The effect of respiratory activity, non-invasive respiratory support and facemasks on aerosol generation and its relevance to COVID-19.

Respirable aerosols (< 5 µm in diameter) present a high risk of SARS-CoV-2 transmission. Guidelines recommend using aerosol precautions during aerosol-generating procedures, and droplet (> 5 µm) precautions at other times. However, emerging evidence indicates respiratory activities may be a more important source of aerosols than clinical procedures such as tracheal intubation. We aimed to measure the size, total number and volume of all human aerosols exhaled during respiratory activities and therapies. We used a novel chamber with an optical particle counter sampling at 100 l.min-1 to count and size-fractionate close to all exhaled particles (0.5-25 µm). We compared emissions from ten healthy subjects during six respiratory activities (quiet breathing; talking; shouting; forced expiratory manoeuvres; exercise; and coughing) with three respiratory therapies (high-flow nasal oxygen and single or dual circuit non-invasive positive pressure ventilation). Activities were repeated while wearing facemasks. When compared with quiet breathing, exertional respiratory activities increased particle counts 34.6-fold during talking and 370.8-fold during coughing (p < 0.001). High-flow nasal oxygen 60 at l.min-1 increased particle counts 2.3-fold (p = 0.031) during quiet breathing. Single and dual circuit non-invasive respiratory therapy at 25/10 cm.H2 O with quiet breathing increased counts by 2.6-fold and 7.8-fold, respectively (both p < 0.001). During exertional activities, respiratory therapies and facemasks reduced emissions compared with activities alone. Respiratory activities (including exertional breathing and coughing) which mimic respiratory patterns during illness generate substantially more aerosols than non-invasive respiratory therapies, which conversely can reduce total emissions. We argue the risk of aerosol exposure is underappreciated and warrants widespread, targeted interventions.

Domestic exposure to fungal allergenic particles determined by halogen immunoassay using subject's serum versus particles carrying three non-fungal allergens determined by allergen-specific HIA.

UNLABELLED: Studies that estimate indoor aeroallergen exposure typically measure a pre-selected limited range of allergens. In this study, inhalable aeroallergen particles were quantified using the halogen immunoassay (HIA) to determine the contribution of fungal and non-fungal aeroallergens to total allergen exposure. Bioaerosols from 39 homes of fungal-allergic subjects were sampled using inhalable fraction samplers and immunostained by HIA using resident subject's immunoglobulin E (IgE) to detect allergen-laden particles. Fungal aerosols as well as particles carrying mite, cat, and cockroach allergens were identified and enumerated by HIA. Reservoir dust-mite (Der p 1), cat (Fel d 1), and cockroach (Bla g 1) allergen concentrations were quantified by ELISA. Fungal particles that bound subject's IgE in the HIA were 1.7 (bedroom)- and 1.4 (living room)-fold more concentrated than Der p 1, Fel d 1, and Bla g 1 allergen particles combined. Predominant fungal conidia that bound IgE were derived from common environmental genera including Cladosporium and other fungi that produce amerospores. Airborne mite, cat, and cockroach allergen particle counts were not associated with reservoir concentrations determined by ELISA. This study demonstrates that inhalable fungal aerosols are the predominant aeroallergen sources in Sydney homes and should be considered in future exposure assessments. PRACTICAL IMPLICATIONS: Indoor allergen exposure assessment studies have primarily focused on a limited range of allergen sources in samples derived from reservoir dust samples. Using an innovative immunodiagnostic approach, this study demonstrates that fungal bioaerosols are the dominant source of aeroallergen exposure in the domestic environment, providing unique insight into domestic aeroallergen exposure.

Cloning and sequencing of a cDNA expressing a recombinant house dust mite protein that binds human IgE and corresponds to an important low molecular weight allergen.

A cDNA clone coding for a mite allergen of mol wt approximately 14,000 has been isolated and its DNA sequence determined. The native component from mite extracts encoded by this DNA was identified by immunoprobing blots of mite body extract with human IgE eluted from the electroblotted cloned fusion polypeptides derived from the expressed cDNA clone. The clone encodes a polypeptide with a deduced mol wt of 17,460. The deduced amino acid sequence was not homologous to any known protein sequences and it contains no cysteine or tryptophan. On blots, 21 of 38 sera from mite-allergic subjects recognized the cloned material, and this recognition strongly correlated with IgE-binding to the native component on protein blots of mite extract.

Der p 1 facilitates transepithelial allergen delivery by disruption of tight junctions.

House dust mite (HDM) allergens are important factors in the increasing prevalence of asthma. The lung epithelium forms a barrier that allergens must cross before they can cause sensitization. However, the mechanisms involved are unknown. Here we show that the cysteine proteinase allergen Der p 1 from fecal pellets of the HDM Dermatophagoides pteronyssinus causes disruption of intercellular tight junctions (TJs), which are the principal components of the epithelial paracellular permeability barrier. In confluent airway epithelial cells, Der p 1 led to cleavage of the TJ adhesion protein occludin. Cleavage was attenuated by antipain, but not by inhibitors of serine, aspartic, or matrix metalloproteinases. Putative Der p 1 cleavage sites were found in peptides from an extracellular domain of occludin and in the TJ adhesion protein claudin-1. TJ breakdown nonspecifically increased epithelial permeability, allowing Der p 1 to cross the epithelial barrier. Thus, transepithelial movement of Der p 1 to dendritic antigen-presenting cells via the paracellular pathway may be promoted by the allergen's own proteolytic activity. These results suggest that opening of TJs by environmental proteinases may be the initial step in the development of asthma to a variety of allergens.

Localization of antigens and allergens in thin sections of the house dust mite, Dermatophagoides pteronyssinus (Acari: Pyroglyphidae).

Cryostat sections of the house dust mite, Dermatophagoides pteronyssinus, were probed in fluorescent microscopy studies with rabbit polyclonal and mouse monoclonal antibodies specific for mite allergens including the major allergen, Der p I. Sections also were probed for allergens with sera from human mite-allergic subjects containing IgE antibodies to mite allergens and with lectins. Antibody binding was mainly to the gut lining and gut contents of the mite, although some specific labeling also was associated with the head region and cuticle. This is the first detailed localization of mite allergens in situ. The morphology of the mite was investigated using plastic embedded thin sections and was found to be similar to that previously described for D. farinae.

Protein binding to nitrocellulose, nylon and PVDF membranes in immunoassays and electroblotting.

A selection of different membranes commonly used to bind proteins in blotting and dot binding assays were investigated for a range of properties which would influence their performance. Large differences were observed in the membranes' ability to bind increasing amounts of protein, the effect of incubation times on the quantity of protein bound and the loss of proteins from the membranes following their incubation with different detergents or protein blocking agents. These differences could only partially explain the observed performance of the membranes when used as protein adsorbants in immunoassays and when different buffers were used for the electro-transfer of several different proteins to a range of membranes.

Comparison of different blocking agents and nitrocelluloses in the solid phase detection of proteins by labelled antisera and protein A.

Five different brands of nitrocellulose (NC), each of pore size 0.45 micron and without adsorbed antigen, bound different amounts of two labelled antisera and labelled protein A. Experiments with some non-ionic surface active agents and proteins showed that milk powder and bovine serum albumin were the most effective agents for blocking non-specific binding of labelled protein to NC. With some of the NCs, Nonidet P-40 (NP-40) and Tween 20 were almost as effective as milk powder. The protein-binding capacity of unblocked NC and the level of protein binding after blocking were found to be inversely proportional to the pore size of the NC. A comparison of blocking agents in an immunoassay with pollen proteins adsorbed to NC discs revealed that the highest specific uptakes of antiserum occurred with NP-40 and Tween and not with any of the protein blocking agents such as milk powder. Hence, for the detection of proteins using NC-based assays (but not necessarily following electroblotting), the best choices would appear to be: NC of pore size 0.45 micron; a brand of NC that provides a suitable balance between protein binding capacity and non-specific uptake of protein after blocking; a non-ionic detergent such as NP-40 or Tween 20.

Protein blotting on nitrocellulose: some important aspects of the resolution and detection of antigens in complex extracts.

The resolution and detection of individual components in complex extracts by protein blotting have been investigated. By probing nitrocellulose transfers with monospecific and multispecific antisera, it was demonstrated that dissociating conditions were required for the maximum resolution of antigens by polyacrylamide gel electrophoresis, a conclusion reinforced by results from 2-D electrophoresis. The dissociating and reducing treatments employed, however, were both shown to be responsible for some loss of total antigenicity and included the complete loss of at least one important antigen. Assays with nitrocelluloses of different pore sizes demonstrated that both higher protein-binding capacities and higher backgrounds were associated with the use of the smallest pore size, while the sensitivity of the assay was greatest when a non-ionic detergent, and not proteins, were used for blocking. Nitrocellulose-bound proteins may be stained with amido black, India ink, toluidine blue, Ponceau S or a gold sol, but these agents do not always give identical staining patterns. While detection of components with immuno-enzyme staining methods had some advantages, problems with non-specific binding were encountered. These did not occur with affinity purified radiolabelled second antibodies, which in combination with scanning of autoradiographs allowed a quantitative approach to be adopted.

A modern miasma hypothesis and back-to-school asthma exacerbations.

A sudden increase in the rate of asthma exacerbations has been observed among young children in many countries 2-3 weeks after their return-to-school following the summer holidays. These exacerbations are frequently associated with human rhinovirus (hRV) infections, with possible interactions with allergen sensitisation, allergen exposure and medication use. It was originally proposed that the sudden increase resulted from new strains of respiratory viruses acquired during the holidays spreading rapidly on return to school. While there is compelling evidence implicating hRV in these exacerbations, recent observations on virus transmission, infection patterns and immune responses to both viruses and allergens have led us to propose an additional hypothesis for this increase in exacerbations. We propose that classrooms typically provide persistent exposure to a mixture of airborne viruses, viral proteins, endotoxin, community allergens and other human-derived aerosols - a modern miasma. During the preceding school term, this exposure establishes and maintains a level of immune tolerance and herd immunity, which then declines during the two-month holidays due to lack of such exposure, creating a transitory window of susceptibility to viral infections and asthma. The return to school re-establishes exposure to these aerosols resulting in an acceleration of exacerbations, until the tolerance and herd immunity are re-established. Thus, the peak in return-to-school asthma is more a function of a transitory increase in susceptibility due to a temporary lack of this complex exposure, than it is to novel, locally endemic strains of hRV.

Do immune responses to inhaled skin flakes modulate the expression of allergic disease?

We examine the nature of the immune responses to inhaled skin particles and query whether early exposure could play a role in providing protection against the development of allergic disease. Currently, the main hypothesis used to explain environmental modulation of allergic diseases, the 'hygiene hypothesis', is linked exclusively to microbial exposures acting upon the innate immune system. However, many of the exposures sustaining this hypothesis also involve co-exposure to skin flakes from humans or animals. Such skin flakes contain a complex mixture of antigens, glycolipids and small peptides that may induce immune responses. Should these responses prove relevant to the modulation of allergic diseases, it provides new opportunities to better understand the epidemic of allergic disease and to develop new interventions for its prevention.

Early predictors for developing allergic disease and asthma: examining separate steps in the 'allergic march'.

BACKGROUND: Sensitization and symptoms of allergic disease are strongly correlated, but little is known about the early clinical precursors of the development of allergen sensitization in childhood. The aim of this study was to identify these predictors, and to examine separately the effect of early sensitization on subsequent wheeze, asthma, rhinitis and eczema. METHODS: In the Childhood Asthma Prevention Study, children with a family history of asthma were assessed for allergen sensitization, total serum IgE, wheeze, asthma, eczema and rhinitis at ages 18 months and 5 years. To examine predictors, at 18 months, for subsequent sensitization, children who were non-sensitized at 18 months and had data on sensitization at 5 years were investigated, n=375. To examine the predictors, at age 18 months, of subsequent onset of symptoms, children who did not have wheeze, asthma, eczema or rhinitis at 18 months were followed-up at 5 years, n=177. RESULTS: Among children who were non-sensitized at age 18 months, the presence of eczema [adjusted relative risk (aRR), 1.67, 95% confidence interval (CI) 1.20-2.33], but not wheeze, asthma or rhinitis, was an independent predictor of the onset of sensitization by age 5 years. Among children who were asymptomatic at age 18 months, sensitization to any allergen at 18 months was an independent predictor for the presence of wheeze (aRR 2.41, 95% CI 1.28-4.55), asthma (aRR 4.66, 95% CI 1.88-11.54) and rhinitis (aRR 1.77, 95% CI 1.08-2.90), but not for the development of eczema (aRR 0.78, 95% CI 0.23-2.64) at 5 years. CONCLUSION: In non-sensitized children, eczema, but not wheeze, asthma or rhinitis is a predictor for subsequent development of sensitization. This suggests that early childhood eczema, rather than wheeze and rhinitis, may promote subsequent allergen sensitization and raises the possibility that early management of eczema may reduce the prevalence of sensitization in children.

Measuring environmental fungal exposure.

Airborne fungi are ubiquitous in the environment and human exposure is inevitable. Such fungi differ greatly in their taxonomic, physical, ecological, behavioral, and pathogenic characteristics. Many strategies have evolved to sample, identify and interpret fungal exposure and their choice is determined by the hypotheses involved. While fungi can be sampled directly from surfaces, results do not generally reflect human exposure. For this reason, airborne spores are commonly sampled, by either filtration or impaction, using volumetric air samplers. Identification is commonly performed by either culture on nutrient medium or light microscopy using morphological criteria, although new techniques using DNA probes or characteristic antigens or toxins continue to be developed. Interpretation of such exposure data is both complex and contentious, but while there are numerous recommendations there is no consensus on exposure thresholds. A better understanding of the complex pathogenic roles of fungi and susceptibilities of their hosts will enable refinement of techniques for sampling and interpretation.

Evaluation of home allergen sampling devices.

BACKGROUND: Simple, inexpensive methods of sampling from allergen reservoirs are necessary for large-scale studies or low-cost householder-operated allergen measurement. METHODS: We tested two commercial devices: the Indoor Biotechnologies Mitest Dust Collector and the Drager Bio-Check Allergen Control; two devices of our own design: the Electrostatic Cloth Sampler (ECS) and the Press Tape Sampler (PTS); and a Vacuum Sampler as used in many allergen studies (our Reference Method). Devices were used to collect dust mite allergen samples from 16 domestic carpets. Results were examined for correlations between the sampling methods. RESULTS: With mite allergen concentration expressed as microg/g, the Mitest, the ECS and the PTS correlated with the Reference Method but not with each other. When mite allergen concentration was expressed as microg/m2 the Mitest and the ECS correlated with the Reference Method but the PTS did not. In the high allergen conditions of this study, the Drager Bio-Check did not relate to any methods. CONCLUSIONS: The Mitest Dust Collector, the ECS and the PTS show performance consistent with the Reference Method. Many techniques can be used to collect dust mite allergen samples. More investigation is needed to prove any method as superior for estimating allergen exposure.

The reduction of rhinitis symptoms by nasal filters during natural exposure to ragweed and grass pollen.

BACKGROUND: Prototype nasal filters were developed to collect inhaled pollen. This study evaluated the efficacy of the filters for prevention of rhinitis symptoms during acute outdoor pollen exposure. METHODS: A randomized double-blind design was used. Subjects (n=46) with a history of autumn exacerbation of rhinitis and positive skin test to ragweed, Bermuda and/or Bahia grass wore either active or placebo nasal filters for 2 h in autumn in a park containing these species. Major and Total Symptoms scores were recorded at 0, 30, 60, 90 and 120 min. RESULTS: Subjects wearing active nasal filters had significantly reduced scores, at all time-points compared with placebo group (all P <0.05). Of 14 individual symptoms measured, seven were significantly reduced (number of sneezes, runny nose, itchy nose, sniffles, itchy throat; itchy eyes and watery eyes) and another three showed a trend towards lower severity. The nasal filters also enabled the resolution of existing symptoms. Maximal difference in symptoms was seen immediately after subjects had spent 20 min sitting beside a large patch of ragweed. CONCLUSION: This is the first clinical trial of a nasal filter. The results suggest it has potential for enhancing rhinitis management during acute allergen exposure.

Allergen exposure and control.

Allergens produced by the house dust mites (family Pyroglyphidae) are probably the single most important allergens associated with asthma world wide. If exposure to these allergens in houses could be sufficiently reduced, then asthma symptoms may be markedly reduced and even prevented from being initiated. Only about half of the many attempts to reduce mite allergens in houses have shown any clinical benefit. One reason may be that exposure was not reduced enough--however exposure to mite allergens has never been measured in any trial. This review summarises previous allergen control trials and then provides an outline of allergen exposure, including the nature of exposure, the analytical methods available and the recognised risks of allergen exposure. This provides a perspective to evaluate the individual methods used to kill mites and to reduce exposure to the allergens. The object is to provide a framework to improve and develop allergen avoidance as an effective component of asthma management.

An on-line computer data bank of monoclonal antibodies that recognize allergens--invitation for entries.

Monoclonal antibodies that recognize allergens are potentially extremely useful reagents for use in a variety of applications, including highly specific and sensitive assays for the standardization of allergen extracts and the measurement of environmental allergens, epitope mapping of allergens, comparison of IgE-binding determinants, and the immunolocalization of allergens in thin sections. Although monoclonal antibodies have many properties that enable them to be shared internationally, it is difficult to know from the literature what monoclonal antibodies are available and, in some cases, what are the individual characteristics of the antibodies. We are organizing an on-line data base of information about allergen-reactive monoclonal antibodies, which can be accessed by computer with international telecommunications networks. Each record of a monoclonal antibody includes details, if they are known, of allergen source and name, antibody production, complimentary antigenic determinant, names and addresses of the owners, availability of the antibody, and other important information. Investigators who have produced monoclonal antibodies that react with allergens are invited to submit details of the antibodies for inclusion in the data base.

Detection of house dust mite allergens and frequency of IgE binding following electroblotting and enzyme immunoassay.

Mite allergens, fractionated by polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes, were identified using 45 mite atopic sera and an enzyme immunostaining assay for IgE. More than 20 different mite components bound IgE and almost every serum showed a different pattern of binding. 7 of the 20 components were bound by half of the sera and 70% were bound by at least 20% of the sera. These results demonstrate a greater number of house dust mite allergens and a far greater diversity of the IgE antibody response to mite allergens than has previously been described.

Comparison by electroblotting of IgE-binding components in extracts of house dust mite bodies and spent mite culture.

Eight Dermatophagoides pteronyssinus extracts (three culture extracts, four mite body extracts, and the World Health Organization International Standard [IS]) were investigated by side-by-side sodium dodecyl sulfate-polyacrylamide gel electrophoresis, by electrotransfer to nitrocellulose, and by probing with a pooled serum from mite-allergic subjects. Representative body and mite culture extracts were compared by probing with individual sera, and both types of extract were also compared by RAST-inhibition studies. Extracts from the same source differed in the molecular weight (MW) of some of their IgE-binding components. In general, most IgE-binding components in culture extracts and the IS were in the 14 to 35 kd MW region, whereas extracts from mite bodies and one culture extract contained more IgE-binding components of higher MW (35 to 110 kd). Comparison of representative mite body and culture extracts by use of 22 separate sera resolved 26 and 19 IgE-binding components in the two extracts, respectively. Patterns of RAST inhibition produced by both types of extracts when they were used either as the inhibitor or as the allergosorbent demonstrated qualitative differences between the two types of extracts. These results demonstrate that mite extracts may differ considerably in their allergenic composition and emphasize the need for standardization of mite allergenic extracts and the reexamination of the suitability of the D. pteronyssinus IS.

Effect of reagins and allergen extracts on radioallergosorbent assays for mite allergen.

The reproducibility of the radioallergosorbent (RAST) inhibition and direct binding assys with mite allergen were investigated in the presence of heterogeneous extracts and non-mite-sensitive atopic sera. Both contain components similar to potential contaminants which would occur in the assay of mite allergen and dust allergen and dust allergen extracts. The standardized inhibition and direct binding assays employed had a day to day (n = 4) coefficient of variation [(s.d. x 100)/mean] of 15% and 24% respectively. The inhibition assay for mite allergen was reproducible in the presence of protein concentrations of added plant, fungal, arthropod and animal extracts in excess of the protein concentrations that occur under the operational mite assay conditions. The mite inhibition assay was also reproducible in the presence of non-mite allergen extracts, with and without additional sera containing IgE specific for the non-mite allergens. The binding of a additional sera containing IgE specific for the non-mite allergens. The binding of a constant quantity of mite allergen to the activated solid phase in the direct binding assay was reproducible in the presence of added bovine serum albumin, and of a fungal or arthropod extract, representing the heterogeneous components of an allergen extract at the concentrations of total protein known to occur in the direct binding assay of mite extracts.