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The Murine Pulmonary Proteomic Profile Associated with Allergic Aspergillus Fumigatus Exposure
Abstracts AB181
J ALLERGY CLIN IMMUNOL VOLUME 137, NUMBER 2
Fungal Metagenomic Analysis of Indoor Evaporative Cooler Environments in the Great Basin Desert Region
Angela R. Lemons, MS1, Mary Beth Hogan, MD, FAAAAI2, Ruth A. Gault, PhD3, Kathleen J. Holland, MD4, Edward Sobek, PhD5, Kimberly A. Olsen-Wilson, MSHS6, Brett J. Green, PhD, FAAAAI1; 1Allergy and Clinical Immunology Branch, Health Effects Laboratory Division, National Institute for Occupational Safety and Health, Centers for Disease Control and Prevention, Morgantown, WV, 2Department of Pediatrics, University of Nevada School of Medicine, Las Vegas, NV, 3Department of Microbiology and Immunology, University of Nevada School of Medicine, Reno, NV, 4Department of Pediatrics, Indiana University, Indianapolis, IN, 5Assured Bio Labs, Oak Ridge, TN, 6Department of Pediatrics, University of Nevada School of Medicine, Reno, NV. RATIONALE: Personal exposure to fungal bioaerosols can increase the risk of respiratory disease, including allergy and asthma. Skin test reactivity to fungal and dust mite allergens has recently been shown to be increased in children living in homes cooled by evaporative coolers in the Great Basin Desert region. The objective of this study was to determine if the increased humidity reported in these homes leads to different fungal populations compared to homes cooled using traditional systems. METHODS: Children from Reno, Nevada with physician diagnosed asthma and/or allergy that lived in environments cooled by evaporative coolers (n510) or air conditioner systems (n59) were recruited into the study. Air samples were collected from indoor living environments for genomic DNA extraction and metagenomic analysis of fungi using the next-generation sequencing platform, Illumina Mi-Seq. RESULTS: The fungal populations observed in homes that utilized evaporative coolers differed greatly from air conditioned homes. The most prevalent species discovered in air conditioned environments belonged to the genera Cryptococcus (20%) and Aspergillus (20%). In contrast, the most predominant fungi identified in homes that utilized evaporative coolers included Alternaria alternata (32%) and Phoma spp. (21%). Outdoor air in the region revealed a high prevalence of Cladosporium spp. (25%) and Davidiella spp. (43%). CONCLUSIONS: Metagenomic analysis of air samples derived from air conditioned and evaporative cooler environments revealed differences in fungal populations. These variations could explain the increased skin test reactivity and disease severity reported in pediatric populations that live in evaporative cooler environments located in this region.
593
Internal Transcribed Spacer rRNA Gene Sequencing Analysis of Dustborne Fungi in a Water-Damaged Office Building
Brett J. Green, PhD, FAAAAI1, Angela R. Lemons, MS1, Yeonmi Park, MS2, Jean M. Cox-Ganser, PhD2, Ju-Hyeong Park, ScD, MPH, CIH2; 1Allergy and Clinical Immunology Branch, Health Effects Laboratory Division, National Institute for Occupational Safety and Health, Centers for Disease Control and Prevention, Morgantown, WV, 2Field Studies Branch, Division of Respiratory Disease Studies, National Institute for Occupational Safety and Health, Centers for Disease Control and Prevention, Morgantown, WV. RATIONALE: Viable fungal composition in dust is used to assess exposure in indoor and occupational environments. In this study, internal transcribed spacer (ITS) rRNA gene sequencing was used to further elucidate the fungal profiles in floor dust derived from a water-damaged office building. METHODS: Floor dust samples (n524) were analyzed as part of an earlier investigation of an office building located in the northeastern United States that had well characterized water intrusion. Genomic DNA was extracted from 5 mg of floor dust. Fungal ITS regions were amplified and sequenced using Sanger sequencing. Sequences were clustered into organizational taxonomic units (OTUs) and sequence identifications
were determined by searching the National Center for Biotechnology Information nucleotide database. RESULTS: A total of 1028 ITS sequences were identified and placed in 288 OTUs. The dust samples were composed of sequences derived from the fungal phyla, Ascomycota (58%), Basidiomycota (38%), Zygomycota (3%), Glomeromycota (0.4%), Chytridiomycota (0.2%) and unclassified fungi (0.5%). The predominant Ascomycota classes included the Dothideomycetes (33%) and Eurotiomycetes (14%). In contrast, the Basidiomycota were primarily composed of the Ustilaginomycetes (13%), Tremellomycetes (10%), and Agaricomycetes (8%). Species that represented greater than 5% of all identified fungal sequences included Pithomyces chartarum, Aureobasidium microstictum, Aspergillus penicillioides, and Ustilago syntherismae. CONCLUSIONS: The Sanger sequencing dataset demonstrated a broad spectrum of fungi that were recovered from the dust of a water damaged office environment. These data identify a previously overlooked fungal profile composed of Basidiomycota species. These fungi may contribute to occupants’ exposure in moisture damaged indoor environments.
594
The Murine Pulmonary Proteomic Profile Associated with Allergic Aspergillus Fumigatus Exposure
Ajay P. Nayak, PhD1, Tara L. Croston, PhD2, Angela R. Lemons, MS2, W. Travis Goldsmith, BSCpE3, Michael L. Kashon, PhD4, Dori M. Germolec, PhD5, Donald H. Beezhold, PhD, FAAAAI6, Brett J. Green, PhD, FAAAAI1; 1Allergy and Clinical Immunology Branch, Health Effects Laboratory Division, National Institute for Occupational Safety and Health, Centers for Disease Control and Prevention, Morgantown, WV, 2CDC/NIOSH/ACIB, Morgantown, WV, 3Engineering and Control Technology Branch, Health Effects Laboratory Division, National Institute for Occupational Safety and Health, Centers for Disease Control and Prevention, Morgantown, WV, 4Biostatistics and Epidemiology Branch, Health Effects Laboratory Division, National Institute for Occupational Safety and Health, Centers for Disease Control and Prevention, Morgantown, WV, 5Toxicology Branch, Division of the National Toxicology Program, National Institute of Environmental Health Sciences, Research Triangle Park, NC, 6Health Effects Laboratory Division, National Institute for Occupational Safety and Health, Centers for Disease Control and Prevention, Morgantown, WV. RATIONALE: Quantitative proteomics is an approach that complements existing methodologies available to examine disease state and enable biomarker discovery. We aimed to examine the alterations in the lung proteome following repeated inhalation of Aspergillus fumigatusconidia in a murine model of fungal allergy. METHODS: Na€ıve B6C3F1/n mice inhaled viable A. fumigatus conidia (105) or HEPA-filtered air (control) twice a week for 13 weeks. Proteins from lung homogenates were separated by SDS-PAGE, and analyzed by nano LC/MS/MS with a Waters NanoAcquity system by MS Bioworks. Acquired peptide-ions were searched against the Mus musculusdatabase. The resultant spectral count (SpC) dataset was assessed to evaluate differences in abundance between viable and air control group. RESULTS: In mice exposed to viable conidia, 143 proteins were identified in increased abundance. Chitinase like-proteins 3 and 4, were identified as most abundant proteins (10-50 fold higher SpC vs control) associated with allergic inflammation. Antimicrobial proteins S100-A9 and surfactant protein D were also significantly more abundant in lungs of these mice (3-5 fold higher Spc vs control). Repeated microbial insult resulted in hypoxic microenvironment as evident by increased abundance of hypoxia regulated protein. Vigilin, a novel marker with uncharacterized role was also significantly abundant (3 fold higher Spc vs control) in exposed mice. CONCLUSIONS: This study demonstrates shifts in pulmonary proteomic profile following chronic fungal exposure. The study also highlights the utility of SpC metrics in determining the abundance of specific protein markers and underlines a significant role for identified proteins in disease progression in the described model.
SUNDAY
592
J ALLERGY CLIN IMMUNOL VOLUME 137, NUMBER 2
Fungal Metagenomic Analysis of Indoor Evaporative Cooler Environments in the Great Basin Desert Region
Angela R. Lemons, MS1, Mary Beth Hogan, MD, FAAAAI2, Ruth A. Gault, PhD3, Kathleen J. Holland, MD4, Edward Sobek, PhD5, Kimberly A. Olsen-Wilson, MSHS6, Brett J. Green, PhD, FAAAAI1; 1Allergy and Clinical Immunology Branch, Health Effects Laboratory Division, National Institute for Occupational Safety and Health, Centers for Disease Control and Prevention, Morgantown, WV, 2Department of Pediatrics, University of Nevada School of Medicine, Las Vegas, NV, 3Department of Microbiology and Immunology, University of Nevada School of Medicine, Reno, NV, 4Department of Pediatrics, Indiana University, Indianapolis, IN, 5Assured Bio Labs, Oak Ridge, TN, 6Department of Pediatrics, University of Nevada School of Medicine, Reno, NV. RATIONALE: Personal exposure to fungal bioaerosols can increase the risk of respiratory disease, including allergy and asthma. Skin test reactivity to fungal and dust mite allergens has recently been shown to be increased in children living in homes cooled by evaporative coolers in the Great Basin Desert region. The objective of this study was to determine if the increased humidity reported in these homes leads to different fungal populations compared to homes cooled using traditional systems. METHODS: Children from Reno, Nevada with physician diagnosed asthma and/or allergy that lived in environments cooled by evaporative coolers (n510) or air conditioner systems (n59) were recruited into the study. Air samples were collected from indoor living environments for genomic DNA extraction and metagenomic analysis of fungi using the next-generation sequencing platform, Illumina Mi-Seq. RESULTS: The fungal populations observed in homes that utilized evaporative coolers differed greatly from air conditioned homes. The most prevalent species discovered in air conditioned environments belonged to the genera Cryptococcus (20%) and Aspergillus (20%). In contrast, the most predominant fungi identified in homes that utilized evaporative coolers included Alternaria alternata (32%) and Phoma spp. (21%). Outdoor air in the region revealed a high prevalence of Cladosporium spp. (25%) and Davidiella spp. (43%). CONCLUSIONS: Metagenomic analysis of air samples derived from air conditioned and evaporative cooler environments revealed differences in fungal populations. These variations could explain the increased skin test reactivity and disease severity reported in pediatric populations that live in evaporative cooler environments located in this region.
593
Internal Transcribed Spacer rRNA Gene Sequencing Analysis of Dustborne Fungi in a Water-Damaged Office Building
Brett J. Green, PhD, FAAAAI1, Angela R. Lemons, MS1, Yeonmi Park, MS2, Jean M. Cox-Ganser, PhD2, Ju-Hyeong Park, ScD, MPH, CIH2; 1Allergy and Clinical Immunology Branch, Health Effects Laboratory Division, National Institute for Occupational Safety and Health, Centers for Disease Control and Prevention, Morgantown, WV, 2Field Studies Branch, Division of Respiratory Disease Studies, National Institute for Occupational Safety and Health, Centers for Disease Control and Prevention, Morgantown, WV. RATIONALE: Viable fungal composition in dust is used to assess exposure in indoor and occupational environments. In this study, internal transcribed spacer (ITS) rRNA gene sequencing was used to further elucidate the fungal profiles in floor dust derived from a water-damaged office building. METHODS: Floor dust samples (n524) were analyzed as part of an earlier investigation of an office building located in the northeastern United States that had well characterized water intrusion. Genomic DNA was extracted from 5 mg of floor dust. Fungal ITS regions were amplified and sequenced using Sanger sequencing. Sequences were clustered into organizational taxonomic units (OTUs) and sequence identifications
were determined by searching the National Center for Biotechnology Information nucleotide database. RESULTS: A total of 1028 ITS sequences were identified and placed in 288 OTUs. The dust samples were composed of sequences derived from the fungal phyla, Ascomycota (58%), Basidiomycota (38%), Zygomycota (3%), Glomeromycota (0.4%), Chytridiomycota (0.2%) and unclassified fungi (0.5%). The predominant Ascomycota classes included the Dothideomycetes (33%) and Eurotiomycetes (14%). In contrast, the Basidiomycota were primarily composed of the Ustilaginomycetes (13%), Tremellomycetes (10%), and Agaricomycetes (8%). Species that represented greater than 5% of all identified fungal sequences included Pithomyces chartarum, Aureobasidium microstictum, Aspergillus penicillioides, and Ustilago syntherismae. CONCLUSIONS: The Sanger sequencing dataset demonstrated a broad spectrum of fungi that were recovered from the dust of a water damaged office environment. These data identify a previously overlooked fungal profile composed of Basidiomycota species. These fungi may contribute to occupants’ exposure in moisture damaged indoor environments.
594
The Murine Pulmonary Proteomic Profile Associated with Allergic Aspergillus Fumigatus Exposure
Ajay P. Nayak, PhD1, Tara L. Croston, PhD2, Angela R. Lemons, MS2, W. Travis Goldsmith, BSCpE3, Michael L. Kashon, PhD4, Dori M. Germolec, PhD5, Donald H. Beezhold, PhD, FAAAAI6, Brett J. Green, PhD, FAAAAI1; 1Allergy and Clinical Immunology Branch, Health Effects Laboratory Division, National Institute for Occupational Safety and Health, Centers for Disease Control and Prevention, Morgantown, WV, 2CDC/NIOSH/ACIB, Morgantown, WV, 3Engineering and Control Technology Branch, Health Effects Laboratory Division, National Institute for Occupational Safety and Health, Centers for Disease Control and Prevention, Morgantown, WV, 4Biostatistics and Epidemiology Branch, Health Effects Laboratory Division, National Institute for Occupational Safety and Health, Centers for Disease Control and Prevention, Morgantown, WV, 5Toxicology Branch, Division of the National Toxicology Program, National Institute of Environmental Health Sciences, Research Triangle Park, NC, 6Health Effects Laboratory Division, National Institute for Occupational Safety and Health, Centers for Disease Control and Prevention, Morgantown, WV. RATIONALE: Quantitative proteomics is an approach that complements existing methodologies available to examine disease state and enable biomarker discovery. We aimed to examine the alterations in the lung proteome following repeated inhalation of Aspergillus fumigatusconidia in a murine model of fungal allergy. METHODS: Na€ıve B6C3F1/n mice inhaled viable A. fumigatus conidia (105) or HEPA-filtered air (control) twice a week for 13 weeks. Proteins from lung homogenates were separated by SDS-PAGE, and analyzed by nano LC/MS/MS with a Waters NanoAcquity system by MS Bioworks. Acquired peptide-ions were searched against the Mus musculusdatabase. The resultant spectral count (SpC) dataset was assessed to evaluate differences in abundance between viable and air control group. RESULTS: In mice exposed to viable conidia, 143 proteins were identified in increased abundance. Chitinase like-proteins 3 and 4, were identified as most abundant proteins (10-50 fold higher SpC vs control) associated with allergic inflammation. Antimicrobial proteins S100-A9 and surfactant protein D were also significantly more abundant in lungs of these mice (3-5 fold higher Spc vs control). Repeated microbial insult resulted in hypoxic microenvironment as evident by increased abundance of hypoxia regulated protein. Vigilin, a novel marker with uncharacterized role was also significantly abundant (3 fold higher Spc vs control) in exposed mice. CONCLUSIONS: This study demonstrates shifts in pulmonary proteomic profile following chronic fungal exposure. The study also highlights the utility of SpC metrics in determining the abundance of specific protein markers and underlines a significant role for identified proteins in disease progression in the described model.
SUNDAY
592