Transcript Profiling of the Murine Immune Response to Invasive Aspergillosis
Invasive aspergillosis is an opportunistic infection for which complex host–pathogen interactions determine infection outcome. In particular, immunosuppressive therapies and other host factors, such as neutropenia, need to be taken into account when model
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1. Introduction Invasive aspergillosis is now the most common fungal cause of death in modern health-care systems (1). This rise in mortality appears to have been driven by increasing numbers of patients at risk from invasive disease, as a consequence of a rapidly expanding number of novel immunosuppressive agents (2). The greatest risk factors for invasive aspergillosis are currently neutropenia and steroid exposure (3). Classically, two standard models of murine invasive aspergillosis have been used, both for studies of fungal virulence and for host response to disease. The first model is a cyclophosphamidehydrocortisone-based model, which leads to rapid induction of Alexandra C. Brand and Donna M. MacCallum (eds.), Host-Fungus Interactions: Methods and Protocols, Methods in Molecular Biology, vol. 845, DOI 10.1007/978-1-61779-539-8_30, © Springer Science+Business Media, LLC 2012
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profound neutropenia. Whilst profound neutropenia is most commonly encountered during intensive conditioning for haematological allogenic stem cell transplantation, it is notable that the immunosuppressive regimen used in this model is most reflective of classical regimens used for solid malignancies (4). The second classic model of invasive aspergillosis is a high-dose steroid model based entirely on hydrocortisone immunosuppression (5). This immunosuppressive regimen does not result in neutropenia, and is more representative of aspergillosis in the context of graft-versushost disease, long-term steroid therapy (e.g. obstructive pulmonary disease), and solid organ transplant rejection, where steroid immunosuppression is employed routinely. In vivo transcript profiling is an attractive approach to understanding the pathogen or host response during invasive aspergillosis, as it enables an overview of the transcriptional response during this biological process (6). However this approach is limited, first because transcript abundance does not always predict protein concentration or activity, and second because organ-level transcript profiling does not easily yield information on the relative abundance of immune cell types at the site of infection, or which cells are responsible for the transcript signal in question. Here we describe a protocol for the analysis of host pulmonary immune responses that employs genomic oligo microarrays to assay organ-level transcriptional responses in neutropenic invasive aspergillosis.
2. Materials 2.1. Preparation of Aspergillus fumigatus inoculum
1. Aspergillus complete medium (for 20 mL slopes in 50-mL Falcon Tubes). 2. Aspergillus minimal medium (7). 3. Sealable plastic box. 4. Sterile agar plates. 5. Aspergillus fumigatus strains Af 239 and AfCEA10 (recommended pathogenic sequenced strains). 6. Sterile 50-mL Falcon tubes. 7. Autoclaved miracloth (Calbiochem). 8. Sterile saline (Baxter Healthcare Ltd). 9. Centrifuge.
2.2. Immunosuppression of CD1 Mice with Hydrocortisone and Cyclophosphamide
1. Cyclophosphamide 20 mg/mL in normal saline (150 mg/kg, ENDOXANA, Asta Medica). 2.
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