Description:
In 1998, a team of eight EPA researchers from NERL, NHEERL, NRMRL and NCEA organized to examine the role of indoor molds/fungi in children's health. The make-up of the indoor environment is critical to the health of children in general and has a particularly important influence on the incidence and exacerbation of asthma. Asthma is the most common chronic disease of childhood. About 75% of asthma is associated with allergy. In nearly 100% of elementary school children with asthma, allergens are the primary trigger for asthma and their disease is thought to result from early exposure and sensitization to common allergens in their environment (e.g., dust mite, cockroach, pollen, molds, animal dander). It appears that early in life, in some individuals, the immune system is programmed to promote asthmatic responses to certain antigens. Some of these allergens have been studied extensively (e.g. dust mite and cockroach), and specific antigens responsible for sensitization have been characterized. However, almost none of the mold allergens have been characterized, despite their widespread distribution and potential importance in the induction and exacerbation of asthma. This project focuses on the antigens, particularly protein components of molds, that initiate allergic sensitization.
Although childhood asthma remains the focus of our efforts, there is growing awareness that other health problems may be associated with exposure to various molds. Visual contrast sensitivity (VCS) testing provides a noninvasive, rapid, sensitive, reliable and portable, indicator of neurological dysfunction which has previously been associated with biotoxin exposure. This study will report deficits in VCS.
Perhaps, the most severe potential response to fungal exposure is what was originally called pulmonary hemosiderosis (PH) in infants. In 1994 and 1997, CDC reported clusters of acute pulmonary hemorrhage in infants. Reviews by internal CDC and external expert panels of these investigations identified shortcomings in the conduct of the studies. The panels concluded that the investigations did not prove an association between acute pulmonary hemorrhage in infants and exposure to molds, specifically Stachybotrys chartarum (atra).
Subtasks include: 1. Develop more mold assays for use in quantitative polymerase chain reaction technology (QPCR). PI: Haugland; 2. Develop database of mold concentrations in homes, hospitals, schools, business across the US and the world. PI: Haugland; 3. Identify the IgE inducing proteins from three fungi (Metarhizium anisopliae, Stachybotrys chartarum, and Penicillium chrysogenum) by characterizing these immunologically and by advanced proteomics and identifying any common characteristics. PIs:Selgrade/Marsh; 4. Confirm that the proteins identified as IgE inducers are the relevant allergens in the mouse disease model and to determine if these proteins are allergenic in humans.PI: Selgrade/Marsh; 5: To characterize the physio-chemical properties of the allergenic protein(s) from the three fungi and compare to other well-characterized proteins allergens such as dust mite allergens. PI:Shoemaker/Donohue; 6. To determine the relative potency of the fungal allergens compared to other protein allergens such as dust mite, cockroach, alcalase, etc. PI: Selgrade/Marsh; 7. Evaluate through epidemiological studies and clinical evaluations (being completed by Case Western Reserve University Medical School through our cooperative agreement) the exposures of children to fungi that might lead to asthma, neurological dysfunction and potentially pulmonary hemosiderosis. PI: Vesper; 7B. To determine if exposure to specific fungi is correlated with increased asthma incidence. PI: Hudnell; 7C. To determine if exposure to some fungi is correlated with neurological dysfunction. 7D. To determine if exposure to fungal toxins, e.g. to S. chartarum toxins, can be measured by ELISA assays of serum samples from exposed children and other clinical and environmental samples. PI: Van Emon; 8. Develop an exposure estimate for children under "real-world" conditions and test methods or agents to prevent the exposure. PI: Menetrez; 9. Provide comprehensive information to be used by the Office of Air and Radiation and by the Regional Offices in helping our clients, the American parent, in dealing with this major environmental problem.
PI: Frederick/Furtaw/Kolb
Keywords:
MOLD, ASTHMA, FUNGUS, STACHYBOTRYS, RISK ASSESSMENT, CHILDREN, CHILDREN'S HEALTH, CUMULATIVE RISK, AGING INITIATIVE, ENVIRONMENTAL JUSTICE, GENOMICS,
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Project Information:
Progress
:Subtask 1. Develop more mold assays for use in quantitative polymerase chain reaction technology (QPCR)(PIs: Haugland/Vesper). We have now identified specific DNA sequences that are unique to more than 70 species or groups of species of potentially pathogenic fungi. These sequences were used to develop primers and probes for a quantitative PCR assay using the TaqManTM system. The TaqManTM assay signals the formation of PCR amplicons by a process involving the nucleolytic degradation of a double-labeled fluorogenic probe that hybridizes to the target template at a site between the two primer recognition sequences. The model 7700 automates the detection and quantitative measurement of these signals. Samples can be analyzed in as little as two hours.
Subtask 3. Identify the IgE inducing proteins from three fungi (Metarhizium anisopliae, Stachybotrys chartarum, and Penicillium chrysogenum) by characterizing these immunologically and by advanced proteomics and identifying any common characteristics.
Subtask 4: Confirm that the proteins identified as IgE inducers are the relevant allergens in the mouse disease model and to determine if these proteins are allergenic in humans.
Subtask 5: To characterize the physio-chemical properties of the allergenic protein(s) from the three fungi and compare to other well-characterized proteins allergens such as dust mite allergens. (PIs: Selgrade/Marsh/Donohue) Mice were hyperimmunized with aqueous extracts from both S. chartarum, and P. chrysogenum. The serum was collected from these mice and total IgE titers (and IgA or IgG) were determined by ELISA. Iso-electric focusing gel electrophoresis, 2-D gel electrophoresis and second dimension SDS-PAGE gel were used to separate proteins in the extracts. Western blot analysis was then used to identify four allergens (proteins recognized by immunoglobulins in the immune serum) in the protein extracts from these mold species. The allergenic protein spots on other 2-D gels were harvested and transferred for peptide and protein structure analysis using mass spectrometry (MS). The two primary techniques utilized for proteomic research are electrospray mass spectrometry (ESI-MS) and matrix assisted laser desorption mass spectrometry (MALDI-MS). These MS techniques can provide accurate molecular weights for peptides and proteins with masses up to approximately 300,000 daltons using low femtomole to low picomole quantities. The sequence data obtained in this fashion led to the identification of one allergenic protein as having catalase activity. will be used to mine databases for potential sequence and function homology.
Western blot analysis using this serum identified four allergens in protein extracts from these mold species. One of the allergens was identified as having catalase activity. From this work, S. chartarum, P. chrysogenum, or M. anisopliae allergen specific ELISAs (NHEERL/NERL collaboration) were developed to provide a possible method of determining exposure of humans to these mold species.
Subtask 2. Develop database of mold concentrations in homes, hospitals, schools, business across the US and the world.
Subtask 7. Evaluate through epidemiological studies and clinical evaluations (being completed by Case Western Reserve University Medical School through our cooperative agreement) the exposures of children to fungi that might lead to asthma, neurological dysfunction and potentially pulmonary hemosiderosis (PIs: Vesper/Hudnell/Van Emon. ) Studies at Case Western Reserve University (CWRU) in Cleveland were supported in conjunction with NIH and HUD funding. CWRU recognized that the EPA fungal identification/quantification technology was unique and asked EPA to participate in a joint study. The children (and their homes) in this study come from those selected in a HUD supported remediation program in Cleveland, OH. These children are also being recruited from an ongoing NIH asthma study
Relevance
:Relevance: The Office of Air and Radiation, US EPA Regions are the primary EPA clients for this research. Other interested groups include the US General Services Administration, Insurance Companies and the Mortgage Industry.
Significance: The significance of this effort is already showing. We now have companies using a standardized technology for quantifying most of the molds. Soon we hope to have technologies for monitoring the levels of exposure through quantifiable "biomakers" for use by physicians and industry. Next we will have reagents available for quantification of the actually allergenic agents. And we expect to have a non-invasive test demonstrated that can help understand if children are having neurological problems as a result of mold exposures.
Impact: The results of this study will lay the foundation for understanding the role of the indoor molds/fungi as inducers of asthma and neurological defects in children and as the cause of pulmonary hemorrhaging in infants. We are already changing an entire industry for mold analysis. We expect to have a similar impact in the medical community. Most importantly this research will result in the protection of human health.
Clients
:Physicians, indoor air community, US EPA Office of Air and Radiation, Department of Housing and Urban Development
Project IDs:
ID Code
:6175
Project type
:OMIS