A cohort study of cucumber greenhouse workers' exposure to microorganisms as measured using NGS and MALDI-TOF MS and biomarkers of systemic inflammation.

Work in greenhouses entails exposure to airborne fungi and bacteria. The aims of this study are to obtain knowledge about whether exposure to fungal and bacterial genera and species during work in a cucumber greenhouse is affected by work tasks, and whether a cohort of greenhouse workers' serum levels of serum amyloid A (SAA) and C-reactive protein (CRP), biomarkers of systemic inflammation, are associated with this. Data on personal exposure to airborne fungal and bacterial species measured over 4 years as well as serum levels of SAA and CRP sampled over two years were analyzed. For data analysis, the main work tasks were grouped into three different groups, called 'grouped work task'. Microorganisms were identified using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF MS) and next-generation sequencing (NGS). The 'daily exposure' of greenhouse workers' were as follows: 4.8 × 104 CFU bacteria/m3, 1.4 × 106 CFU fungi/m3, and 392 EU/m3 of endotoxin. Workers were exposed to many different microbial species including several species within the genera Acinetobacter, Bacillus, Microbacterium, Pseudomonas, Staphylococcus, and Streptomyces. The genera Ralstonia and Cladosporium were found in most samples. The exposure levels as well as the microbial composition were associated significantly with grouped work task and season with high exposures during tasks in close contact with mature and old plants and in the autumn. CRP and SAA levels were also associated with exposure level and grouped work tasks. The Shannon-Wiener indices were not different in the 3 'grouped work tasks'. Several specific species including e.g. Halomonas elongata, Stenotrophomonas maltophilia, Podosphaera fusca, and Wallemia spp. were found frequently or in high concentrations in the exposures associated with the highest levels of CRP and SAA. The microorganisms S. maltophilia, P. fusca, and Wallemia spp. were also found on the cucumber plant leaves. In conclusion, both exposure level and the species composition seem to have an effect on the serum levels of CRP and SAA of exposed workers. The greenhouse workers were exposed to only a few species characterized as human pathogens.

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.

ß-Glucan exacerbates allergic asthma independent of fungal sensitization and promotes steroid-resistant TH2/TH17 responses.

BACKGROUND: Allergic sensitization to fungi has been associated with asthma severity. As a result, it has been largely assumed that the contribution of fungi to allergic disease is mediated through their potent antigenicity. OBJECTIVE: We sought to determine the mechanism by which fungi affect asthma development and severity. METHODS: We integrated epidemiologic and experimental asthma models to explore the effect of fungal exposure on asthma development and severity. RESULTS: We report that fungal exposure enhances allergen-driven TH2 responses, promoting severe allergic asthma. This effect is independent of fungal sensitization and can be reconstituted with ß-glucan and abrogated by neutralization of IL-17A. Furthermore, this severe asthma is resistant to steroids and characterized by mixed TH2 and TH17 responses, including IL-13+IL-17+CD4+ double-producing effector T cells. Steroid resistance is dependent on fungus-induced TH17 responses because steroid sensitivity was restored in IL-17rc-/- mice. Similarly, in children with asthma, fungal exposure was associated with increased serum IL-17A levels and asthma severity. CONCLUSION: Our data demonstrate that fungi are potent immunomodulators and have powerful effects on asthma independent of their potential to act as antigens. Furthermore, our results provide a strong rationale for combination treatment strategies targeting IL-17A for this subgroup of fungus-exposed patients with difficult-to-treat asthma.

Indoor Fungal Exposure and Allergic Respiratory Disease.

A gathering body of evidence has repeatedly revealed associations between indoor fungi and initiation, promotion, and exacerbation of allergic respiratory disease. The relationship between the exposure and outcome are complicated by the difficulties in measuring both exposure and outcome, the multifactorial nature of the disease, and the wide range of potential confounders. New technologies are becoming available that may enable better measurement of exposure and tighter case definitions so as to build more confidence in the associations discovered. The growing strength of the evidence base will aid the design of future public health interventions and generate new hypotheses on the cause of the rapid increase in allergic respiratory disease prevalence.

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.

Occupational Exposure to ß-d-Glucans, Mould Allergens, Endotoxins and Cultivable Fungi in Pig Farms.

Airborne concentrations of organic dust on animal farms are known to be very high. This dust is partly composed of microorganisms such as bacteria, fungi and their components [endotoxins, (1→3)-ß-d-glucans, mould allergens, mycotoxins], recognised as being responsible for numerous health effects. Several cross-sectional studies have measured levels of airborne bacteria, fungi and endotoxins on pig farms. However, the temporal dynamics of organic dust's components throughout the year have rarely been assessed, and airborne concentrations of (1→3)-ß-d-glucans and mould allergens remain poorly understood in these work environments. This longitudinal, four-season study measured cultivable fungi, endotoxins, (1→3)-ß-d-glucans, Aspergillus versicolor (AveX), Aspergillus fumigatus (Asp f1) and Alternaria sp (Alt a1) allergens on 31 pig farms in Switzerland. Results showed that exposure to AveX occurred in all four seasons. Total mean airborne concentration of endotoxins were between 3 and 4 times higher than the Swiss recommended limit value of 1000 EU m-3 and mean airborne concentrations of fungi were between 30 and 50 times higher than the Swiss recommended limit value of 1000 cfu m-3. Finally, accumulations of faecal matter on floors, humidity and dusty pathways were associated with increased concentrations of (1→3)-ß-d-glucans. In conclusion, pig farmers require better information about biological occupational risks, and measures to improve air quality should be implemented, especially in winter.

Comparison of serum interleukin-10 level of fungal exposure among patients with pulmonary sarcoidosis and healthy people.

Introduction: Sarcoidosis is a chronic systemic inflammatory disease with unknown etiology. Fungal exposure has been assumed as one of many possible causes of the disease. The prevalence of sarcoidosis is likely to be higher in the Northern Iran compared with other regions. Environmental studies have shown higher levels of fungal spores in the air of this area. Some studies have shown that fungal exposure in patients with sarcoidosis is associated with decreased levels of interleukin-10 (IL-10) serum levels. The aim of present study was comparison of the serum levels of IL-10 in patients with pulmonary sarcoidosis and healthy people. Objectives and Methods: In this current analytical, cross-sectional study, 40 patients with pulmonary sarcoidosis compared with 34 healthy individuals as a control group, who had been visited in a pulmonary referral clinic in Rasht (Guilan-Iran). Demographic data were collected by a questionnaire. Serum IL-10 levels were measured by ELISA kit. The data were analyzed by using the SPSS software (version 19). Results: The mean concentration of IL-10 serum levels were reported 10.96±9.48 pg/ml-1 and 3.77±1.47 pg/ml-1 among the patients with pulmonary sarcoidosis and healthy individuals, respectively. The significance difference was demonstrated between patients with pulmonary sarcoidosis and control group (p<0.0001). The IL-10 showed a significant difference between the patients older than 40 and those younger than 40. In statistical analysis, 4.75 pg.ml-1 was considered the cutoff point to separate patients and control group. Conclusion: The results showed that IL-10 was greater among patients who diagnosed as pulmonary sarcoidosis. There was a contrary opinion of the expectations for the role of fungal exposure as a possible cause of greater prevalence of sarcoidosis in Northern Iran. Age and stage of disease showed a significant relationship with the IL-10 serum level and requires further investigation. IL-10 might be a possible predictor of sarcoidosis along with other factors. (Sarcoidosis Vasc Diffuse Lung Dis 2018; 35: 294-298).

MicroRNA Regulation of Host Immune Responses following Fungal Exposure.

Fungal bioaerosols are ubiquitous in the environment and human exposure can result in a variety of health effects ranging from systemic, subcutaneous, and cutaneous infections to respiratory morbidity including allergy, asthma, and hypersensitivity pneumonitis. Recent research has focused on the role of microRNAs (miRNAs) following fungal exposure and is overlooked, yet important, group of regulators capable of influencing fungal immune responses through a variety of cellular mechanisms. These small non-coding ribose nucleic acids function to regulate gene expression at the post-transcriptional level and have been shown to participate in multiple disease pathways including cancer, heart disease, apoptosis, as well as immune responses to microbial hazards and occupational allergens. Recent animal model studies have characterized miRNAs following the exposure to inflammatory stimuli. Studies focused on microbial exposure, including bacterial infections, as well as exposure to different allergens have shown miRNAs, such as miR-21, miR-146, miR-132, miR-155, and the let-7 family members, to be involved in immune and inflammatory responses. Interestingly, the few studies have assessed that the miRNA profiles following fungal exposure have identified the same critical miRNAs that have been characterized in other inflammatory-mediated and allergy-induced experimental models. Review of available in vitro, animal and human studies of exposures to Aspergillus fumigatus, Candida albicans, Cryptococcus neoformans, Paracoccidioides brasiliensis, and Stachybotrys chartarum identified several miRNAs that were shared between responses to these species including miR-125 a/b (macrophage polarization/activation), miR-132 [toll-like receptor (TLR)2-mediated signaling], miR-146a (TLR mediated signaling, alternative macrophage activation), and miR-29a/b (natural killer cell function, C-leptin signaling, inhibition of Th1 immune response). Although these datasets provide preliminary insight into the role of miRNAs in fungal exposed models, interpretation of miRNA datasets can be challenging for researchers. To assist in navigating this rapidly evolving field, the aim of this review is to describe miRNAs in the framework of host recognition mechanisms and provide initial insight into the regulatory pathways in response to fungal exposure.

A next generation sequencing approach with a suitable bioinformatics workflow to study fungal diversity in bioaerosols released from two different types of composting plants.

Composting is used all over the world to transform different types of organic matter through the actions of complex microbial communities. Moving and handling composting material may lead to the emission of high concentrations of bioaerosols. High exposure levels are associated with adverse health effects among compost industry workers. Fungal spores are suspected to play a role in many respiratory illnesses. There is a paucity of information related to the detailed fungal diversity in compost as well as in the aerosols emitted through composting activities. The aim of this study was to analyze the fungal diversity of both organic matter and aerosols present in facilities that process domestic compost and facilities that process pig carcasses. This was accomplished using a next generation sequencing approach that targets the ITS1 genomic region. Multivariate analyses revealed differences in the fungal community present in samples coming from compost treating both raw materials. Furthermore, results show that the compost type affects the fungal diversity of aerosols emitted. Although 8 classes were evenly distributed in all samples, Eurotiomycetes were more dominant in carcass compost while Sordariomycetes were dominant in domestic compost. A large diversity profile was observed in bioaerosols from both compost types showing the presence of a number of pathogenic fungi newly identified in bioaerosols emitted from composting plants. Members of the family Herpotrichiellaceae and Gymnoascaceae which have been shown to cause human diseases were detected in compost and air samples. Moreover, some fungi were identified in higher proportion in air compared to compost. This is the first study to identify a high level of fungal diversity in bioaerosols present in composting plants suggesting a potential exposure risk for workers. This study suggests the need for creating guidelines that address human exposure to bioaerosols. The implementation of technical and organizational measure should be a top priority. However, skin and respiratory protection for compost workers could be used to reduce the exposure as a second resort.

Innate and Adaptive Immune Response to Fungal Products and Allergens.

Exposure to fungi and their products is practically ubiquitous, yet most of this is of little consequence to most healthy individuals. This is because there are a number of elaborate mechanisms to deal with these exposures. Most of these mechanisms are designed to recognize and neutralize such exposures. However, in understanding these mechanisms it has become clear that many of them overlap with our ability to respond to disruptions in tissue function caused by trauma or deterioration. These responses involve the innate and adaptive immune systems usually through the activation of nuclear factor kappa B and the production of cytokines that are considered inflammatory accompanied by other factors that can moderate these reactivities. Depending on different genetic backgrounds and the extent of activation of these mechanisms, various pathologies with resulting symptoms can ensue. Complicating this is the fact that these mechanisms can bias toward type 2 innate and adaptive immune responses. Thus, to understand what we refer to as allergens from fungal sources, we must first understand how they influence these innate mechanisms. In doing so it has become clear that many of the proteins that are described as fungal allergens are essentially homologues of our own proteins that signal or cause tissue disruptions.