Fungal diversity in homes and asthma morbidity among school-age children in New York City.

BACKGROUND: Asthma development has been inversely associated with exposure to fungal diversity. However, the influence of fungi on measures of asthma morbidity is not well understood. OBJECTIVES: This study aimed to test the hypothesis that fungal diversity is inversely associated with neighborhood asthma prevalence and identify specific fungal species associated with asthma morbidity. METHODS: Children aged 7-8 years (n = 347) living in higher (11-18%) and lower (3-9%) asthma prevalence neighborhoods were recruited within an asthma case-control study. Fungal communities were analyzed from floor dust using high-throughput DNA sequencing. A subset of asthmatic children (n = 140) was followed to age 10-11 to determine asthma persistence. RESULTS: Neighborhood asthma prevalence was inversely associated with fungal species richness (P = 0.010) and Shannon diversity (P = 0.059). Associations between neighborhood asthma prevalence and diversity indices were driven by differences in building type and presence of bedroom carpet. Among children with asthma at age 7-8 years, Shannon fungal diversity was inversely associated with frequent asthma symptoms at that age (OR 0.57, P = 0.025) and with asthma persistence to age 10-11 (OR 0.48, P = 0.043). Analyses of individual fungal species did not show significant associations with asthma outcomes when adjusted for false discovery rates. DISCUSSION: Lower fungal diversity was associated with asthma symptoms in this urban setting. Individual fungal species associated with asthma morbidity were not detected. Further research is warranted into building type, carpeting, and other environmental characteristics which influence fungal exposures in homes.

Efficacy of Do-It-Yourself air filtration units in reducing exposure to simulated respiratory aerosols.

Many respiratory diseases, including COVID-19, can be spread by aerosols expelled by infected people when they cough, talk, sing, or exhale. Exposure to these aerosols indoors can be reduced by portable air filtration units (air cleaners). Homemade or Do-It-Yourself (DIY) air filtration units are a popular alternative to commercially produced devices, but performance data is limited. Our study used a speaker-audience model to examine the efficacy of two popular types of DIY air filtration units, the Corsi-Rosenthal cube and a modified Ford air filtration unit, in reducing exposure to simulated respiratory aerosols within a mock classroom. Experiments were conducted using four breathing simulators at different locations in the room, one acting as the respiratory aerosol source and three as recipients. Optical particle spectrometers monitored simulated respiratory aerosol particles (0.3-3 µm) as they dispersed throughout the room. Using two DIY cubes (in the front and back of the room) increased the air change rate as much as 12.4 over room ventilation, depending on filter thickness and fan airflow. Using multiple linear regression, each unit increase of air change reduced exposure by 10%. Increasing the number of filters, filter thickness, and fan airflow significantly enhanced the air change rate, which resulted in exposure reductions of up to 73%. Our results show DIY air filtration units can be an effective means of reducing aerosol exposure. However, they also show performance of DIY units can vary considerably depending upon their design, construction, and positioning, and users should be mindful of these limitations.

Mycobiota and the Contribution of Yeasts in Floor Dust of 50 Elementary Schools Characterized with Sequencing Internal Transcribed Spacer Region of Ribosomal DNA.

The assemblage of fungi including unicellular yeasts in schools is understudied. We conducted an environmental study to characterize fungal communities in classroom floor dust. We collected 500 samples from 50 elementary schools in Philadelphia, PA, and evaluated room dampness/mold conditions. Genomic DNA from dust was extracted for internal transcribed spacer 1 Illumina MiSeq sequencing to identify operational taxonomic units (OTUs) organized from DNA sequences. Differential abundance analyses were performed to examine significant differences in abundance among groups. We identified 724 genera from 1490 OTUs. The genus Epicoccum was not diverse but the most abundant (relative abundance = 18.9%). Fungi were less diverse but most dissimilar in composition in the most water-damaged classrooms compared to the least water-damaged, indicating differential effects of individual classroom water-damage on fungal compositions. We identified 62 yeast genera, representing 19.6% of DNA sequences. Cyberlindnera was the most abundant (6.1%), followed by Cryptococcus, Aureobasidium, Rhodotorula, and Candida. The average relative abundance of yeasts tended to increase with increasing dampness and mold score and was significantly (p-value = 0.048) higher in the most water-damaged classrooms (22.4%) than the least water-damaged classrooms (18.2%). Our study suggests the need for further research on the potential health effects associated with exposures to yeasts in schools.

Reduction of exposure to simulated respiratory aerosols using ventilation, physical distancing, and universal masking.

To limit community spread of SARS-CoV-2, CDC recommends universal masking indoors, maintaining 1.8 m of physical distancing, adequate ventilation, and avoiding crowded indoor spaces. Several studies have examined the independent influence of each control strategy in mitigating transmission in isolation, yet controls are often implemented concomitantly within an indoor environment. To address the influence of physical distancing, universal masking, and ventilation on very fine respiratory droplets and aerosol particle exposure, a simulator that coughed and exhaled aerosols (the source) and a second breathing simulator (the recipient) were placed in an exposure chamber. When controlling for the other two mitigation strategies, universal masking with 3-ply cotton masks reduced exposure to 0.3-3 µm coughed and exhaled aerosol particles by >77% compared to unmasked tests, whereas physical distancing (0.9 or 1.8 m) significantly changed exposure to cough but not exhaled aerosols. The effectiveness of ventilation depended upon the respiratory activity, that is, coughing or breathing, as well as the duration of exposure time. Our results demonstrate that a layered mitigation strategy approach of administrative and engineering controls can reduce personal inhalation exposure to potentially infectious very fine respiratory droplets and aerosol particles within an indoor environment.

Spring is associated with increased total and allergenic fungal concentrations in house dust from a pediatric asthma cohort in New York City.

Introduction: Asthma and allergy symptoms vary seasonally due to exposure to environmental sources of allergen, including fungi. However, we need an improved understanding of seasonal influence on fungal exposures in the indoor environment. We hypothesized that concentrations of total fungi and allergenic species in vacuumed dust vary significantly by season. Objective: Assess seasonal variation of indoor fungi with greater implications related to seasonal asthma control. Methods: We combined next-generation sequencing with quantitative polymerase chain reaction (qPCR) to measure concentrations of fungal DNA in indoor floor dust samples (n = 298) collected from homes participating in the New York City Neighborhood Asthma and Allergy Study (NAAS). Results: Total fungal concentration in spring was significantly higher than the other three seasons (p ≤ 0.005). Mean concentrations for 78% of fungal species were elevated in the spring (26% were significantly highest in spring, p < 0.05). Concentrations of 8 allergenic fungal species were significantly (p < 0.5) higher in spring compared to at least two other seasons. Indoor relative humidity and temperature were significantly highest in spring (p < 0.05) and were associated with total fungal concentration (R2 = 0.049, R2 = 0.11, respectively). Conclusion: There is significant seasonal variation in total fungal concentration and concentration of select allergenic species. Indoor relative humidity and temperature may underlie these associations.

Gaseous and Particulate Content of Laser Tattoo Removal Plume.

BACKGROUND: There is increasing awareness of the potential hazards of surgical plumes. The plume associated with laser tattoo removal remains uncharacterized. OBJECTIVE: To determine the gaseous, particulate, and microbiological content of the laser tattoo removal plume. MATERIALS AND METHODS: Air sampling was performed during laser tattoo removal from pig skin and from patients. Measurement of metals, volatile organic compounds (VOCs), carbon monoxide (CO), hydrogen sulfide (HS), and ultrafine particulates (UPs) as well as bacterial 16S ribosomal DNA sequencing were performed. RESULTS: Metals were identified in the plume from both pig and human skin. Volatile organic compounds were found at similar levels within and outside the treatment room. Several bacterial phyla were detected in the treatment room, but not outside. High levels of UPs were measured throughout the treatment room during tattoo removal from pig skin. Ultrafine particulates were detected at low levels in the room periphery during tattoo removal from human skin, but at higher levels in the immediate treatment zone. HS and CO were not detected. CONCLUSION: Metals, VOCs, HS, and CO were found at levels below applicable occupational exposure limits. The presence of bacteria is of uncertain significance, but may be hazardous. High levels of UPs require further investigation.

Inhalation of Stachybotrys chartarum Fragments Induces Pulmonary Arterial Remodeling.

Stachybotrys chartarum is a fungal contaminant within the built environment and a respiratory health concern in the United States. The objective of this study was to characterize the mechanisms influencing pulmonary immune responses to repeatedly inhaled S. chartarum. Groups of B6C3F1/N mice repeatedly inhaled viable trichothecene-producing S. chartarum conidia (strain A or strain B), heat-inactivated conidia, or high-efficiency particulate absolute-filtered air twice per week for 4 and 13 weeks. Strain A was found to produce higher amounts of respirable fragments than strain B. Lung tissue, serum, and BAL fluid were collected at 24 and 48 hours after final exposure and processed for histology, flow cytometry, and RNA and proteomic analyses. At 4 weeks after exposure, a T-helper cell type 2-mediated response was observed. After 13 weeks, a mixed T-cell response was observed after exposure to strain A compared with a T-helper cell type 2-mediated response after strain B exposure. After exposure, both strains induced pulmonary arterial remodeling at 13 weeks; however, strain A-exposed mice progressed more quickly than strain B-exposed mice. BAL fluid was composed primarily of eosinophils, neutrophils, and macrophages. Both the immune response and the observed pulmonary arterial remodeling were supported by specific cellular, molecular, and proteomic profiles. The immunopathological responses occurred earlier in mice exposed to high fragment-producing strain A. The rather striking induction of pulmonary remodeling by S. chartarum appears to be related to the presence of fungal fragments during exposure.

Cultivation and aerosolization of Stachybotrys chartarum for modeling pulmonary inhalation exposure.

Objective:Stachybotrys chartarum is a hydrophilic fungal species commonly found as a contaminant in water-damaged building materials. Although several studies have suggested that S. chartarum exposure elicits a variety of adverse health effects, the ability to characterize the pulmonary immune responses to exposure is limited by delivery methods that do not replicate environmental exposure. This study aimed to develop a method of S. chartarum aerosolization to better model inhalation exposures. Materials and methods: An acoustical generator system (AGS) was previously developed and utilized to aerosolize and deliver fungal spores to mice housed in a multi-animal nose-only exposure chamber. In this study, methods for cultivating, heat-inactivating, and aerosolizing two macrocyclic trichothecene-producing strains of S. chartartum using the AGS are described. Results and discussion: In addition to conidia, acoustical generation of one strain of S. chartarum resulted in the aerosolization of fungal fragments (<2 µm aerodynamic diameter) derived from conidia, phialides, and hyphae that initially comprised 50% of the total fungal particle count but was reduced to less than 10% over the duration of aerosolization. Acoustical generation of heat-inactivated S. chartarum did not result in a similar level of fragmentation. Delivery of dry, unextracted S. chartarum using these aerosolization methods resulted in pulmonary inflammation and immune cell infiltration in mice inhaling viable, but not heat-inactivated S. chartarum. Conclusions: These methods of S. chartarum growth and aerosolization allow for the delivery of fungal bioaerosols to rodents that may better simulate natural exposure within water-damaged indoor environments.

Potential occupational and respiratory hazards in a Minnesota cannabis cultivation and processing facility.

BACKGROUND: Cannabis has been legalized in some form for much of the United States. The National Institute for Occupational Safety and Health (NIOSH) received a health hazard evaluation request from a Minnesota cannabis facility and their union to undertake an evaluation. METHODS: NIOSH representatives visited the facility in August 2016 and April 2017. Surface wipe samples were collected for analysis of delta-9 tetrahydrocannabinol (Δ9-THC), delta-9 tetrahydrocannabinol acid (Δ9-THCA), cannabidiol, and cannabinol. Environmental air samples were collected for volatile organic compounds (VOCs), endotoxins (limulus amebocyte lysate assay), and fungal diversity (NIOSH two-stage BC251 bioaerosol sampler with internal transcribed spacer region sequencing analysis). RESULTS: Surface wipe samples identified Δ9-THC throughout the facility. Diacetyl and 2,3-pentanedione were measured in initial VOC screening and subsequent sampling during tasks where heat transference was greatest, though levels were well below the NIOSH recommended exposure limits. Endotoxin concentrations were highest during processing activities, while internal transcribed spacer region sequencing revealed that the Basidiomycota genus, Wallemia, had the highest relative abundance. CONCLUSIONS: To the authors' knowledge, this is the first published report of potential diacetyl and 2,3-pentanedione exposure in the cannabis industry, most notably during cannabis decarboxylation. Endotoxin exposure was elevated during grinding, indicating that this is a potentially high-risk task. The findings indicate that potential health hazards of significance are present during cannabis processing, and employers should be aware of potential exposures to VOCs, endotoxin, and fungi. Further research into the degree of respiratory and dermal hazards and resulting health effects in this industry is recommended.

Aspergillus fumigatus viability drives allergic responses to inhaled conidia.

BACKGROUND: Aspergillus fumigatus-induced allergic airway disease has been shown to involve conidial germination in vivo, but the immunological mechanisms remain uncharacterized. OBJECTIVE: A subchronic murine exposure model was used to examine the immunological mediators that are regulated in response to either culturable or nonculturable A fumigatus conidia. METHODS: Female B6C3F1/N mice were repeatedly dosed via inhalation with 1 × 105 viable or heat-inactivated conidia (HIC), twice per week for 13 weeks (26 exposures). Control mice inhaled high-efficiency particulate arrestor-filtered air. The influence of A fumigatus conidial germination on the pulmonary immunopathological outcomes was evaluated by flow cytometry analysis of cellular infiltration in the airways, assessment of lung messenger RNA expression, quantitative proteomics, and histopathology of whole lung tissue. RESULTS: Repeated inhalation of viable conidia, but not HIC, resulted in allergic inflammation marked by vascular remodeling, extensive eosinophilia, and accumulation of alternatively activated macrophages (AAMs) in the murine airways. More specifically, mice that inhaled viable conidia resulted in a mixed TH1 and TH2 (IL-13) cytokine response. Recruitment of eosinophils corresponded with increased Ccl11 transcripts. Furthermore, genes associated with M2 or alternatively activated macrophage polarization (eg, Arg1, Chil3, and Retnla) were significantly up-regulated in viable A fumigatus-exposed mice. In mice inhaling HIC, CD4+ T cells expressing IFN-γ (TH1) dominated the lymphocytic infiltration. Quantitative proteomics of the lung revealed metabolic reprogramming accompanied by mitochondrial dysfunction and endoplasmic reticulum stress stimulated by oxidative stress from repetitive microbial insult. CONCLUSION: Our studies demonstrate that A fumigatus conidial viability in vivo is critical to the immunopathological presentation of chronic fungal allergic disease.

Microbial hazards during harvesting and processing at an outdoor United States cannabis farm.

Cannabis cultivation is an emerging industry within the United States. Organic dust derived in part from naturally occurring microorganisms is known to cause byssinosis in the hemp industry. In this pilot study, bacteria and fungi encountered by workers at an outdoor cannabis farm that utilized organic practices were elucidated by 16 S ribosomal RNA (rRNA) and Internal Transcribed Spacer (ITS) region sequencing, respectively. Area (n = 14) and personal air samples (n = 12) were collected during harvesting and processing activities. 16 S rRNA and ITS regions of extracted bacterial and fungal genomic DNA were amplified and sequenced using Sanger sequencing. Bacterial sequencing resolved 1,077 sequences that were clustered into 639 operational taxonomic units (OTUs) and predominantly placed in the phylum, Actinobacteria (46%). Personal air samples revealed higher bacterial and Actinobacteria diversity compared to outdoor area samples collected within the facility (p < 0.05). A high degree of dissimilarity between bacteria was identified within and between samples. Fungal sequences (n = 985) were identified and predominantly clustered in the phylum Ascomycota (53%). Of the 216 fungal OTUs elucidated, the cannabis plant pathogenic species, Botrytis cinerea, was the most prevalent and accounted for 34% of all fungal sequences. The relative abundance of B. cinerea was highest in personal air samples (59%) compared to area samples collected in the drying room (19%), greenhouse (18%), and outdoor environment (6%). There was 49% sample similarity between fungi identified within personal air samples, but higher dissimilarity coefficients were observed within and between greenhouse, drying room, and outdoor area air samples. The results of this pilot study suggest that the cannabis farm workers are potentially exposed to Actinobacteria as well as the cannabis plant pathogen, B. cinerea during harvesting, bud-stripping, and hand-trimming processes.

Assessment of fungal diversity in a water-damaged office building.

Recent studies have described fungal communities in indoor environments using gene sequencing-based approaches. In this study, dust-borne fungal communities were elucidated from a water-damaged office building located in the northeastern region of the United States using internal transcribed spacer (ITS) rRNA gene sequencing. Genomic DNA was extracted from 5 mg of floor dust derived from 22 samples collected from either the lower floors (n = 8) or a top floor (n = 14) of the office building. ITS gene sequencing resolved a total of 933 ITS sequences and was clustered into 216 fungal operational taxonomic units (OTUs). Analysis of fungal OTUs at the 97% similarity threshold showed a difference between the lower and top floors that was marginally significant (p = 0.049). Species richness and diversity indices were reduced in the lower floor samples compared to the top floor samples and there was a high degree of compositional dissimilarity within and between the two different areas within the building. Fungal OTUs were placed in the phyla Ascomycota (55%), Basidiomycota (41%), Zygomycota (3%), Glomeromycota (0.4%), Chytridiomycota (0.3%), and unassigned fungi (0.5%). The Ascomycota classes with the highest relative abundances included the Dothideomycetes (30%) and Eurotiomycetes (16%). The Basidiomycota consisted of the classes Ustilaginomycetes (14%), Tremellomycetes (11%), and Agaricomycetes (8%). Sequence reads derived from the plant pathogen Ustilago syntherismae were the most abundant in the analysis as were obligate Basidiomycota yeast species that accounted for 12% and 11% of fungal ITS sequences, respectively. ITS gene sequencing provides additional insight into the diversity of fungal OTUs. These data further highlight the contribution of fungi placed in the phylum Basidiomycota, obligate yeasts, as well as xerophilic species that are typically not resolved using traditional culture methods.

The influence of diisocyanate antigen preparation methodology on monoclonal and serum antibody recognition.

Exposure to diisocyanates (dNCOs), such as methylene diphenyl diisocyanate (MDI) can cause occupational asthma (OA). Currently, lab tests for dNCO specific IgE are specific, but not sensitive, which limits their utility in diagnosing dNCO asthma. This may be due to variable preparation and poor characterization of the standard antigens utilized in these assays. The aim of this study was to produce and characterize a panel of antigens prepared using three different commonly employed methods and one novel method. The conjugates were examined for recognition by anti-MDI monoclonal antibodies (mAbs) in varying enzyme linked immunosorbant assay (ELISA) formats, extent of crosslinking, total amount of MDI, the sites of MDI conjugation, relative shape/charge, and reactivity with human serum with antibodies from sensitized, exposed workers. Results indicate that while there are minimal differences in the total amount of MDI conjugated, the extent of crosslinking, and the conjugation sites, there are significant differences in the recognition of differently prepared conjugates by mAbs. Native and denaturing polyacrylamide gel electrophoresis demonstrate differences in the mobility of different conjugates, indicative of structural changes that are likely important for antigenicity. While mAbs exhibited differential binding to different conjugates, polyclonal serum antibodies from MDI exposed workers exhibited equivalent binding to different conjugates by ELISA. While differences in the recognition of the different conjugates exist by mAb detection, differences in antigenicity could not be detected using human serum from MDI-sensitized individuals. Thus, although dNCO conjugate preparation can, depending on the immunoassay platform, influence binding of specific antibody clones, serologic detection of the dNCO-exposure-induced polyclonal antibody response may be less sensitive to these differences.

Investigating a persistent odor at an aircraft seat manufacturer.

An aircraft seat manufacturing company requested a NIOSH health hazard evaluation to help identify a strong odor that had persisted throughout the facility for over a year. Employees reported experiencing health effects thought to be related to the odor. We collected and analyzed area air samples for volatile organic compounds, endotoxin, bacterial and fungal metagenome, and metalworking fluid aerosol. Bulk metalworking fluid samples were analyzed for endotoxin, bacterial and fungal metagenome, and viable bacteria and fungus. We also evaluated the building ventilation systems and water diversion systems. Employees underwent confidential medical interviews about work practices, medical history, and health concerns. Based on our analyses, the odor was likely 2-methoxy-3,5-dimethylpyrazine. This pyrazine was found in air samples across the facility and originated from bacteria in the metalworking fluid. We did not identify bacteria known to produce the compound but bacteria from the same Proteobacteria order were found as well as bacteria from orders known to produce other pyrazines. Chemical and biological contaminants and odors could have contributed to health symptoms reported by employees, but it is likely that the symptoms were caused by several factors. We provided several recommendations to eliminate the odor including washing and disinfecting the metalworking machines and metalworking fluid recycling equipment, discarding all used metalworking fluid, instituting a metalworking fluid maintenance program at the site, and physically isolating the metalworking department from other departments.

A murine monoclonal antibody with broad specificity for occupationally relevant diisocyanates.

Diisocyanates (dNCOs) used in industrial applications are well known low molecular weight allergens. Occupational exposure is associated with adverse health outcomes including allergic sensitization and occupational asthma. In this study, we report the production and initial characterization of a dNCO-hapten specific murine IgM monoclonal antibody (mAb). Female BALB/c mice were immunized intraperitoneally with 25 µg of 4,4'-methylene diphenyl diisocyanate (MDI)-keyhole limpet hemocyanin. Following six biweekly booster immunizations, splenocytes were recovered and fused to Sp2/0-Ag14 murine myeloma cell line for hybridoma production. Hybridomas were then screened in a solid-phase indirect enzyme-linked immunosorbent assay (ELISA) against 40:1 4,4'-MDI- human serum albumin (HSA). mAb reactivity to dNCO-HSA conjugates and dNCO-HSA spiked human serum were characterized using a sandwich ELISA. One hybridoma produced a multimeric IgM mAb (15D4) that reacted with 4,4'-MDI-HSA. Sandwich ELISA analysis demonstrated comparable reactivity with other occupationally relevant dNCO-HSA adducts, including 2,4-toluene diisocyanate (TDI)-HSA, 2,6-TDI-HSA, and 1,6-hexamethylene diisocyanate (HDI)-HSA, but not other electrophilic chemical HSA conjugates. The limit of quantification (LOQ) of 4,4'-MDI-HSA, 2,4-TDI-HSA, 2,6-TDI-HSA, and 1,6-HDI-HSA sandwich ELISAs were 567.2, 172.7, 184.2, and 403.5 ng/mL (8.67, 2.60, 2.77, and 6.07 pmol/mL), respectively. In contrast, experiments using dNCO-supplemented human sera showed an increase in the detectable limit of the assay. A mAb has been produced that has potential utility for detecting mixed diisocyanate exposures in occupational environments. The mAb may have additional utility in the standardization of specific IgE detection immunoassays as well as chromatographic-mass spectrometric methods to enrich dNCO adducted HSA in the plasma of occupationally exposed workers.

Corrigendum: Persisting Cryptococcus yeast species Vishniacozyma victoriae and Cryptococcus neoformans elicit unique airway inflammation in mice following repeated exposure.

[This corrects the article DOI: 10.3389/fcimb.2023.1067475.].

Characterization of methylene diphenyl diisocyanate-haptenated human serum albumin and hemoglobin.

Protein haptenation by polyurethane industrial intermediate 4,4'-methylene diphenyl diisocyanate (MDI) is thought to be an important step in the development of diisocyanate (dNCO)-specific allergic sensitization; however, MDI-haptenated albumins used to screen specific antibody are often poorly characterized. Recently, the need to develop standardized immunoassays using a consistent, well-characterized dNCO-haptenated protein to screen for the presence of MDI-specific IgE and IgG from workers' sera has been emphasized and recognized. This has been challenging to achieve due to the bivalent electrophilic nature of dNCOs, leading to the capability to produce multiple cross-linked protein species and polymeric additions to proteins. In the current study, MDI was reacted with human serum albumin (HSA) and hemoglobin (Hb) at molar ratios ranging from 1:1 to 40:1 MDI/protein. Adducts were characterized by (i) loss of available 2,4,6-trinitrobenzene sulfonic acid (TNBS) binding to primary amines, (ii) electrophoretic migration in polyacrylamide gels, (iii) quantification of methylene diphenyl diamine following acid hydrolysis, and (iv) immunoassay. Concentration-dependent changes in all of the above noted parameters were observed, demonstrating increases in both number and complexity of conjugates formed with increasing MDI concentrations. In conclusion, a series of bioanalytical assays should be performed to standardize MDI-antigen preparations across lots and laboratories for measurement of specific antibody in exposed workers that in total indicate degree of intra- and intermolecular cross-linking, number of dNCOs bound, number of different specific binding sites on the protein, and degree of immunoreactivity.

Persisting Cryptococcus yeast species Vishniacozyma victoriae and Cryptococcus neoformans elicit unique airway inflammation in mice following repeated exposure.

Background: Allergic airway disease (AAD) is a growing concern in industrialized nations and can be influenced by fungal exposures. Basidiomycota yeast species such as Cryptococcus neoformans are known to exacerbate allergic airway disease; however, recent indoor assessments have identified other Basidiomycota yeasts, including Vishniacozyma victoriae (syn. Cryptococcus victoriae), to be prevalent and potentially associated with asthma. Until now, the murine pulmonary immune response to repeated V. victoriae exposure was previously unexplored. Objective: This study aimed to compare the immunological impact of repeated pulmonary exposure to Cryptococcus yeasts. Methods: Mice were repeatedly exposed to an immunogenic dose of C. neoformans or V. victoriae via oropharyngeal aspiration. Bronchoalveolar lavage fluid (BALF) and lungs were collected to examine airway remodeling, inflammation, mucous production, cellular influx, and cytokine responses at 1 day and 21 days post final exposure. The responses to C. neoformans and V. victoriae were analyzed and compared. Results: Following repeated exposure, both C. neoformans and V. victoriae cells were still detectable in the lungs 21 days post final exposure. Repeated C. neoformans exposure initiated myeloid and lymphoid cellular infiltration into the lung that worsened over time, as well as an IL-4 and IL-5 response compared to PBS-exposed controls. In contrast, repeated V. victoriae exposure induced a strong CD4+ T cell-driven lymphoid response that started to resolve by 21 days post final exposure. Discussion: C. neoformans remained in the lungs and exacerbated the pulmonary immune responses as expected following repeated exposure. The persistence of V. victoriae in the lung and strong lymphoid response following repeated exposure were unexpected given its lack of reported involvement in AAD. Given the abundance in indoor environments and industrial utilization of V. victoriae, these results highlight the importance to investigate the impact of frequently detected fungal organisms on the pulmonary response following inhalational exposure. Moreover, it is important to continue to address the knowledge gap involving Basidiomycota yeasts and their impact on AAD.

Optimization of Aspergillus versicolor Culture and Aerosolization in a Murine Model of Inhalational Fungal Exposure.

Aspergillus versicolor is ubiquitous in the environment and is particularly abundant in damp indoor spaces. Exposure to Aspergillus species, as well as other environmental fungi, has been linked to respiratory health outcomes, including asthma, allergy, and even local or disseminated infection. However, the pulmonary immunological mechanisms associated with repeated exposure to A. versicolor have remained relatively uncharacterized. Here, A. versicolor was cultured and desiccated on rice then placed in an acoustical generator system to achieve aerosolization. Mice were challenged with titrated doses of aerosolized conidia to examine deposition, lymphoproliferative properties, and immunotoxicological response to repeated inhalation exposures. The necessary dose to induce lymphoproliferation was identified, but not infection-like pathology. Further, it was determined that the dose was able to initiate localized immune responses. The data presented in this study demonstrate an optimized and reproducible method for delivering A. versicolor conidia to rodents via nose-only inhalation. Additionally, the feasibility of a long-term repeated exposure study was established. This experimental protocol can be used in future studies to investigate the physiological effects of repeated pulmonary exposure to fungal conidia utilizing a practical and relevant mode of delivery. In total, these data constitute an important foundation for subsequent research in the field.