Brett J. Green

Airborne fungal fragments and allergenicity

Exposure to fungi, particularly in water damaged indoor environments, has been thought to exacerbate a number of adverse health effects, ranging from subjective symptoms such as fatigue, cognitive difficulties or memory loss to more definable diseases such as allergy, asthma and hypersensitivity pneumonitis. Understanding the role of fungal exposure in these environments has been limited by methodological difficulties in enumerating and identifying various fungal components in environmental samples. Consequently, data on personal exposure and sensitization to fungal allergens are mainly based on the assessment of a few select and easily identifiable species. The contribution of other airborne spores, hyphae and fungal fragments to exposure and allergic sensitization are poorly characterized. There is increased interest in the role of aerosolized fungal fragments following reports that the combination of hyphal fragments and spore counts improved the association with asthma severity. These fragments are particles derived from any intracellular or extracellular fungal structure and are categorized as either submicron particles or larger fungal fragments. In vitro studies have shown that submicron particles of several fungal species are aerosolized in much higher concentrations (300-500 times) than spores, and that respiratory deposition models suggest that such fragments of Stachybotrys chartarum may be deposited in 230-250 fold higher numbers than spores. The practical implications of these models are yet to be clarified for human exposure assessments and clinical disease. We have developed innovative immunodetection techniques to determine the extent to which larger fungal fragments, including hyphae and fractured conidia, function as aeroallergen sources. These techniques were based on the Halogen Immunoassay (HIA), an immunostaining technique that detects antigens associated with individual airborne particles >1 microm, with human serum immunoglobulin E (IgE). Our studies demonstrated that the numbers of total airborne hyphae were often significantly higher in concentration than conidia of individual allergenic genera. Approximately 25% of all hyphal fragments expressed detectable allergen and the resultant localization of IgE immunostaining was heterogeneous among the hyphae. Furthermore, conidia of ten genera that were previously uncharacterized could be identified as sources of allergens. These findings highlight the contribution of larger fungal fragments as aeroallergen sources and present a new paradigm of fungal exposure. Direct evidence of the associations between fungal fragments and building-related disease is lacking and in order to gain a better understanding, it will be necessary to develop diagnostic reagents and detection methods, particularly for submicron particles. Assays using monoclonal antibodies enable the measurement of individual antigens but interpretation can be confounded by cross-reactivity between fungal species. The recent development of species-specific monoclonal antibodies, used in combination with a fluorescent-confocal HIA technique should, for the first time, enable the speciation of morphologically indiscernible fungal fragments. The application of this novel method will help to characterize the contribution of fungal fragments to adverse health effects due to fungi and provide patient-specific exposure and sensitization profiles.

Discrimination of Aspergillus isolates at the species and strain level by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry fingerprinting.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was used to generate highly reproducible mass spectral fingerprints for 12 species of fungi of the genus Aspergillus and 5 different strains of Aspergillus flavus. Prior to MALDI-TOF MS analysis, the fungi were subjected to three 1-min bead beating cycles in an acetonitrile/trifluoroacetic acid solvent. The mass spectra contain abundant peaks in the range of 5 to 20kDa and may be used to discriminate between species unambiguously. A discriminant analysis using all peaks from the MALDI-TOF MS data yielded error rates for classification of 0 and 18.75% for resubstitution and cross-validation methods, respectively. If a subset of 28 significant peaks is chosen, resubstitution and cross-validation error rates are 0%. Discriminant analysis of the MALDI-TOF MS data for 5 strains of A. flavus using all peaks yielded error rates for classification of 0 and 5% for resubstitution and cross-validation methods, respectively. These data indicate that MALDI-TOF MS data may be used for unambiguous identification of members of the genus Aspergillus at both the species and strain levels.

Fungal pigments inhibit the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis of darkly pigmented fungi

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been used to discriminate moniliaceous fungal species; however, darkly pigmented fungi yield poor fingerprint mass spectra that contain few peaks of low relative abundance. In this study, the effect of dark fungal pigments on the observed MALDI mass spectra was investigated. Peptide and protein samples containing varying concentrations of synthetic melanin or fungal pigments extracted from Aspergillus niger were analyzed by MALDI-TOF and MALDI-qTOF (quadrupole TOF) MS. Signal suppression was observed in samples containing greater than 250ng/μl pigment. Microscopic examination of the MALDI sample deposit was usually heterogeneous, with regions of high pigment concentration appearing as black. Acquisition of MALDI mass spectra from these darkly pigmented regions of the sample deposit yielded poor or no [M+H](+) ion signal. In contrast, nonpigmented regions within the sample deposit and hyphal negative control extracts of A. niger were not inhibited. This study demonstrated that dark fungal pigments inhibited the desorption/ionization process during MALDI-MS; however, these fungi may be successfully analyzed by MALDI-TOF MS when culture methods that suppress pigment expression are used. The addition of tricyclazole to the fungal growth media blocks fungal melanin synthesis and results in less melanized fungi that may be analyzed by MALDI-TOF MS.

The Macroecology of Airborne Pollen in Australian and New Zealand Urban Areas

The composition and relative abundance of airborne pollen in urban areas of Australia and New Zealand are strongly influenced by geographical location, climate and land use. There is mounting evidence that the diversity and quality of airborne pollen is substantially modified by climate change and land-use yet there are insufficient data to project the future nature of these changes. Our study highlights the need for long-term aerobiological monitoring in Australian and New Zealand urban areas in a systematic, standardised, and sustained way, and provides a framework for targeting the most clinically significant taxa in terms of abundance, allergenic effects and public health burden.

Bioaerosol sampling for the detection of aerosolized influenza virus

Background Influenza virus was used to characterize the efficacy of a cyclone‐based, two‐stage personal bioaerosol sampler for the collection and size fractionation of aerosolized viral particles.

Analysis of Airborne Betula Pollen in Finland; a 31-Year Perspective

In this 31-year retrospective study, we examined the influence of meteorology on airborne Betula spp. (birch) pollen concentrations in Turku, Finland. The seasonal incidence of airborne birch pollen in Turku occurred over a brief period each year during spring (April 30 – May 31). Mean peak concentrations were restricted to May (May 5 to 13). Statistically significant increases in the annual accumulated birch pollen sum and daily maximum values were observed over the study period. Birch pollen counts collected in April were retrospectively shown to increase over the duration of the study. Increases in April temperature values were also significantly associated with the earlier onset of the birch pollen season. Furthermore, the number of days where daily birch pollen concentrations exceeded 10 and 1,000 grains/m3 also increased throughout the study period. These data demonstrate that increases in temperature, especially during months preceding the onset of the birch pollen season, favor preseason phenological development and pollen dispersal. Birch pollen derived from other geographical locations may also contribute to the aerospora of Turku, Finland. To date, the public health burden associated with personal exposure to elevated birch pollen loads remains unclear and is the focus of future epidemiological research.

Characterization of Cannabis sativa allergens

BACKGROUND Allergic sensitization to Cannabis sativa is rarely reported, but the increasing consumption of marijuana has resulted in an increase in the number of individuals who become sensitized. To date, little is known about the causal allergens associated with C sativa. OBJECTIVE To characterize marijuana allergens in different components of the C sativa plant using serum IgE from marijuana sensitized patients. METHODS Serum samples from 23 patients with a positive skin prick test result to a crude C sativa extract were evaluated. IgE reactivity was variable between patients and C sativa extracts. IgE reactivity to C sativa proteins in Western blots was heterogeneous and ranged from 10 to 70 kDa. Putative allergens derived from 2-dimensional gels were identified. RESULTS Prominent IgE reactive bands included a 23-kDa oxygen-evolving enhancer protein 2 and a 50-kDa protein identified to be the photosynthetic enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase. Additional proteins were identified in the proteomic analysis, including those from adenosine triphosphate synthase, glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase, and luminal binding protein (heat shock protein 70), suggesting these proteins are potential allergens. Deglycosylation studies helped refine protein allergen identification and demonstrated significant IgE antibodies against plant oligosaccharides that could help explain cross-reactivity. CONCLUSION Identification and characterization of allergens from C sativa may be helpful in further understanding allergic sensitization to this plant species.

Industrial Fungal Enzymes: An Occupational Allergen Perspective

Occupational exposure to high-molecular-weight allergens is a risk factor for the development and pathogenesis of IgE-mediated respiratory disease. In some occupational environments, workers are at an increased risk of exposure to fungal enzymes used in industrial production. Fungal enzymes have been associated with adverse health effects in the work place, in particular in baking occupations. Exposure-response relationships have been demonstrated, and atopic workers directly handling fungal enzymes are at an increased risk for IgE-mediated disease and occupational asthma. The utilization of new and emerging fungal enzymes in industrial production will present new occupational exposures. The production of antibody-based immunoassays is necessary for the assessment of occupational exposure and the development of threshold limit values. Allergen avoidance strategies including personal protective equipment, engineering controls, protein encapsulation, and reduction of airborne enzyme concentrations are required to mitigate occupational exposure to fungal enzymes.

Prevalence of allergic sensitization to indoor fungi in West Virginia

Exposure to indoor fungi is of growing concern in residential and occupational environments in the United States. The purpose of this study was to determine the prevalence of sensitization to common indoor fungal species in an atopic population. We evaluated 102 patients (73 female and 29 male patients) for immunoglobulin E (IgE) reactivity to a panel of skin-prick test (SPT) reagents used for routine allergy testing. Patients also were tested for six additional fungi that are common indoor contaminants. All patients had symptoms consistent with allergic rhinitis or asthma. The presence of specific IgE against the fungal species was determined using immunoblotting. Of the 102 eligible patients, 68% had at least one positive skin test. The most prevalent positive SPTs were to dust mites, cats, vernal grass, and short ragweed. Overall, 21/102 (21%) patients with asthma or allergic rhinitis were skin test positive to at least one fungal extract. Of the patients with a positive SPT to fungi, 12/21 (58%) showed sensitivity to one or more of the newly tested species; most notably Trichoderma viride (8%), Chaetomium globosum (7%), Paecilomyces variotii (7%), and Acremonium strictum (6%). Immunoblotting revealed specific IgE against a number of protein bands belonging to these fungal species. The prevalence of fungal sensitization was common, particularly for indoor fungal contaminants that are not routinely included in SPT panels. Cross-reactivity with other fungi may partially explain our results; however, skin testing for these indoor fungi may provide useful diagnostic information.

The streptococcal collagen-like protein-1 (Scl1) is a significant determinant for biofilm formation by group a Streptococcus

BackgroundGroup A Streptococcus (GAS) is a human-specific pathogen responsible for a number of diseases characterized by a wide range of clinical manifestations. During host colonization GAS-cell aggregates or microcolonies are observed in tissues. GAS biofilm, which is an in vitro equivalent of tissue microcolony, has only recently been studied and little is known about the specific surface determinants that aid biofilm formation. In this study, we demonstrate that surface-associated streptococcal collagen-like protein-1 (Scl1) plays an important role in GAS biofilm formation.ResultsBiofilm formation by M1-, M3-, M28-, and M41-type GAS strains, representing an intraspecies breadth, were analyzed spectrophotometrically following crystal violet staining, and characterized using confocal and field emission scanning electron microscopy. The M41-type strain formed the most robust biofilm under static conditions, followed by M28- and M1-type strains, while the M3-type strains analyzed here did not form biofilm under the same experimental conditions. Differences in architecture and cell-surface morphology were observed in biofilms formed by the M1- and M41-wild-type strains, accompanied by varying amounts of deposited extracellular matrix and differences in cell-to-cell junctions within each biofilm. Importantly, all Scl1-negative mutants examined showed significantly decreased ability to form biofilm in vitro. Furthermore, the Scl1 protein expressed on the surface of a heterologous host, Lactococcus lactis, was sufficient to induce biofilm formation by this organism.ConclusionsOverall, this work (i) identifies variations in biofilm formation capacity among pathogenically different GAS strains, (ii) identifies GAS surface properties that may aid in biofilm stability and, (iii) establishes that the Scl1 surface protein is an important determinant of GAS biofilm, which is sufficient to enable biofilm formation in the heterologous host Lactococcus. In summary, the GAS surface adhesin Scl1 may have an important role in biofilm-associated pathogenicity.