Tracking seasonal changes in diversity of pollen allergen exposure: Targeted metabarcoding of a subtropical aerobiome.

The importance of grass pollen to the global burden of allergic respiratory disease is well established but exposure to subtropical and temperate pollens is difficult to discern. Current monitoring of airborne pollen relies on light microscopy, limiting identification of taxa to family level. This informs seasonal fluctuations in pollen aerobiology but restricts analysis of aerobiological composition. We aimed to test the utility of DNA metabarcoding to identify specific taxa contributing to the aerobiome of environmental air samples, using routine pollen and spore monitoring equipment, as well as assess temporal variation of Poaceae pollen across an entire season. Airborne pollen concentrations were determined by light microscopy over two pollen seasons in the subtropical city of Brisbane (27°32'S, 153°00E), Australia. Thirty daily pollen samples were subjected to high throughput sequencing of the plastid rbcL amplicon. Amplicons corresponded to plants observed in the local biogeographical region with up to 3238 different operational taxonomic units (OTU) detected. The aerobiome sequencing data frequently identified pollen to genus levels with significant quantitative differences in aerobiome diversity between the months and seasons detected. Moreover, multiple peaks of Chloridoideae and Panicoideae pollen were evident over the collection period confirming these grasses as the dominant Poaceae pollen source across the season. Targeted high throughput sequencing of routinely collected airborne pollen samples appears to offer utility to track temporal changes in the aerobiome and shifts in pollen exposure. Precise identification of the composition and temporal distributions of airborne pollen is important for tracking biodiversity and for management of allergic respiratory disease.

IgE+ B cells are scarce, but allergen-specific B cells with a memory phenotype circulate in patients with allergic rhinitis.

BACKGROUND: Despite the critical role of immunoglobulin E (IgE) in allergy, circulating IgE+ B cells are scarce. Here, we describe in patients with allergic rhinitis B cells with a memory phenotype responding to a prototypic aeroallergen. METHODS: Fifteen allergic rhinitis patients with grass pollen allergy and 13 control subjects were examined. Blood mononuclear cells stained with carboxyfluorescein diacetate succinimidyl ester (CFSE) were cultured with Bahia grass pollen. Proliferation and phenotype were assessed by multicolour flow cytometry. RESULTS: In blood of allergic rhinitis patients with high serum IgE to grass pollen, most IgE(hi) cells were CD123+ HLA-DR(-) basophils, with IgE for the major pollen allergen (Pas n 1). Both B and T cells from pollen-allergic donors showed higher proliferation to grass pollen than nonallergic donors (P = 0.002, and 0.010, respectively), whereas responses to vaccine antigens and mitogen did not differ between groups. Allergen-driven B cells that divided rapidly (CD19(mid) CD3(-) CFSE(lo) ) showed higher CD27 (P = 0.008) and lower CD19 (P = 0.004) and CD20 (P = 0.004) expression than B cells that were slow to respond to allergen (CD19(hi) CD3(-) CFSE(mid) ). Moreover, rapidly dividing allergen-driven B cells (CD19(mid) CFSE(lo) CD27(hi) ) showed higher expression of the plasmablast marker CD38 compared with B cells (CD19(hi) CFSE(mid) CD27(lo) ) that were slow to divide. CONCLUSION: Patients with pollen allergy but not control donors have a population of circulating allergen-specific B cells with the phenotype and functional properties of adaptive memory B-cell responses. These cells could provide precursors for allergen-specific IgE production upon allergen re-exposure.

On the mechanism of cogenotoxic action between ingested amphibole asbestos fibres and benzo[a]pyrene: II. Tissue specificity studies using comet assay.

Epidemiological data seem to be equivocal on the probable increase in cancer incidence in populations exposed to asbestos-fibre contaminated drinking water. Although animal experiments failed to demonstrate carcinogenicity of oral asbestos exposure, the large surface area of the fibres, however, creates the possibility of cogenotoxic action with adsorbed water-borne organics. In our animal model, rats were gavaged with untreated UICC crocidolite and anthophyllite fibres and fibres that had been allowed to adsorb benzo[a]pyrene molecules from aqueous solutions. Peritoneal macrophages and intestine, parietal peritoneum and omentum samples were obtained from the animals after 24 h. The alkaline single-cell microgel electrophoresis assay (comet assay) was performed on cells isolated from the solid tissues. Tail moment was applied as a basis of evaluation following image analysis. Our results indicate high levels of DNA strand breaks in the cells prepared from the omentum and intestine. We could also demonstrate a significant potentiating effect of the adsorbed carcinogen on the induction of DNA damage in the omentum. The parietal peritoneum and macrophages were not involved in the early genotoxic alterations under study. Our results support the molecular model of asbestos cocarcinogenesis, including both asbestos-induced deletions and mutations caused by a mutagen carried by the same fibres.

On the mechanism of cogenotoxic action between ingested amphibole asbestos fibres and benzo[a]pyrene: I. Urinary and serum mutagenicity studies with rats.

Recently, there has been concern that ingested asbestos may cause an increase in cancer incidence in populations exposed to fibre-contaminated drinking water. Although animal experiments failed to demonstrate carcinogenicity of the oral asbestos exposure, the high adsorption capacity of the fibres creates the possibility of cocarcinogenic action with adsorbed organics. In a simple in vivo model we demonstrated earlier that UICC crocidolite and anthophyllite asbestos fibres were able to adsorb carcinogen molecules from aqueous solutions. When orally administered, these fibres increased the sister chromatid exchange frequency in bone marrow cells of rats. In the present study we tried to follow the desorption and metabolization processes of carcinogenic benzo[a]pyrene molecules transported by the ingested fibres using the highly sensitive Salmonella/Ames mutagenicity assay. The bacterial test was performed on concentrated serum and urine samples of the treated animals by using the TA98 and 100 strains in the presence and absence of liver microsomal and deconjugating enzymes. All sets of urine and serum samples failed to show mutagenic activity indicating a lack of both desorption in the serum and the ability of the liver to metabolize. Considering our results, the cytogenetic impact demonstrated earlier in the bone marrow can be explained by a local action of accumulated and transported carcinogen molecules.

In vivo studies on genotoxicity and cogenotoxicity of ingested UICC anthophyllite asbestos.

Early cytogenetic action of oral exposure to UICC anthophyllite, an amphibole type of asbestos, was studied in Fischer-344 rats. The animals were gavaged with a suspension of untreated fibres (50 mg/kg) and fibres which had been allowed to adsorb benzo[alpha]pyrene molecules from aqueous solutions of 0.25-2.5 micrograms/ml. HPLC measurements indicated effective adsorption of the benzo[alpha]pyrene molecules on the fibres. The authors consider this system a suitable model for the drinking of water containing asbestos fibres and organic micropollutants. The formation of micronuclei and sister chromatid exchanges was studied in bone marrow samples taken from animals 24 h after oral administration of suspensions. Whereas anthophyllite fibres failed to induce cytogenetic alterations, fibres pretreated with the polycyclic aromatic solutions caused dose-dependent increase in the sister chromatid exchange frequencies. The observed cytogenetic impact can be explained by a local action of carcinogen molecules accumulated and subsequently transported. The results support the hypothesis that epidemiological evidence of carcinogenicity of asbestos in potable water may rather be explained by cogenotoxic action of the asbestos fibres and biologically active organic micropollutants adsorbed on their surface.

Studies on genotoxicity of orally administered crocidolite asbestos in rats: implications for ingested asbestos induced carcinogenesis.

The early genotoxic action of oral exposure to UICC crocidolite asbestos fibres was studied in different short-term tests. Fischer-344 rats were gavaged with 50 mg/b.w.kg untreated asbestos fibres and fibres which had been allowed to adsorb benzo(a)pyrene molecules from extremely low concentration (0.25-2.5 microg/ml) aqueous solutions. This system can be considered a model for the drinking of potable water contaminated by asbestos fibres together with biologically active organic micro-pollutants. The Ames Salmonella mutagenicity assay was performed on concentrated urine and serum samples of treated animals. The formation of micronuclei and sister chromatid exchanges was also studied in the bone marrow of the exposed rats. The micronucleus analysis indicated marginal genotoxic activity only upon treatment with crocidolite prepared from the solution of 1 microg/ml. A dose-dependent increase was, however, demonstrated in the sister chromatid exchange frequency upon treatment with benzo(a)pyrene coated fibres. These experiments suggest the acute cogenotoxic activity of such fibres in orally exposed animals.

Review of the significance of fibre size in fibre-related lung disease: a centrifuge cell for preparing accurate microscope-evaluation specimens from slurries used in inoculation studies.

Intratracheal, intrapleural and intraperitoneal inoculation studies in animals are widely used for identifying important factors in the pathogenicity of fine fibrous particles and estimating the potential of new materials to produce human pulmonary disease. Evidence on the significance of fibre size is reviewed, with emphasis on direct data derived from airborne fibres in asbestos mines and fibres retained in the mineworkers' lungs. This evidence indicates a need in mesothelioma-related inoculation experiments for means of measuring fibres down to 0.01 microns in diameter. A test cell, developed for preparing microscope-evaluation specimens from injection slurries, has a sector-shaped sedimentation chamber and is used in a swing-rotor centrifuge. To minimize re-formation of aggregates that are dispersed by shearing forces during sedimentation, a sample of the slurry is diluted beforehand to a degree indicated by the length of the longest fibres seen in the light microscope. Fibres and other particles are collected as a uniform deposit on a collodion film enveloping a microscope cover-glass. Current techniques are used to prepare specimens from films for light microscopy, scanning electron microscopy and the transmission electron microscopy which is so necessary for measurement of very fine fibres. Applications of the cell to fibre samples from other sources are outlined.

Simple method of estimating severity of pulmonary fibrosis on a numerical scale.

A continuous numerical scale for determining the degree of fibrosis in lung specimens was devised for correlation with other pulmonary variables such as lung function tests or mineral burden. Grading was scored on a scale from 0 to 8, using the average of microscope field scores. The system allows fibrosis to be measured in small samples of tissue (1 cm) which can provide a detailed description of the changes in a lung, currently not possible with most existing methods.

Measurement of fibres in human lung tissue.

The paper outlines application of a method based on magnetic alignment and light scattering to assessment of anthophyllite fibres in a large series of autopsy lung tissue specimens. Data are presented on fibre size and concentration. A child showed thicker and much longer fibres than did adults.

Uses of the UICC samples.

The UICC samples are now used in a wider variety of applications than was originally envisaged. They have been employed in inhalation, ingestion, inoculation and in vitro experiments. Other applications are in identification of asbestos type in aerosols and tissues; standardization of conventional fibre counting; development and calibration of techniques and instruments for fibre assessment; and studies on influence of fibre size and shape on inhalation and retention. The paper outlines applications and gives literature references.

Practical application of magnetic alignment of mineral fibres for hazard evaluation.

Techniques and instrumentation have been developed employing the magnetic alignment of fibrous dust collected on membrane filters coupled with light-scatter measurement for the accurate and reproducible assessment of airborne fibre hazard. This equipment, specifically designed to measure asbestos hazard, is built into a standard microscope, which also incorporates a facility for manual fibre counting and for bulk sample identification. With this new technique, up to 100 samples per day can be analysed automatically, compared with some 10-15 samples per day by manual counting. Collected fibres are assessed from areas of membrane filters some 30 times greater than those with manual methods; consequently, a more representative result ensues. These techniques lend themselves to considerable and varied application and involve relatively inexpensive instrumentation.

The effect of fibre size on the in vitro biological activity of three types of amphibole asbestos.

Three standard (UICC) samples of amphibole asbestos were subjected to ball-milling; the main effect of this procedure was to reduce the length of the fibres present in each sample. The numbers of fibres in unit masses, and the distribution of fibre sizes in all the samples, both parent and milled, were estimated from electron micrographs. The ability of all the samples to reduce the plating efficiency of V79-4 cells is compared, on the basis of mass, fibre number and fibre number number above various length thresholds. This biological activity of all the samples correlated best with the number of fibres above a threshold length of 6.5 micron. This is compared with the sizes of fibre previously reported to induce mesotheliomata when implanted into the pleural cavities of rats.

The influence of fibre shape in lung deposition-mathematical estimates.

Inhaled fibres deposit by sedimentation, diffusion, impaction, and interception in airways of the respiratory system. Long straight fibres may exhibit periodic motiion with ordered orientation in those airways having laminar flow. We assume that irregular fibres are randomly oriented in airways. Mathematical models based on respiratory system architecture, respiratory airflow, and mathematical expressions for deposition mechanisms have been developed to predict deposition in respiratory compartments of fibres in ordered orientation and of various fibre confirgurations in random orientations. The size and shape characteristics of chamber aerosols generated with U.I.C.C. asbestos specimens have been determined. Combinations of the chamber aerosol data with mathematical estimates of deposition suggest that fibre shape as well as size influences the magnitude of deposition in pulmonary spaces. For size distributions such as those of the U.I.C.C. asbestos chamber aerosols, the mathematical models predict pulmonary spaces deposition for straight fibres in ordered orientation about twice as great as for irregular fibres in random orientation.

The effects of the inhalation of asbestos in rats.

Two experiments in which SPF Wistar rats were exposed by inhalation to dust clouds of the UICC standard reference samples for periods of between one day and 2 years are described. All the samples of asbestos produced asbestosis which continued to progress after removal from exposure but only a little fibrosis was observed in control rats. Lung tumours, ranging in severity from adenomata to squamous carcinomata, were produced by all samples but in the controls there were only a few adenomata and none of the more serious tumours. Of the 20 tumours which metastasized, 16 occurred after exposure to one or other of the 2 chrysotile samples. In addition, a total of 11 mesotheliomata occurred, 4 of which were with crocidolite and 4 with Canadian chrysotile. Two of the mesotheliomata occurred with only one day's exposure to asbestos. There was a positive association between asbestosis and lung tumours.