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    Post: Rand & Miller~Inflammation From Fungi~Toxins

    Posted by Sharon on 12/23/09

    Arch Toxicol. 2009 Nov 11. [Epub ahead of print]

    Dectin-1 and inflammation-associated gene transcription and
    expression in mouse lungs by a toxic (1,3)-beta-D: glucan.

    Rand TG, Sun M, Gilyan A, Downey J, Miller JD.

    Department of Biology, Saint Mary's University, 923 Robie
    St., Halifax, NS, B3H 3C3, Canada,

    The form of (1-3)-beta-D: glucan found in the cell walls of
    the anamorphic Trichocomaceae that grow on damp building
    materials is considered to have potent toxic and
    inflammatory effects on cells of the respiratory system. It
    is also considered to have a potential role in the
    development of non-allergenic respiratory health effects.
    While human studies involving experimental exposures all
    point to the inflammatory potential of pure curdlan, a
    linear (1-3)-beta-D: glucan in a triple helix
    configuration, animal experiments result in conflicting
    conclusions concerning the inflammatory potency of this
    glucan. However, because mice appear to be a better model
    than guinea pigs for exploring the respiratory effects of
    curdlan and because molecular mechanisms associated with
    this glucan remain largely unknown, we conducted further
    work to clarify the role of curdlan on the inflammatory
    response using our mouse model of lung disease. This study
    used in situ hybridization (ISH) to probe dectin-1 mRNA
    transcription with a digoxigenin-labeled cDNA probe, with
    reverse transcription (RT)-PCR based arrays used to measure
    inflammation gene and receptor transcriptional responses.
    Also, immunohistochemistry (IHC) was used to probe dectin-1
    as well as anti-mouse Ccl3, Il1-alpha, and TNF-alpha
    expression to evaluate dose and time-course (4 and 12 h)
    postexposure (PE) response patterns in the lungs of
    intratracheally instilled mice exposed to a single 50 mul
    dose of curdlan at 10(-7), 10(-8), 10(-9), and 10(-10)
    M/animal (=4 mug to 4 ng curdlan/kg lung wt). Dectin-1 mRNA
    transcription and expression was observed in bronchiolar
    epithelium, alveolar macrophages (AMs), and alveolar type
    II cells (ATIIs) of lungs exposed to 4 mug to 40 ng
    curdlan/kg lung wt, at both time points. Compared to
    controls, array analysis revealed that 54 of 83 genes
    assayed were significantly modulated by curdlan. mRNA
    transcription patterns showed both dose and time
    dependency, with highest transcription levels in 10(-7) and
    10(-8) M treatment animals, especially at 4-h PE. Nine gene
    mRNA transcripts (Ccl3, Ccl11, Ccl17, Ifng, Il1alpha, Il-
    20, TNF-alpha, Tnfrsf1b, and CD40lg) were significantly
    expressed at all doses suggesting they may have a central
    role in immunomodulating curdlan exposures. IHC revealed
    Ccl3, Il1-alpha, and TNF-alpha expression in bronchiolar
    epithelium, AMs and ATIIs illustrate the important
    immunomodulatory role that these cells have in the
    recognition of, and response to glucan. Collectively, these
    results confirm the inflammatory nature of curdlan and
    demonstrate the complex of inflammation-associated gene
    responses induced by (1-3)-beta-D: glucan in triple helical
    forms. These observations also provide a biological basis
    for the irritant and inflammatory response to curdlan
    observed in humans and animals in experimental studies.

    Chem Biol Interact. 2010 Jan 5;183(1):113-24.

    Inflammation-associated gene transcription and expression
    in mouse lungs induced by low molecular weight compounds
    from fungi from the built environment.

    Miller JD, Sun M, Gilyan A, Roy J, Rand TG.

    Department of Chemistry, Carleton University, Ottawa,
    Ontario, Canada K1S 5B6.

    Few metabolites from fungi found indoors have been tested
    for inflammatory mediators endpoints in primary cultures of
    alveolar macrophages or in vivo. In this study, mice were
    intratracheally instilled with a single dose comprising 4x10
    (-5)moletoxin/kg lung wt dose of either atranone C,
    brevianamide, cladosporin, mycophenolic acid, neoechinulin
    A & B, sterigmatocystin or TMC-120A. These toxins are from
    fungi common on damp building materials. The dose used was
    comparable to the estimated doses of possible human
    exposure. Hematoxylin and eosin (H&E) histology and Alcian
    Blue/Periodic Acid Schiff (AB/PAS) histochemistry were used
    to evaluate lungs for time course (4h and 12h post-exposure
    (PE)) inflammatory and toxic changes. Reverse-transcription
    (RT)-PCR based arrays were also employed to evaluate time
    course inflammation-associated gene transcription in lung
    tissues of the different toxins. Immunohistochemistry (IHC)
    was used to probe MIP-2 and Tnf-alpha protein expression in
    treatment lungs to determine whether responses correspond
    with gene transcription data. Both histology and
    histochemistry revealed that toxin exposed lungs at 12h PE
    showed evidence of inflammation. H&E revealed that
    bronchioli were lined with irregularly thickened and
    sometimes sloughing epithelium and bronchiolar spaces
    supported infiltration of leukocytes, cellular and mucus-
    like debris while alveolar spaces supported swollen
    macrophages and modest amorphous debris accumulations. All
    toxin-instilled lungs exhibited copious mucus production
    and alveolar macrophages with red stained cytoplasm on
    bronchiolar surfaces, especially at 12h PE. Array analysis
    of 83 inflammation-associated genes extracted from lung
    tissue demonstrated a number of patterns, compared to
    controls. 82 genes assayed at 4h PE and 75 genes at 12h PE
    were significantly altered (p< or =0.05; >or =1.5-fold or <
    or =-1.5-fold change) in the different treatment animal
    groups. Expression of transcriptionally regulated genes was
    confirmed using immunohistochemistry that demonstrated MIP-
    2 and Tnf-alpha staining in respiratory bronchiolar
    epithelia, alveolar macrophages and alveolar type II cells.
    The transcriptional regulation in these genes in the
    treatment groups suggests that they may serve central roles
    in the immunomodulation of toxin-induced pro-inflammatory
    lung responses. Hierarchical cluster analysis revealed
    significant patterns of gene transcription linking the
    response of the toxins at equimolar doses in three groups:
    (1) brevianamide, mycophenolic acid and neoechinulin B, (2)
    neoechinulin A and sterigmatocystin, and (3) cladosporin,
    atranone C and TMC-120. The results further confirm the
    inflammatory nature of metabolites/toxins from such fungi
    can contribute to the development of non-allergenic
    respiratory health effects

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