1. PURPOSE OF RESEARCH  Project as a whole: By using several methods of measuring fungal biomass and speciating fungi, we wish to compare existing and modified tests to determine which one or combination of tests, best correlate with health measures  Personal Project: Quantitatively measure 1,3-  -D-glucan by developing chemical procedures to adapt the Limulus amebocyte lysate assay which is commonly used for LPS endotoxin

2. INTRODUCTION Research to Date  Since 1982, ~30 questionnaire studies on the association of dampness, mold and respiratory health in Europe and North America  Dose-Effect has shown that more visible mold yielded more symptoms in children  Unfortunately, acceptance of association between indoor fungal growth and health as causal is prevented by uncertainty in measuring the exposure

3. Types of Measurement  Airborne Ergosterol - information on total mold biomass in air  Settled Dust Collection - information on taxa  Measurement of Fungi and Fungal Products in Dust -1,3-  -D-glucan and ergosterol  Spore Collection - examined by microscopy with a number of stains used to visualize a) sticky surface b) impaction on agar  Indoor Air Indicator Biochemicals - for either a) bacteria  endotoxin and peptidyl glycan b) fungi  1,3-  -D-glucan and ergosterol

4. 1,3-  -D-glucan  A polyglucose compound found in the cell wall of fungi, certain bacteria, and plants  Can form triple helices, single helices and single chains  Some forms show anti-tumor activity, while others show a relationship between glucan concentration and the extent of airways inflammation  Treatment of 1,3-  -D-glucan with urea, NaOH, or by heating causes a change in conformation

5. EXPERIMENTAL  Use a modified form of the classical limulus amebocyte lysate test for determination of 1,3-  -D-glucan

6.  Perform various control tests using LAL on glucan and glucan + endotoxin standards to obtain optimal modified test specific to 1,3-  -D-glucan  Extract 1,3-  -D-glucan from field samples using the above optimal conditions (exposure expressed as ng/m 3 )  Determine 1,3-  -D-glucan content of common fungal species encountered in indoor air using modified LAL  Remove and extract ergosterol from field sample filters using microwave-digestion technique (Young, 1995)  Analyze extract by means of HPLC or GC-MS to determine amounts of ergosterol (exposure expressed as ng/m 3 ) Young, JC., “Microwave-assisted extraction of the fungal metabolites ergosterol and total fatty acids” in J.Agric. Food Chem., 43:2904-2910 (1995)

7. RESULTS  LAL chromogenic test is approximately 1000 times more sensitive to endotoxin (LPS) than to 1,3-  -D-glucan  Curdlan (unbranched linear 1,3-  -D-glucan) retains activity after NaIO 4 oxidation  indicates std is pure within LOD of test  Upon addition of Tween, no significant change in glucan activity over a range of 0.1-0.005% added  Determined that endotoxins’ reactivity (10 ng) with LAL could be inactivated up to 100% using various NaOH concentrations (i.e. 0.15N - 0.50N)  0.15N (100%), 0.3N (78%), 0.5N (52%)

8.  From above, determined time scale of endotoxin destruction for 0.15 N NaOH

9.  Curdlan in 0.15N NaOH shows increase in LAL activity of up to 5x (for 2.5hrs) compared to neutralized curdlan  therefore, greater test sensitivity  LAL activity of curdlan showed no increase after 2.5 hrs, up to 25 hours in length  After neutralization, a decrease in LAL activity was observed at 10 and 23 hours, at which time activity was equal to that of controls (0.3125-2.5 ng/ml)  LAL test provides r 2 values of greater than.990 consistently for 1,3-  -D-glucan standard curves

11. 1,3-  -D-glucan structure in linear form A proposed mechanism of conformational change involving partially opened triple-helix for 1,3-  -D- glucan. Young, S.H., Dong, W.J., Jacobs, R.R., Journal of Biological Chemistry, v.275, n.16, pg. 11874-11879 (2000)

12.  Pure endotoxin + curdlan showed upto a 350% increase in expected glucan activity, resulting in a “leveling effect” of the reagent  2:1 reagent:sample mix eliminated this effect  Longer hydrolysis times (upto 25 hrs) did not affect the destruction of endotoxin in the presence of curdlan  Preliminary tests of extracting 1,3-  -glucan from real fungal spores (Cladosporium cladosporioides) indicated a glucan content of 3.09 ±0.6 pg/spore

13.  Using above result, a known number of C. cladosporioides spores were added to filters followed by endotoxin  0.15N NaOH not sufficient to destroy endotoxin activity, therefore increased [NaOH]  No significant change (p<0.05) below 1.25ng and 5ng endotoxin added for 0.3N and 0.5N NaOH, respectively

14.  Curdlan in 0.5N NaOH showed a further increase in LAL activity (and hence in sensitivity) compared to 0.15N  Using result for C. cladosporioides, LAL method was applied to outdoor air samples to determine mean number of spores/m 3 (=2400 spores/m 3 )  comparable with results obtained by microscopy  Other experiments demonstrating LAL applicability for accurate measurement of 1,3-  -D-glucan were performed on pollen, Ambrosia atemisiifolia, and yeast  Pollen - 83ng/mg is comparabe to results by Rylander et al. using 1,3-  -D-glucan specific test called the Fungitec G-Test  Yeast - 0.321ng/spore is comparable to fungal spores Rylander, R., et al. “1,3-  -D-glucan may contribute to pollen sensitivity” Clin Exp Immunol 1999; 115:383-384

15.  Correlation between ergosterol and 1,3-  -D-glucan from field samples was 0.654 (n=108, Pearson Correlation)  Glucan content of 12 different common indoor fungal species was obtained using LAL method, followed by spore dimensioning using digital images Digital image of P.variotii used for spore dimensioning and glucan content of different indoor fungal species

16. CONCLUSIONS This presentation will probably involve audience discussion, which will create action items. Use PowerPoint to keep track of these action items during your presentation In Slide Show, click on the right mouse button Select “Meeting Minder” Select the “Action Items” tab Type in action items as they come up Click OK to dismiss this box This will automatically create an Action Item slide at the end of your presentation with your points entered. ã 0.5 N NaOH showed best results as an extracting solvent for samples containing both endotoxin and 1,3-  -D-glucan, with no significant difference in LAL activity below 5ng of endotoxin  satisfactory for indoor and outdoor air samples as endotoxin rarely exceeds the 5ng range, based on previous results ã Modified LAL method demonstrated good applicability with pollen results that are comparable with 1,3-  -D-glucan specific test ã Physical methods are currently being explored (ie. MALDI-MS, SEC) with curdlan standards coupled with the LAL method to confirm concentration of the insoluble 1,3-  -D-glucans ã Statistical analysis of normalized spore dimensions and glucan content show a correltion of 0.604 (0.939 without C. cladosporioides) ACKNOWLEDGMENTS: Funding provided by NSERC, CHRP, TSIR, Health Canada. We thank John Bissett (AAFC) for his assistance with the digital imaging.