Metabarcoding airborne pollen from subtropical and temperate eastern Australia over multiple years reveals pollen aerobiome diversity and complexity.

eDNA metabarcoding is an emergent tool to inform aerobiome complexity, but few studies have applied this technology with real-world environmental pollen monitoring samples. Here we apply eDNA metabarcoding to assess seasonal and regional differences in the composition of airborne pollen from routine samples collected across successive years. Airborne pollen concentrations over two sampling periods were determined using a continuous flow volumetric impaction air sampler in sub-tropical (Mutdapilly and Rocklea) and temperate (Macquarie Park and Richmond), sites of Australia. eDNA metabarcoding was applied to daily pollen samples collected once per week using the rbcL amplicon. Composition and redundancy analysis of the sequence read counts were examined. The dominant pollen families were mostly consistent between consecutive years but there was some heterogeneity between sites and years for month of peak pollen release. Many more families were detected by eDNA than counted by light microscopy with 211 to 399 operational taxonomic units assigned to family per site from October to May. There were 216 unique and 119 taxa shared between subtropics (27°S) and temperate (33°S) latitudes, with, for example, Poaceae, Myrtaceae and Causurinaceae being shared, and Manihot, Vigna and Aristida being in subtropical, and Ceratodon and Cerastium being in temperate sites. Certain genera were observed within the same location and season over the two years; Chloris at Rocklea in autumn of 2017-18 (0.625, p ≤ 0.004) and 2018-19 (0.55, p ≤ 0.001), and Pinus and Plantago at Macquarie Park in summer of 2017-18 (0.58, p ≤ 0.001 and 0.53, p ≤ 0.003, respectively), and 2018-19 (0.8, p ≤ 0.003 and 0.8, p ≤ 0.003, respectively). eDNA metabarcoding is a powerful tool to survey the complexity of pollen aerobiology and distinguish spatial and temporal profiles of local pollen to a far deeper level than traditional counting methods. However, further research is required to optimise the metabarcode target to enable reliable detection of pollen to genus and species level.

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.

Grass pollen allergens globally: the contribution of subtropical grasses to burden of allergic respiratory diseases.

Grass pollens of the temperate (Pooideae) subfamily and subtropical subfamilies of grasses are major aeroallergen sources worldwide. The subtropical Chloridoideae (e.g. Cynodon dactylon; Bermuda grass) and Panicoideae (e.g. Paspalum notatum; Bahia grass) species are abundant in parts of Africa, India, Asia, Australia and the Americas, where a large and increasing proportion of the world's population abide. These grasses are phylogenetically and ecologically distinct from temperate grasses. With the advent of global warming, it is conceivable that the geographic distribution of subtropical grasses and the contribution of their pollen to the burden of allergic rhinitis and asthma will increase. This review aims to provide a comprehensive synthesis of the current global knowledge of (i) regional variation in allergic sensitivity to subtropical grass pollens, (ii) molecular allergenic components of subtropical grass pollens and (iii) allergic responses to subtropical grass pollen allergens in relevant populations. Patients from subtropical regions of the world show higher allergic sensitivity to grass pollens of Chloridoideae and Panicoideae grasses, than to temperate grass pollens. The group 1 allergens are amongst the allergen components of subtropical grass pollens, but the group 5 allergens, by which temperate grass pollen extracts are standardized for allergen content, appear to be absent from both subfamilies of subtropical grasses. Whilst there are shared allergenic components and antigenic determinants, there are additional clinically relevant subfamily-specific differences, at T- and B-cell levels, between pollen allergens of subtropical and temperate grasses. Differential immune recognition of subtropical grass pollens is likely to impact upon the efficacy of allergen immunotherapy of patients who are primarily sensitized to subtropical grass pollens. The literature reviewed herein highlights the clinical need to standardize allergen preparations for both types of subtropical grass pollens to achieve optimal diagnosis and treatment of patients with allergic respiratory disease in subtropical regions of the world.

Functional immunoglobulin E cross-reactivity between Pas n 1 of Bahia grass pollen and other group 1 grass pollen allergens.

BACKGROUND: Grass pollens are major triggers of allergic rhinitis and asthma, but the immunological relationships between pollen allergens of the subtropical Bahia grass, Paspalum notatum, and temperate grasses are unresolved. OBJECTIVE: To assess serum IgE cross-reactivity between subtropical P. notatum and temperate Lolium perenne (Ryegrass) pollen allergens. METHODS: Serum IgE reactivities of grass pollen-allergic patients with P. notatum, L. perenne and Cynodon dactylon (Bermuda grass) pollen extracts and their respective purified group 1 allergens, Pas n 1, Lol p 1 and Cyn d 1, were compared by immunoblotting, ELISA and basophil activation. RESULTS: In a cohort of 51 patients from a temperate region, a high frequency of IgE reactivity with each grass pollen was detected, but reactivity with L. perenne pollen was substantially greater than with P. notatum and C. dactylon pollen. Similarly, serum IgE reactivity with Lol p 1 was greater than with Pas n 1 or Cyn d 1. For seven of eight sera studied in detail, asymmetric serum IgE cross-reactivity was observed; L. perenne pollen inhibited IgE reactivity with P. notatum pollen but not the converse, and IgE reactivity with Pas n 1 was inhibited by Lol p 1 but IgE reactivity with Lol p 1 was not inhibited by Pas n 1 or Cyn d 1. Importantly, P. notatum pollen and Pas n 1 activated basophils in grass pollen-allergic patients from a temperate region, although stimulation was greater by pollen of L. perenne than P. notatum or C. dactylon, and by Lol p 1 than Pas n 1 or Cyn d 1. In contrast, a cohort of 47 patients from a subtropical region showed similar IgE reactivity with P. notatum and L. perenne pollen, and reciprocal cross-inhibition of IgE reactivity between L. perenne and P. notatum. CONCLUSIONS: Pollen allergens of the subtropical P. notatum, including Pas n 1, show clinically relevant IgE cross-reactivity with pollen allergens of L. perenne but also species-specific IgE reactivity.

Bahia grass pollen specific IgE is common in seasonal rhinitis patients but has limited cross-reactivity with Ryegrass.

BACKGROUND: Perennial Ryegrass is a major cause of rhinitis in spring and early summer. Bahia grass, Paspalum notatum, flowers late into summer and could account for allergic rhinitis at this time. We determined the frequency of serum immunoglobulin (Ig)E reactivity with Bahia grass in Ryegrass pollen allergic patients and investigated IgE cross-reactivity between Bahia and Ryegrass. METHODS: Serum from 33 Ryegrass pollen allergic patients and 12 nonatopic donors were tested for IgE reactivity with Bahia and Ryegrass pollen extracts (PE) by enzyme-linked immunosorbent assay (ELISA), western blotting and inhibition ELISA. Allergen-specific antibodies from a pool of sera from allergic donors were affinity purified and tested for IgE cross-reactivity. RESULTS: Seventy-eight per cent of the sera had IgE reactivity with Bahia grass, but more weakly than with Ryegrass. Antibodies eluted from the major Ryegrass pollen allergens, Lol p 1 and Lol p 5, showed IgE reactivity with allergens of Ryegrass and Canary but not Bahia or Bermuda grasses. Timothy, Canary and Ryegrass inhibited IgE reactivity with Ryegrass and Bahia grass, whereas Bahia, Johnson and Bermuda grass did not inhibit IgE reactivity with Ryegrass. CONCLUSIONS: The majority of Ryegrass allergic patients also showed serum IgE reactivity with Bahia grass PE. However, Bahia grass and Ryegrass had only limited IgE cross-reactivity indicating that Bahia grass should be considered in diagnosis and treatment of patients with hay fever late in the grass pollen season.

VH gene usage in immunoglobulin E responses of seasonal rhinitis patients allergic to grass pollen is oligoclonal and antigen driven.

BACKGROUND: IgE is the pivotal-specific effector molecule of allergic reactions yet it remains unclear whether the elevated production of IgE in atopic individuals is due to superantigen activation of B cell populations, increased antibody class switching to IgE or oligoclonal allergen-driven IgE responses. OBJECTIVES: To increase our understanding of the mechanisms driving IgE responses in allergic disease we examined immunoglobulin variable regions of IgE heavy chain transcripts from three patients with seasonal rhinitis due to grass pollen allergy. METHODS: Variable domain of heavy chain-epsilon constant domain 1 cDNAs were amplified from peripheral blood using a two-step semi-nested PCR, cloned and sequenced. RESULTS: The VH gene family usage in subject A was broadly based, but there were two clusters of sequences using genes VH 3-9 and 3-11 with unusually low levels of somatic mutations, 0-3%. Subject B repeatedly used VH 1-69 and subject C repeatedly used VH 1-02, 1-46 and 5a genes. Most clones were highly mutated being only 86-95% homologous to their germline VH gene counterparts and somatic mutations were more abundant at the complementarity determining rather than framework regions. Multiple sequence alignment revealed both repeated use of particular VH genes as well as clonal relatedness among clusters of IgE transcripts. CONCLUSION: In contrast to previous studies we observed no preferred VH gene common to IgE transcripts of the three subjects allergic to grass pollen. Moreover, most of the VH gene characteristics of the IgE transcripts were consistent with oligoclonal antigen-driven IgE responses.

Origins and implications of drinking water odours in lakes and reservoirs of British Columbia, Canada.

The relationship between commonly measured limnological parameters and odours was examined in 16 reservoirs and lakes used as sources for drinking water and three reference lakes. Odour analysis was conducted using flavour profile analysis (FPA) and, on select lakes, gas chromatography ion-trap mass spectrometry (GC-ITMS) for target compounds. Total phosphorus (TP) was the best single predictor of FPA intensity and multiple regression models accounted for 37-39% of intensity variance in the epilimnion and metalimnion, respectively. Earthy odours were more prevalent in reservoirs and lakes with higher TP, whereas decomposing vegetation and green vegetation+grassy odours almost exclusively occurred when TP was lower (<13 microg P/L). Only geosmin was identified with GC-ITMS, and it was found to occur in lakes and reservoirs of higher trophic status (e.g. more algal biomass). Infrequent episodic events in the Greater Victoria's principal reservoir (e.g. algal blooms) have previously been linked with taste and odour problems in their tap water. However, analysis of odours under the conditions of this study (i.e. no strong odour episodes in the source reservoir) suggest that typical odours prevalent in tap water originating from Sooke Lake Reservoir are derived from treatment processes or the distribution system, not directly from the reservoir. This study demonstrates the utility of employing relatively simple and established methods to better understand management issues of a drinking water system.

A novel grass pollen allergen mimotope identified by phage display peptide library inhibits allergen-human IgE antibody interaction.

The aim of this study was to investigate the molecular basis of human IgE-allergen interaction by screening a phage-displayed peptide library with an allergen-specific human IgE-mimicking monoclonal antibody (mAb). A mAb that reacted with major grass pollen allergens was successfully identified and shown to inhibit human IgE-allergen interaction. Biopanning of a phage-displayed random peptide library with this mAb yielded a 12 amino acid long mimotope. A synthetic peptide based on this 12-mer mimotope inhibited mAb and human IgE binding to grass pollen extracts. Our results indicate that such synthetic peptide mimotopes of allergens have potential as novel therapeutic agents.

Novel BTB/POZ domain zinc-finger protein, LRF, is a potential target of the LAZ-3/BCL-6 oncogene.

BTB/POZ-domain C2H2 zinc(Zn)-finger proteins are encoded by a subfamily of genes related to the Drosophila gap gene krüppel. To date, two such proteins, PLZF and LAZ-3/BCL-6, have been implicated in oncogenesis. We have now identified a new member of this gene subfamily which encodes a 62 kDa Zn-finger protein, termed LRF, with a BTB/POZ domain highly similar to that of PLZF. Both human and mouse LRF genes, which localized to syntenic chromosomal regions (19p13.3 and 10B5.3, respectively), were widely expressed in adult tissues and cell lines. At approximately 9.5-10.0 days of embryonic development, the mouse LRF gene was expressed in the limb buds, pharyngeal arches, tail bud, placenta and neural tube. The LRF protein associated in vivo with LAZ-3/BCL-6, but not with PLZF to which it was more related. Although the LRF, or LAZ-3/BCL-6, BTB/POZ domain could readily homodimerize, no heterodimerization was detected in vivo between the LRF and LAZ-3/BCL-6 BTB/POZ domains and interaction between full length LRF and LAZ-3/BCL-6 required the presence of both the BTB/POZ domain and Zn-fingers in each partner protein. As expected from the above results, LRF and LAZ-3/BCL-6 also colocalized with each other in the nucleus. Taken together, our findings suggest that BTB/ POZ-domain Zn-finger proteins may function as homo and heterodimeric complexes whose formation, and hence the resultant effect on transcription of their downstream target genes, is determined by the levels and expression domains of a given partner protein.

Pure white cell aplasia and health food products.

The safety of 'health' foods and complementary medicine products is increasingly questioned. We report a case of pure white cell aplasia developing in a patient who took a variety of such products leading to an excessive intake of zinc. Recovery was complete following the withdrawal of her 'medications'. The aetiology of the pure white cell aplasia is discussed with respect to the ingredients of the products ingested.

Accident analysis of large-scale technological disasters applied to an anaesthetic complication.

The occurrence of serious accidents in complex industrial systems such as at Three Mile Island and Bhopal has prompted development of new models of causation and investigation of disasters. These analytical models have potential relevance in anaesthesia. We therefore applied one of the previously described systems to the investigation of an anaesthetic accident. The model chosen describes two kinds of failures, both of which must be sought. The first group, active failures, consists of mistakes made by practitioners in the provision of care. The second group, latent failures, represents flaws in the administrative and productive system. The model emphasizes the search for latent failures and shows that prevention of active failures alone is insufficient to avoid further accidents if latent failures persist unchanged. These key features and the utility of this model are illustrated by application to a case of aspiration of gastric contents. While four active failures were recognized, an equal number of latent failures also became apparent. The identification of both types of failures permitted the formulation of recommendations to avoid further occurrences. Thus this model of accident causation can provide a useful mechanism to investigate and possibly prevent anaesthetic accidents.

Amiloride prevents amphotericin B related hypokalaemia in neutropenic patients.

Twenty neutropenic patients with various haematological disorders were randomised prospectively to receive either intravenous amphotericin B alone or amphotericin B and oral amiloride 5 mg twice a day for treatment of confirmed or suspected fungal infection. Patients receiving amiloride had a significantly higher plasma potassium (p less than 0.01), a significantly lower urinary potassium loss (p less than 0.01), and required significantly less potassium chloride supplementation to maintain their plasma potassium within the normal range (p less than 0.001). Amiloride was well tolerated, had no clinically important side effects, and provided effective control of plasma potassium in patients treated with amphotericin B.