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Characterization of Mold in Indoor Air and Development of Respiratory Illness

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Characterization of Mold in Indoor Air and Development of Respiratory Illness Characterization of Mold in Indoor Air and Development of Respiratory Illness: Developing Co- Culture Techniques to Reveal How Fungal Conidia in Norwegian Indoor Air Might Cause Respiratory Health Problems Anders Benteson Nygaard Characterization of Mold in Indoor Air and Development of Respiratory Illness: Developing Co-Culture Techniques to Reveal How Fungal Conidia in Norwegian Indoor Air Might Cause Respiratory Health Problems By Anders Benteson Nygaard Master’s Degree, Biomedicine Faculty of Health Sciences Thesis submitted for the Master’s degree, 60 ECTS, Oslo and Akershus University College of Applied Sciences May 21st, 2012 ii Acknowledgements The work that is presented in this thesis has been carried out at the Faculty of Health Sciences, Oslo and Akershus University College of Applied Sciences (HIOA) from August 2011 to May 2012. The thesis is for the Master’s degree in Biomedicine at HIOA. The supervisors for this thesis were Associate Professor Jan Inge Herseth and Professor Colin Charnock. I wish to thank my supervisors for providing enthusiastic and encouraging support, and for sharing their knowledge with me in the work with this thesis. By providing fast and constructive feedback about lab routines or results, it has led to many fruitful discussions that have helped to form this thesis. Thanks for your time and excellent supervision during the course of this project. I also want to thank Nanna Winger Steen at the ABI-lab at the University of Oslo for the sequencing of DNA samples used to identify molds, and Bente Hellum at the Institute of Building and Energy Engineering at HIOA for providing the air impactor used to collect air samples in this thesis. Furthermore I wish to thank Hilde Herning and others who work around the laboratories at HIOA for teaching me the way around the lab, and for enlightening conversations by the centrifuge or in the hallways. Thanks for providing an including and friendly working environment. Most of all I am grateful to Sara for her incredible support and patience during the course of this Master’s degree. Without Sara, and our daughter Ella, I would not have been able to complete this degree. Oslo, May 2012 Anders Benteson Nygaard iii Abstract Living in damp or moldy homes has shown consistent associations with respiratory or allergic health effects. However, the causal link for these associations remains unclear. In this study we wished to establish a model in which it is possible to assess damp and moldy indoor environments and then study isolated molds from these environments in in-vitro models to understand how they potentially may cause respiratory health problems. This study collected air samples from damp/water damaged rooms and used polymerase chain reaction (PCR) sequencing along with other tests to determine the identity isolated molds from damp environments. Subsequently, selected mold isolates were selected to be used for in-vitro exposure experiments with cell mono-cultures and co-cultures (THP-1 monocytes (M), THP-1 derived dendritic cells (DCs), A549 pneumocytes (P)). These cell cultures were exposed to mold conidia that were native, heat inactivated or freeze-thaw fragmented. After exposure, the release of cytokines was evaluated with enzyme-linked immunosorbent assay (ELISA). With the use of PCR, identification of isolated molds was in general only possible at the genus level. Some isolates were identified at the species level. Of 12 molds isolated from damp environments, 9 were found to be Aspergillus species. In-vitro models were exposed to conidia from several species of molds. In contact co-cultures with M in contact with P and non-contact with DCs, Aspergillus versicolor was found to induce response of IL-8 whereas in monocultures, several species were found to induce IL-8 release. With use of methods tested and developed in this study, it is possible to study actual isolated molds from damp environments. When combining methods for identification of molds in damp environments with in-vitro methods to study the molds immunological affects, the understanding of damp/moldy indoor environments in relation to respiratory health problems can be improved. iv Sammendrag Å leve i fukt- eller muggskadede hjem har vist en konsekvent assosiasjon til luftveis eller allergiske helse effekter. Men de avgjørende faktorene som utløser disse assosiasjonene er fremdeles ukjente. I denne studien ønsket vi å etablere en modell som gjøre det mulig å vurdere fukt og mugg skadede miljøer, og studere isolere muggsopper fra disse miljøene, for å så bruke isolerte muggsopp i in-vitro forsøks modeller så man kan forstå hvordan de kan skape luftveishelseplager. Denne studien samlet luftprøver fra fuktige og vannskadede rom og brukte polymerase chain reaction (PCR) sekvensering sammen med andre analyser for å identifisere muggsopp isolert fra fuktige rom. Deretter ble utvalgte muggsopp brukt videre i in-vitro eksponering forsøk med celle mono-kulturer og co-kulturer (THP-1 monocytter (M), THP-1 deriverte dendrittiske celler (DCs), A549 pneumocytter (P)). Disse cellekulturene ble eksponert for muggsoppsporer som var naturlige, varmeinaktiverte eller fryse-tine fragmenterte. Etter eksponering ble nivåer at cytokiner målt med enzyme-linked immunosorbent assay (ELISA). Når PCR ble brukt for identifisering av isolerte mugsopp, så var det som regel bare mulig å identifisere til slektsnivå. Noen muggsoppisolater ble identifisert til artsnivå. Av 12 muggsopp isolert fra fuktige innemiljø ble 9 funnet til å være av arten Aspergillus. In-vitro modeller ble eksponert for sporer fra muggsopp av flere arter. I kontakt co-kulturer, med M i kontakt med P og ikke-kontakt med DCs ble det funnet at Aspergillus versicolor induserte utslipp av IL-8, mens i monokulturer fant vi at flere arter av muggsopp induserte utslipp av IL-8. Med metodene som har blitt testet og utviklet i denne studien, så er det mulig å studere muggsopp som er isolert fra steder med fuktskade. Når man kombinerer metodene brukt for å identifisere muggsopp i fuktskadde miljøer med in-vitro metoder for å utforske immunologiske effekter, så kan man øke forståelsen av hvordan fukt eller muggskadede hjem relaterer seg til luftveisplager. v Table of Contents Acknowledgements .................................................................................................................................................................. iii Abstract .......................................................................................................................................................................................... iv Sammendrag ..................................................................................................................................................................................v Table of Contents ....................................................................................................................................................................... vi Abbreviations ................................................................................................................................................................................ x 1 Introduction ........................................................................................................................................................................ 1 1.1 Damp buildings and visible mold .................................................................................................................. 1 1.2 Fungi and molds ..................................................................................................................................................... 4 1.2.1 Conidia ............................................................................................................................................................... 4 1.2.2 General growth conditions ..................................................................................................................... 4 1.2.3 Mycotoxins ...................................................................................................................................................... 4 1.3 Characterization of Fungal Exposure in Homes ..................................................................................... 5 1.3.1 Methods for detecting and sampling fungi in indoor environments ................................ 5 1.3.2 Phenotypic identification of molds ..................................................................................................... 6 1.3.3 Molecular identification of molds ....................................................................................................... 6 1.4 Respiratory Health Effects of Exposure to Molds ................................................................................. 7 1.4.1 Respiratory system ..................................................................................................................................... 7 1.4.2 Deposition of mold particles in the lung .......................................................................................... 8 1.4.3 Cells of the alveolus ................................................................................................................................. 10 1.4.4 Health effects of mold ............................................................................................................................. 11 1.5 Immune response ..............................................................................................................................................
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