergens in dust from low-income homes (N = 22) with evaporative coolers in Utah County, Utah. Overall, 15 homes (68.2%) were positive for either Der p 1 or Der f 1 in at least 1 location. Geometric mean allergen levels in mattresses were 0.107 and 0.087 ug/g dust for Der p 1 and Der f 1, respectively. In furniture, levels were 0.143 and 0.165 ug/g dust for Der p1 and Der f 1, respectively. The percentage of positive homes in this study was much higher than previously reported in larger homes with swamp coolers in the same community. These results suggest socioeconomic factors may play a role in dust mite allergen prevalence in homes with evaporative coolers in dry climates.

Proteases of Dermatophagoides pteronyssinus.

Since the discovery that Der p 1 is a cysteine protease, the role of proteolytic activity in allergic sensitization has been explored. There are many allergens with proteolytic activity; however, exposure from dust mites is not limited to allergens. In this paper, genomic, transcriptomic and proteomic data on Dermatophagoides pteronyssinus (DP) was mined for information regarding the complete degradome of this house dust mite. D. pteronyssinus has more proteases than the closely related Acari, Dermatophagoides farinae (DF) and Sarcoptes scabiei (SS). The group of proteases in D. pteronyssinus is found to be more highly transcribed than the norm for this species. The distribution of protease types is dominated by the cysteine proteases like Der p 1 that account for about half of protease transcription by abundance, and Der p 1 in particular accounts for 22% of the total protease transcripts. In an analysis of protease stability, the group of allergens (Der p 1, Der p 3, Der p 6, and Der p 9) is found to be more stable than the mean. It is also statistically demonstrated that the protease allergens are simultaneously more highly expressed and more stable than the group of D. pteronyssinus proteases being examined, consistent with common assumptions about allergens in general. There are several significant non-allergen outliers from the normal group of proteases with high expression and high stability that should be examined for IgE binding. This paper compiles the first holistic picture of the D. pteronyssinus degradome to which humans may be exposed.