21. Exhibit: WDB There are toxins and inflammagens

Exhibit_5_24_10_if WDB there are toxins and inflammagens
What do we know about biocontaminants in WDB that could make people sick (focus on GAO/WHO)?
The multiple agents (including toxins and inflammagens) found inside WDB each could cause similar inflammatory responses. Trying to separate which of the potential initiators of inflammation, with the inflammatory responses showing no differences, is not possible. So-called, "specific causation" simply doesn't exist as discussed previously. However, there is a significant data base that says that even if not measured specifically, that the multiple inflammagens and toxigens that can cause illness will be found in WDB. The following comments are extracted from the GAO and WHO reports followed by discussions from specific academic papers.
Letter to Senator Kennedy; Pg 1: Several components of mold may cause disease. Fragments may cause adverse health effects. Certain components of cell walls may cause adverse health effects. Page 4: There is difficulty in determining which of several disease-causing agents in damp indoor environments may be responsible for adverse health effects. There is a need for research to determine the health effects of long-term exposure to the toxins that some molds can produce. Two key factors: page 16. A wide variety of potential disease-causing agents are likely to be present in damp indoor environment, which makes it difficult to link health effects with specific agents. Potential disease-causing agents, including many species of mold and other biological agents, such as bacteria, are likely to be present in damp indoor environments. Several different components or products of mold, such as mycotoxins, may function as disease causing agents in indoor environments. Appendix I: Objectives, scope and methodology.
2 GAO searched 19 different terms on PubMed: mold, exposure, health, indoor, glucan, microbial VOCs, mycotoxins, ergosterol, hemolysins, fungal extracellular polysaccharides, fungal/hyphal fragments, allergens, Stachybotrys, acute idiopathic pulmonary hemorrhage, acute pulmonary hemorrhage and infants and hemosiderosis. WHO Abstract: Indoor air pollution is caused by hundreds of species of bacteria and fungi, in particular filamentous fungi (mould), growing indoors when sufficient moisture is available. WHO Executive Summary Pg. xi-xii: There is strong evidence regarding the hazards posed by several biological agents that pollute indoor air; however, the WHO working group convened in October 2006 concluded that the individual species of microbes and other biological agents that are responsible for health effects cannot be identified. This is due to the fact that people are often exposed to multiple agents simultaneously, to complexities in accurately estimating exposure and to the large numbers of symptoms and health outcomes due to exposure. "The presence of many biological agents in the indoor environment is due to dampness and inadequate ventilation. Excess moisture on almost all indoor materials leads to growth of microbes, such as mould, fungi and bacteria, which subsequently emit spores, cells fragments and volatile organic compounds into indoor air." WHO Exec Summary Pg. xiii: Toxicological evidence obtained in vivo and in vitro supports these findings, showing the occurrence of diverse inflammatory and toxic responses after exposure to microorganisms isolated from damp buildings, including their spores, metabolites and components. WHO Exec Summary xiv: The amount of water on or in materials is the most important trigger of the growth of microorganisms, including fungi, actinomycetes and other bacteria. "Health hazards result from a complex chain of events that link penetration of water indoors, excessive moisture to biological growth, physical and chemical degradation, and emission of hazardous biological and chemical agents." Executive Summary, page XIII Microbial growth may result in greater numbers of spores, cell fragments, allergens, mycotoxins, endotoxins, B-glucans and volatile organic compounds in indoor air. The causative agents of adverse health effects have not been identified conclusively, but an excess level of any of these agents in the indoor environment is a potential health hazard. "Toxicological evidence obtained in vivo and in vitro supports these findings, showing the occurrence of diverse inflammatory and toxic responses after exposure to microorganisms isolated from damp buildings, including their spores, metabolites and components."
WHO Exec Summary Pg. xv "As the relations between dampness, microbial exposure and health effects cannot be quantified precisely, no quantitative health-based guideline values or thresholds can be recommended for acceptable levels of contamination with microorganisms. Instead, it is recommended that dampness and mould-related problems be prevented." WHO Introduction Pg. 5: Mechanisms of injury include exposure to B-glucans, toxins, spores, cell fragments and chemicals followed by immune stimulation, suppression and autoimmunity as well as neurotoxic effects. WHO Chapter 2, Pg. 9. Indoor environments contain a complex mixture of live (viable) and dead (non-viable) microorganisms, fragments thereof, toxins, allergens, volatile microbial organic compounds and other chemicals. The indoor concentrations of some of these organisms and agents are known or suspected to be elevated in damp indoor environments and may affect the health of people living or working there. WHO Chapter 2, Pg. 15: "Many fungi and some yeast replicate by producing numerous spores that are well adapted to airborne dispersal....They can stay airborne for long periods and may deposit in the respiratory system, some smaller spores reaching the alveoli. Fungi can release even smaller fungal fragments, which are derived from broken or fractured spores and hyphae and can be categorized into submicron particles...Even more fungal fragments than spores may be deposited in the respiratory tract; like spores, they are known to contain allergens and mycotoxins." WHO Chapter 2, Pg. 16: Mycobacteria have also been shown to be common in moisture damaged buildings, their presence increasing with the degree of fungal damage (Torvinen et al., 2006). Cell wall components of mycobacteria are known to be highly immunogenic, and exposure to mycobacteria may cause inflammatory responses (Huttunen et al, 2000, 2001). In the environment, airborne endotoxins are usually associated with dust particles or aqueous aerosols. "Indoor fungal fragments are not commonly measured in field studies, but a study with an aerosolization chamber showed that submicron fungal fragments from culture plates and mould-contaminated ceiling tiles aerosolized simultaneously with spores but at substantially higher concentrations (320 - 524 times higher). This suggests that indoor exposure to fungal fragments is at least as important as exposure to fungal spores." WHO Chapter 2, pg 17.
4 "Fungal (13) - -D-glucans. (13)- -D-glucans are non-allergenic, water-insoluble structural cell-wall components of most fungi,...and may account for up to 60% of the dry weight of the cell wall of fungi....(13)- -D-glucans have immunomodulating properties and may affect respiratory health." WHO chapter 2, pg 18 "Mycotoxins, or fungal toxins, are low-relative-molecular-mass biomolecules produced by fungi, some of which are toxic to animals and human beings. Mycotoxins are known to interfere with RNA synthesis and may cause DNA damage. Some fungal species may produce various mycotoxins...Several mycotoxins, e.g. aflatoxin from Aspergillus flavus and Aspergillus parasiticus, are potent carcinogens. Many mycotoxins are immunotoxic....The mycotoxins that have perhaps received most attention are the trichothecenes, produced by Stachybotrys chartarum.....[Mycotoxins] could be present in most samples of materials and settled dust from buildings with current or past damage from damp or water." WHO Chapter 2, Pg. 19: These studies demonstrate that mycotoxins are present in the indoor environment and that the levels may be higher in buildings affected by mold and damp. "S. chartarum trichothecene mycotoxins can become airborne in association with both intact conidia and smaller fungal fragments....These studies demonstrate that mycotoxins are present in the indoor environment and that the levels may be higher in buildings affected by mould or damp." "Several fungi produce volatile metabolites, which are a mixture of compounds....Microbial volatile organic compounds are often similar to common industrial chemicals. To date, more than 200 of these compounds derived from different fungi have been identified, including various alcohols, aldehydes, ketones, terpenes, esters, aromatic compounds, amines and sulfur-containing compounds." "Some exposures with adverse health effects associated with damp indoor environments include emissions of volatile organic compounds from damp and mouldy building materials." WHO Chapter 4, Pg. 63 "Microbiological organisms are considered among the most plausible explanations for the health risks associated with indoor dampness." WHO Chapter 5, Pg. 75: The exposures that cause dampness-related illness have not yet been determined. A study of an association between health effects and the concentration of a specific
5 microorganism or microbial compound is in fact testing a hypothesis. In the studies in our review, such hypothetical causal exposures included all culturable fungi, all fungal spores, species-specific spores, all fungal biomass (ergosterol) ( Robine et al., 2005), the total mass of specific organism (Aspergillus and Penicillium extracellular polysaccharides) and specific toxic compounds (endotoxins, B-glucans). WHO Chapter 4, Pg. 78: Numerous studies have shown that B-glucans have important effects on the human immune system. WHO Chapter 4, Pg. 80: Concomitant exposure to endotoxins and curdlan, a (1-3) B-glucan, was shown to diminish the acute neutrophil response but to augment chronic inflammatory effects (Fogelmark, Sjostrand, Rylander, 1994; Rylander, Fogelmark, 1994). Thus, the effects of inhalation of B-glucans apparently depend on the type of glucans as well as on concomitant exposures. WHO Chapter 4, Pg. 85: In damp buildings, people are exposed to constantly changing concentrations of different microbial species, their spores, metabolites and components, and other compounds in indoor air, including chemical emissions from building materials. This complex mixture of exposures inevitably leads to interactions, which affects outcomes in different situations. Furthermore, the effects of microorganisms, microbial substances or dampness  related chemical compounds seen in experimental animals, or cells often result from exposure that are orders of magnitude higher than the average doses that reach the human lungs under normal conditions in indoor air. Nevertheless, the surface doses within the lungs of patients with respiratory conditions can vary a thousand fold, due to uneven particle deposition (Phalen et al., 2006), thus resulting in even larger maximal surface doses in human lungs than in those used in experimental toxicological studies. Moreover, many other factors, such as exercise, can result in larger-than-average doses in the human lung. Many of the health effects may result from recurrent activation of immune defence, leading to exaggerated immune responses and prolonged production of inflammatory mediators. Over production of these compounds damages the surrounding tissue and may manifest itself as chronic inflammation and inflammation-related diseases. WHO Chapter 4, Pg. 86: Furthermore, it has been shown in an animal model that immunological status plays an important role in airway inflammation induced by Stachybotrys Chartarum, enhancing the effects of the mold (Leino et al., 2006). The results imply that sensitized people are more susceptible to exposure to mold than non-atopic people. Different microbial species
6 differ significantly in their immunostimulatory potency in both mouse and human cells in vitro (e.g. Huttunen et al., 2003). Furthermore, it has been clearly demonstrated that different growth conditions and competition between microorganism for the same habitat in vitro change their inflammatory potency, protein expression and toxin production (Ehrlich, 1987) "The immunostimulatory activity of Gram-negative bacterial lipopolysaccharide is well established, but several other bacteria, fungi and isolated mycotoxins associated with damp buildings have been shown to induce inflammatory responses in vitro. In line with the findings in vitro, the same microbial species activate acute and sustained inflammation in the lungs of experimental animals." WHO chapter 4, Pg 87 "Fungal spores appear to have toxic effects other than those that cause the inflammatory reaction. Studies of Gram-positive and -negative bacteria (e.g. Streptomyces californicus, Pseudomonas fluorescens, Mycobacterium terrae, Bacillus cereus) have shown that the significant difference in cytoxicity among strains is due at least partly to differences in inflammatory activity. Spores and toxins of the fungus S. chartarum have been shown to activate the apoptotic pathway....Studies in experimental animals with the same fungal or bacterial species confirm the in vitro findings for cytotoxic effects...as well as lung tissue damage." "Microbial fragments can...cause autoimmune reactions by molecular mimicry, acting as microbial superantigens or by enhancing the presentation of autoantigens." "Spores and other particulate material, as well as volatile organic compounds produced by microorganisms, building materials, paints and solvents, are potentially irritating. In epidemiological studies, the prevalence of respiratory and irritative symptoms has been associated with perceived mould odour, possible indicating the presence of microbial volatile organic compounds." WHO Chapter 4, Pg. 88: Such health effects as fatigue, headache and difficulties in concentration (Johanning et al., 1996; Koskinen et al., 1999b) indicate that microbes or other agents present in damp buildings have neurological effects. WHO Chapter 4, Pg. 89: The immunostimulatory properties of the fungal and bacterial strains typically found in moisture-damaged buildings are synergistically potentiated by microbial interactions during concomitant exposure in vitro (Huttunen et al., 2004). Interactions during co-cultivation stimulate these microbes to produce highly toxic compounds, which can damage DNA and provoke genotoxicity (Penttinen et al., 2007).
7 In addition, concomitant exposure in vitro with amoebae potentiates the cytotoxic and inflammatory properties of the microbial spores of S. californicus or Penicillium spinolosum isolated from damp buildings (Yli-Pirila et al., 2007). These findings point to the importance of considering microbial interactions when investigating the causative agents and mechanisms of the adverse health effects observed in damp buildings. WHO Chapter 4, Pg. 90: It is clear, however, that no single mechanism can explain the wide variety of effects associated with dampness and mold. Toxicological studies, by investigating the ability of microbial agents associated with damp buildings to activate certain toxicological mechanisms, provide insight into the multiple biological mechanisms that might underlie the observed associations between health effects and dampness and mold. In vitro and in vivo studies have demonstrated diverse inflammatory, cytotoxic and immunosuppressive responses after exposure to the spores, metabolites and components of microbial species found in damp buildings, lending plausibility to the epidemiological findings. WHO Chapter 4, Pg. 91: Various microbial agents with diverse, fluctuating inflammatory and toxic potential are present simultaneously with other airborne compounds, inevitably resulting in interactions in indoor air. Such interactions may lead to unexpected responses, even at low concentrations. Therefore, the detection of individual exposures, such as certain microbial species, toxins or chemical agents, cannot always explain any associated adverse health effects. In the search for causative constituents, toxicological studies should be combined with comprehensive microbiological and chemical analyses of indoor samples.
Literature from non-governmental agencies:
The synergistic interactions among microbial agents present in damp buildings suggest that the immunotoxic effects of the fungal and bacterial strains typically found can be potentiated during concomitant exposure, leading, for instance, to increased cell death or cytotoxic or inflammatory effects. Such interactions can give rise to unexpected responses, even at low concentrations of microbial (or chemical) agents, so that it is difficult to detect and implicate specific exposures in the causation of damp buildingassociated adverse health effects. Thus, microbial interactions must be carefully considered when evaluating the possible health effects of exposure in damp buildings. The following is an annotated list of references confirming presence of mycotoxins and other sources of inflammation found in WDB, if the testing is done. Skaug MA, Eduard W, Stormer FC. Ochratoxin A in airborne dust and fungal conidia. Mycopathologia 2001; 151(2): 93-98 This paper from 2001 looks at the question of whether or not mycotoxins are present in
8 airborne dust in fungal fragments in a water-damaged building. They looked at the toxin, ochratoxin, and were able to reproducibly identify it in picogram quantities from extracts of fungal conidia. Conclusion is that airborne dust and fungal conidia can be sources of ochratoxin. Bloom E, Bal K, Nyman E, Must A, Larsson L. Mass spectrometry-based strategy for direct detection and quantification of some mycotoxins produced by Stachybotrys and Aspergillus spp. in indoor environments. Applied and Environmental Microbiology 2007; 73(13): 4211-4217. This is another paper looking at whether or not mycotoxins are found in water-damaged buildings. The authors use mass spectroscopy to confirm that mycotoxins produced by Aspergillus and Stachybotrys are found in buildings with ongoing dampness and a history of water-damage. The direct detection of highly toxic sterigmatocystin and macrocyclic trichothecene mycotoxins in indoor environments is important to their potential health impacts. Direct indication of presence of mycotoxins in 45 of 62 building material samples, three of eight sets of dust samples and five of eight cultures of airborne dust samples confirm the benefit of high performance liquid chromatography-tandem mass spectrometry. Gottschalk C, Bauer J, Meyer K. Detection of satratoxin G and H in indoor air from a water-damaged building. Mycopathologia 2008; 166: 1103-7. This study reported in 2008 used a 0.8 micron fitter to trap particulates from the indoor air of a WDB. Satratoxin g and H were found to be airborne at concentrations of 0.25 and 0.43 ng/cubic meter. These finding support those of Brasel et al. Engelhart S, Loock A, Skutlarek D, Sagunski H, Lommel A, Farber H, Exner M. Occurrence of toxigenic Aspergillus versicolor isolates and sterigmatocystin in carpet dust from damp indoor environments. Applied and Environmental Microbiology 2002; 68(8): 3886-3890. This is a paper published is August 2002, again well before the ACOEM statement was released, that confirms finding of mycotoxins in carpet tests in areas of homes with water-damage. The authors conclude that the Aspergillus versicolor that are isolated from carpet dust was able to produce sterigmatocystin and that this toxin itself was readily isolated from carpet dust. 98% of such isolates of Aspergillus versicolor were toxigenic. Sebastian A, Larson L. Characterization of the microbial community in indoor environments: a chemical-analytical approach. Applied and Environmental Microbiology 2003; 69(6): 3103-3109. This paper is a follow-up on the use of ergosterol as a marker for fungal biomass. Integrated procedures using gas chromatography and ion trap mass spectrometry were able to show differences in house dust for a variety of compounds. This method could be used to characterize microbial communities in environmental samples. NOTE: the
9 technology available for exposure assessment was published in the early 1990 as well as the early years of 2000. Researchers are still arguing on how to do exposure assessments. Martinez KF, Rao CY, Burton NC. Exposure assessment and analysis for biological agents. Grana 2004; 43: 193-208. This review paper from NIOSH focuses on exposure assessments from biological agents. This is a wide-ranging paper that looks at microbes in water damaged buildings together with microbes that could be used as biowarfare agents. The separation of sampling techniques together with analytical techniques includes discussion of ergosterol, a robust indicator of total fungal biomass, beta glucans, mycotoxins and endotoxins. They call for enhanced use of PCR and DNR microarray (such as discussed in the genomic sections of references in Exhibits I and II). Smoragiewicz W, Cossette B, Boutard A, Krzystyniak K. Trichothecene mycotoxins in the dust of ventilation systems in office buildings. Int Arch Occup Environ Health 1993; 65: 113-117. This 1993 paper analyzes trichothecene mycotoxins and dust samples from ventilation systems in office buildings. Duct samples were found to have at least four separate trichothecenes, confirmed by high performance liquid chromatography analysis. These were found with a detectable limit of 0.4 ng/ml of dust, consistent with prior isolation studies showing ease of detection of mycotoxins in buildings with water intrusion. Tuomi T, Reijula K, Johnsson T, Hemminki K, Hintikka EL, Lindroos O, Kalso S, Koukila-Kahkola P, Mussalo-Rauhamaa H, Haajtela T. Mycotoxins in crude building materials from water-damaged buildings. Applied and Environmental Microbiology 2000; 66(5): 1899-1904. This 2000 study from Finland demonstrated the ability to isolate 17 different mycotoxins from 79 bulk samples of interior walls from buildings in Finland with moisture problems and mold growth. Identification and numeration of fungal species present in both materials is possible. Nikulin M, Pasanen AL, Berg S, Hintikka EL. Stachybotrys atra growth and toxin production in some building material and fodder under different relative humidities. Applied and Environmental Microbiology 1994; 60(9): 3421-3424. This 1993 paper confirmed that in the presence of Stachybotrys growing on building materials toxins made by Stachybotrys could be detected by biological assays in chemical methods. Strong growth of Stachybotrys on wall paper and gypsum board has well as in hay and straw resulted in satratoxin production; toxin was not observed from Stachybotrys growing on wood panels.
10 Nielsen KF, Holm G, Uttrup LP, Nielsen PA. Mould growth on building materials under low water activities. Influence of humidity and temperature on fungal growth and secondary metabolism. International Biodeterioration and Biodegradation 2004; 54: 325-336. This paper shows that there is a difference in fungal growth depending on humidity and on particular media with maximum growth of 80% humidity but at 5 centigrade still significant fungal growth at 90% relative humidity. Significant mycotoxin production was noted at room temperature, consistent with the hypothesis that toxigenic fungi that grow will make toxins. Charpin-Kadouch C, Maurel G, Felipo R, Queralt J, Ramadour M, Henri D, Garans M, Botta A, Charpin D. Mycotoxin identification in moldy dwellings. Journal of Applied Toxicology 2006; 26: 475-479. This important paper provides direct evidence for the presence of macrocyclic trichothecenes in water damaged buildings. 15 buildings with floods and water damage contaminated with Stachybotrys or Chaetomium were compared to a control group of 9 buildings without water damage. There were significant differences in wall samples but not any differences for air samples. Significant correlations were observed between levels of wall surfaces and floor dust. Nieminen SM, Karki R, Auriola S, Toivola M, Laatsch H, Laatikainen R, Hyvarinen A, von Wright A. Isolation and identification of Aspergillus fumigatus mycotoxins on growth medium and some building materials. Applied and Environmental Microbiology 2002; 68(10): 4871-4875. Using pure cultures of Aspergillus humicotis, gliotoxin was readily identified through use of selective growth media as well as on building materials. This paper was published in 2002 well before the ACOEM paper; one that insisted that such toxin formation did not occur. Brasel TL, Martin JM, Carriker CG, Wilson SC, Straus DC. Detection of airborne Stachybotrys chartarum macrocyclic trichothecene mycotoxins in the indoor environment. Applied and Environmental Microbiology 2005; 71(11): 7376-7388. In this 2005 paper Brasel, working with Straus's group in Texas Tech, was able to readily identify airborne macrocyclic trichothecene mycotoxins in indoor environments with water damage; these toxins were not found in buildings without water damage. Panaccione DG, Coyle CM. Abundant respirable ergot alkaloids from the common airborne fungus Aspergillus fumigatus. Applied and Environmental Microbiology 2005; 71(6): 3106-3111. Investigators have questioned whether Aspergillus fumigatis becomes airborne or respirable. In this paper from the genetics and developmental biology program at West Virginia, authors were able to show production by four separate toxins associated with
11 conidia (smaller fragments of growing fungi). These conidia contained fungal toxins. The values for physical properties of conidia likely to affect their respirability were lower from Aspergillus fumigatus then for other toxin formers including Stachybotrys. Sorenson WG, Frazer DG, Jarvis BB, Simpson J, Robinson VA. Trichothecene mycotoxins in aerosolized conidia of Stachybotrys atra. Applied and Environmental Microbiology 1987; 53(6): 1370-1375. This 1988 NIOSH paper confirms that particles made by Stachybotrys are essentially all respirable and that 85% of the dust particles were conidia and 6% were hyphal fragments. Methanol extracts from these fragments showed significant toxicity equal to that from satratoxin H and G. This paper establishes conidia from Stachybotrys contain trichothecene mycotoxins and that inhalation of aerosols with these conidia are a potential health hazard. Nielsen KF, Gravesen S, Nielsen PA, Andersen B, Thrane U, Frisvad JC. Production of mycotoxins on artificially and naturally infested building materials. Mycopathologia 1999; 145(1): 43-56. This 1999 paper clearly shows quantities of secondary metabolites, including but not limited to mycotoxins, made by a variety of toxin formers. This is a finding consistent with the finding that mycotoxins are not the only secondary metabolite made by actively growing fungi; contributory aspects of other inflammagens must be analyzed as well. Fog Nielsen K. Mycotoxin production by indoor molds. Fungal Genet Biol 2003; 39(2): 103-117. This 2003 from Denmark analyzes the "worst case scenario," for homeowners produced by consecutive episodes of water damage that provoke fungal growth and mycotoxin synthesis followed by drier conditions that facilitate liberation of spores and hyphal fragments. We have evidence of relative humidity being a problem, as evidence by Nielsen's work (co-author of this paper) and also now intermittent water intrusion consistent with flood scenarios. Brasel TL, Douglas DR, Wilson SC, Straus DC. Detection of airborne Stachybotrys chartarum macrocyclic trichothecene mycotoxins on particulates smaller than conidia. Applied and Environmental Microbiology 2005; 71(1): 114122. This 2005 paper adds to the importance of assessment of particulates found in indoor air. This paper confirms that trichothecene mycotoxins do become air borne in association with intact conidia and smaller particles independent of those toxins found on spores. Implications here are that any air sample taken looking for Stachybotrys or Stachybotrys toxins that does not include assessment of fragments down to 0.6 microns is inadequate and inaccurate.
12 Gorny RL, Reponen T, Willeke K, Schmechel D, Robine E, Boissier M, Grinshpun SA. Fungal fragments as indoor air biocontaminants. Applied and Environmental Microbiology 2002; 3522-3531. This is one of a series of papers from Cincinnati and NIOSH; this paper looks at fungal fragments found in indoor air as being released in up to 320 times the number of spores for a given volume of air. A previous paper (Cho), showed a relationship of 514 for the relationship of fragments to spores; this paper cites 320. The actual number is of lesser importance than the fact that small particle size of fungal fragments makes contributions to adverse human health effects. Gorny RL. Filamentous microorganisms and their fragments in indoor air- a review. Ann Agric Environ Med 2004; 11: 185-197. Gorny follows up his 2002 paper with this published in 2004 showing that fragments of fungi found in indoor air are of a much greater importance than spores. This finding strongly argues against any possible consideration of dose response relationships. Gorny states a clear description of dose response relationship with a majority of biological agents cannot be established. Gorny RL, Mainelis G, Grinshpun SA, Willeke K, Dutkiewicz J, Reponen T. Release of Streptomyces albus propagules from contaminated surfaces. Environmental Research 2003; 91: 45-53. This paper confirms that there is release of elements from Streptomyces, a form of actinomycetes, from surfaces in indoor environments contaminated with growth of these organisms. These fragments can be found in the air of the contaminated building as well as from surfaces. The indoor surfaces with resident actinomycetes therefore provide a reservoir for toxigenic elements. Spaan S, Heederik DJJ, Thorne PS, Wouters IM. Optimization of airborne endotoxin exposure assessment: effects of filter type, transport conditions, extraction solutions, and storage of samples and extracts. Applied and Environmental Microbiology 2007; 73(19): 6134-6143 This paper attempts to provide standardization of isolation and laboratory analysis of endotoxins from indoor air environments. It confirms endotoxins have an extremely high pro-inflammatory potency and that airborne exposure to endotoxin has been associated with respiratory tract symptoms, abnormalities in pulmonary function and systemic inflammatory affects. Giovannangelo ME, Gehring U, Nordling E, Oldenwening M, van Rijswijk K, de Wind S, Hoek G, Heinrich J, Bellander T, Brunekreef B. Levels and determinants of beta(1->3)-glucans and fungal extracellular polysaccharides in house dust of (pre-) school children in three European countries. Environ Int 2007; 33(1): 9-16.
13 This 2007 paper confirms presence of beta-glucans and polysaccharides from fungi found in house dust in three separate European countries. This paper answers questions as to whether or not there are persistent sources of inflammation from fungi in water damaged buildings that can be found independent of identification of spores. Gehring U, Douwes J, Doekes G, Koch A, Bischof W, Fahlbusch B, Richter K, Wichmann HE, Heinrich J, INGA Study Group. Indoor factors and genetics in asthma. Beta (1->3)-glucan in house dust of German homes: housing characteristics, occupant behavior, and relations with endotoxins, allergens, and molds. Environ Health Perspect 2001; 109(2): 139-144. This paper from Dr. Douwe's lab extends understanding of beta-glucans finding those compounds in house dust often in homes in Germany. While several possible sources of exposure can raise beta-glucans, presence of mold within the home remains one of the highest potential sources correlated with increased beta-glucans. Chew GL, Douwes J, Doekes G, Higgins KM, van Strien R, Spithoven J, Brunekreef B. Fungal extracellular polysaccharides, beta (1->3)-glucans and culturable fungi in repeated sampling of house dust. Indoor Air 2001; 11(3): 171-178. This paper is a result of collaboration by US authority Dr. Chew and Dr. Douwes, showing that beta-glucans found in house dust are good markers for presence of fungal concentrations and floor dust which in turn is a surrogate for estimating airborne fungal infections. Rand TG, Giles S, Flemming J, Miller JD, Puniani E. Inflammatory and cytotoxic responses in mouse lungs exposed to purified toxins from building isolated Penicillium brevicompactum dierckx and P. chrysogenum thom. Toxicological Sciences 2005; 87(1): 213-222. This paper looks at toxins from Penicillium species. The results show that the toxins commonly found on damp materials in water-damaged buildings provoke inflammatory responses. Halstensen A. Species-specific fungal DNA in airborne dust as surrogate for occupational mycotoxin exposure. Int J Mol Sci. 2008; 9(12): 2543-58. Methods for mycotoxin detection are not sensitive enough for the small dust masses obtained by personal sampling. Mycotoxin concentrations are highly variable, so results of PCR should be interpreted with caution. Halstensen A, Nordby K, Eduard W, Llemsdal S. Real-time PCR detection of toxigenic Fusarium in airborne and settled grain dust and association with trichothecene mycotoxins. J Environ Monit. 2006; 8: 1235-1241.
14 Inhalational of immunomodulating mycotoxins produced by Fusarium may imply health risks for farmers. PCR on personal samples identifies mycotoxins if Fusarium is present. Halstensen A, Nordby, Klemsdal S, elen O, Clasen P, Eduard W. Toxigenic Fusarium spp. as determinants of trichothecene mycotoxins in settled grain dust. J Occup Environ Hyg. 2006; 3: 651-659. PCR-detected Fusarium genes for trichothecenes (tri5) highly correlated with trichothecene presence. Park J, Schleiff P, Attfield M, Cox-Ganser J, Kreiss K. Building-related respiratory symptoms can be predicted with semi-quantitative indices of exposure to dampness and mold. Indoor Air. 2004; 14: 425-433. Semi-quantitative measures dampness/mold exposure indices, based solely by visual and olfactory observations can predict existence of excessive building-related respiratory symptoms and diseases. Rao C, Cox-Ganser J, Chew G, Doekes G, White S. Use of surrogate markers of biological agents in air and settled dust samples to evaluate a water-damaged hospital. Indoor Air. 2005; 15 supple 9: 89-97. Air and floor dust measurements of marker compounds may be better indicators if current health risk in a water-damaged environment than measurements of culturable fungi or bacteria. A recently completed project in Sweden confirms that when testing for mycotoxins is done in WDB that those compounds are invariably found. Dr. Bloom states, "Previously it was claimed that the occurrence of mold does not necessarily mean that there are toxins present. But they are!" (www.sciencedaily.com/releases/2008/12/081209085622.htm, accessed 12/10/08; Bloom; Doctoral Thesis for the Department of Laboratory medicine, Division of Medical Microbiology, Lund University, Sweden; Mycotoxins in Indoor Environments, Determination using Mass Spectroscopy). Microbial Growth and Fine Particles: The papers cited below confirm the observations of Gorny and Brasel regarding fine particles released from microbial (molds and bacteria) growth in WDB. The vast majority of these particles, as measured by 1-3-D-g;lucans derived form old cell walls, are less than one micron and are easily deposited in the alveolar spaces of adults and more readily, children. The older colonies shed more particulates than younger ones. Cho SH, Seo SC, Schmechel D, Grinshpun SA, Reponen A. Aerodynamics and respiratory deposition of fungal fragments. Atmos Enviiron/2005; 39:5454-65.
15 Reponen T, Seo SVC, Grimsley F, Lee T, Crawford C, Grinshpun SA. Fungal fragments in mold houses: A field study in homes in New Orleans and Southrern Ohio. 2007; Atmos Environ 41:8140-9. Seo SC, Reponen T, Levin L, Borchelt T, Grinshpun SA. Aerosolization of particulate (1-3---D-glucan) from moldy materials. Appl Environ Microbiol 200; 74:585-93. Seo SC, Reponen T, Levin L, Grinshpun SA. Size fractionation (1-3)--D-glucan concentrations aerosolized from different moldy building materials. Sci Total Environment. 2009; 407:806-14.

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