In healthy subjects, acute inhalation of swine dust causes an influx of inflammatory cells into the airways and increased bronchial responsiveness. The exposure may also cause fever and generalized symptoms. It seems likely that proinflammatory cytokines are involved in the response to inhaled swine dust.Nasal and bronchoalveolar lavage (BAL) were performed before, and 7 and 24 h after the start of 3 h exposure to swine dust, during a period of work in a swine confinement building, in 22 healthy subjects. Lavage fluids were analysed with regard to the cellular response and concentrations of interleukin (IL)-1α, IL-1β, IL-6 and tumour necrosis factor-α (TNF-α). Each subject carried personal samplers for exposure measurements. Inhalable dust and airborne endotoxin, 3-hydroxylated (2-OH) fatty acid and muramic acid were measured. Bronchial responsiveness to methacholine was investigated 1-2 weeks before and 7 h after the start of the exposure.Exposure caused fever (>38°C) in three subjects, and approximately 25% of the subjects experienced symptoms. Bronchial responsiveness to methacholine increased by 3.5 (1.6-4.8) doubling doses (median (25th-75th percentile)). Following exposure, granulocytes increased more than 50 fold in BAL fluid and more than 40 fold in nasal lavage fluid. IL-1α and IL-1β increased significantly in BAL fluid (p<0.05) and nasal lavage fluid (p<0.01). IL-6 increased 25 fold in BAL and 15 fold in nasal lavage fluid (p<0.001). TNF-α was below detection limit (0.25 ng·L -1 ) in most subjects before exposure and increased following exposure to 3.8 (2.4-5.7) and 1.3 (0.6-2.3) ng·L -1 in BAL and nasal lavage fluid, respectively, (p<0.001). Total inhalable dust was 20.5 (14.6-30.0) mg·m -3 and the concentrations of airborne endotoxin, 3-OH fatty acid and muramic acid were 1.2 (0.8-1.4), 3.5 (2.2-4.5) and 0.9 (0.3-1.9) µg·m -3 , respectively. There was a significant correlation between the IL-6 response in BAL fluid and exposure to dust endotoxin activity and 3-OH fatty acids (p<0.05). Otherwise, no significant correlations were found between exposure and the cytokine response.We conclude that exposure to swine dust causes an intense upper and lower airway inflammation, which involves the proinflammatory cytokines interleukin-1, interleukin-6 and tumour necrosis factor-α.

Could honeybees' most valuable contribution to mankind besides pollination services be alternative tools against infections? Today, due to the emerging antibiotic-resistant pathogens, we are facing a new era of searching for alternative tools against infections. Natural products such as honey have been applied against human's infections for millennia without sufficient scientific evidence. A unique lactic acid bacterial (LAB) microbiota was discovered by us, which is in symbiosis with honeybees and present in large amounts in fresh honey across the world. This work investigates if the LAB symbionts are the source to the unknown factors contributing to honey's properties. Hence, we tested the LAB against severe wound pathogens such as methicillin-resistant Staphylococcus aureus (MRSA), Pseudomonas aeruginosa and vancomycin-resistant Enterococcus (VRE) among others. We demonstrate a strong antimicrobial activity from each symbiont and a synergistic effect, which counteracted all the tested pathogens. The mechanisms of action are partly shown by elucidating the production of active compounds such as proteins, fatty acids, anaesthetics, organic acids, volatiles and hydrogen peroxide. We show that the symbionts produce a myriad of active compounds that remain in variable amounts in mature honey. Further studies are now required to investigate if these symbionts have a potential in clinical applications as alternative tools against topical human and animal infections.

SummaryMoraxella catarrhalis is an emerging human respiratory pathogen in patients with chronic obstructive pulmonary disease (COPD) and in children with acute otitis media. The specific secretion machinery known as outer membrane vesicles (OMVs) is a mechanism by which Gram-negative pathogens interact with host cells during infection. We identified 57 proteins in M. catarrhalis OMVs using a proteomics approach combining two-dimensional SDS-PAGE and MALDI-TOF mass spectrometry analysis. The OMVs contained known surface proteins such as ubiquitous surface proteins (Usp) A1/A2, and Moraxella IgD-binding protein (MID). Most of the proteins are adhesins/virulence factors triggering the immune response, but also aid bacteria to evade the host defence. FITC-stained OMVs bound to lipid raft domains in alveolar epithelial cells and were internalized after interaction with Toll-like receptor 2 (TLR2), suggesting a delivery to the host tissue of a large and complex group of OMV-attributed proteins. Interestingly, OMVs modulated the pro-inflammatory response in epithelial cells, and UspA1-bearing OMVs were found to specifically downregulate the reaction. When mice were exposed to OMVs, a pulmonary inflammation was clearly seen. Our findings indicate that Moraxella OMVs are highly biologically active, transport main bacterial virulence factors and may modulate the epithelial pro-inflammatory response.

An integrated procedure is presented whereby gas chromatography-ion trap mass spectrometry is used to determine chemical markers of gram-negative bacterial lipopolysaccharide (3-hydroxy fatty acids with 10 to 18 carbon atoms), gram-positive bacteria (branched-chain fatty acids with 15 and 17 carbon atoms), bacterial peptidoglycan (muramic acid), and fungal biomass (ergosterol) in samples of settled house dust. A hydrolysate of 13 C-labeled cyanobacterial cells is used as an internal standard for the first three markers. These analyses require two dust samples, one for 3-OH fatty acids, branched-chain fatty acids, and muramic acid and another for ergosterol. The method may be used to characterize microbial communities in environmental samples.Inhalation of airborne microorganisms in indoor environments has been associated with the development of respiratory disorders. Substances such as endotoxins (lipopolysaccharides [LPS]) (14,17,18,28), peptidoglycan (9, 12), and various fungal components and products (3,6,21) are among the suspected causative agents. Culture-based methods are suitable for detection of culturable infectious agents and allow species identification; however, it is widely agreed that only a small fraction (0.1 to 10%) of the total microbial flora in an indoor environment is currently culturable (29). Direct microscopy is at best semiquantitative. Although the Limulus amebocyte lysate test is extremely sensitive to endotoxin and can detect glucans, it measures bioactivity rather than absolute amounts and its reproducibility and specificity have been questioned (5). Nucleic acid-based methods including PCR are very specific, and by using broad-range (universal) probes and primers sets based on 16S rDNA (20) and 18S rRNA (31), most bacteria and fungi present in a sample can be identified. To date, such universal probes and primers have not been used to characterize the microbiology of indoor environments; rather, PCR has been used to detect specific fungi and bacteria in such environments (4,11,22).In our laboratory we have developed and applied gas chromatography-mass spectrometry (GC-MS) methods to determine biomarker molecules in complex matrices including organic dust. Microorganisms contain unique compounds not found elsewhere in nature, which can be used as chemical markers of larger, bioactive structures (7). Endotoxins (LPS) are major constituents of the outer membrane of gram-negative bacteria. A backbone of lipid A, the toxic component of the LPS molecule, carries in general 4 mol of unique 3-hydroxy fatty acids (3-OH FAs) (23-26). Certain branched-chain FAs are found in most gram-positive bacteria (13,30). Muramic acid is a unique marker of peptidoglycan (2,8,9,15), which is the cross-linked macromolecular structure responsible for the rigidity of bacterial cell walls and is sometimes referred to as "gram-positive bacterial endotoxin" (27). Ergosterol is a common fungal membrane lipid that is widely used as a marker of fungal biomass, although the concentration depends on the species and ...

Dampness in buildings has been linked to adverse health effects, but the specific causative agents are unknown. Mycotoxins are secondary metabolites produced by molds and toxic to higher vertebrates. In this study, mass spectrometry was used to demonstrate the presence of mycotoxins predominantly produced by Aspergillus spp. and Stachybotrys spp. in buildings with either ongoing dampness or a history of water damage. Verrucarol and trichodermol, hydrolysis products of macrocyclic trichothecenes (including satratoxins), and trichodermin, predominately produced by Stachybotrys chartarum, were analyzed by gas chromatographytandem mass spectrometry, whereas sterigmatocystin (mainly produced by Aspergillus versicolor), satratoxin G, and satratoxin H were analyzed by high-performance liquid chromatography-tandem mass spectrometry. These mycotoxin analytes were demonstrated in 45 of 62 building material samples studied, in three of eight settled dust samples, and in five of eight cultures of airborne dust samples. This is the first report on the use of tandem mass spectrometry for demonstrating mycotoxins in dust settled on surfaces above floor level in damp buildings. The direct detection of the highly toxic sterigmatocystin and macrocyclic trichothecene mycotoxins in indoor environments is important due to their potential health impacts.Microorganisms are thought to be involved in health problems connected to damp buildings. However, the causative microbiological agents are unknown (22). Many molds that thrive in damp indoor environments are potent mycotoxin producers and may play a role in the reported adverse health effects ( 1,5,17,23,24,26,30). Mycotoxins are secondary metabolites, e.g., produced to give molds strategic advantages over encroaching organisms. Examples are sterigmatocystin (STRG), a carcinogenic mycotoxin produced mainly by Aspergillus versicolor; satratoxin G (SATG) and satratoxin H (SATH), which are cytotoxic mycotoxins produced by Stachybotrys chartarum; and citrinin, gliotoxin, and patulin, produced by, e.g., Aspergillus spp. and Penicillium spp. The latter three mycotoxins have been shown to be immunomodulatory, causing a polarization in cytokine production towards a Th2 phenotype (36), and citrinin caused depletion of intracellular glutathione at nontoxic concentrations (18). Based on spore counts, the airborne mycotoxin concentrations found in damp buildings have been estimated to be insufficient for causing adverse health effects (20). However, indoor molds may fragment into very small airborne mycotoxin-containing particles, resulting in up to a 500-fold larger exposure than assumed previously (4,11,21,32). In addition, Cho et al. (7) showed that the respiratory deposition of S. chartarum fragments was over 200-fold higher than that of spores in adults and an additional 4 to 5 times higher in infants. These aerosolized fragments could potentially also be the source of allergens (13).S. chartarum and A. versicolor are two commonly encountered molds in buildings with moisture problems (9,12,...

Inhalation of swine-house dust may cause an acute airway inflammatory condition (organic dust toxic syndrome). Thirty-eight healthy subjects were exposed to swine dust while weighing swine for 3 h. We studied the correlation between acute health effects and the inhaled bacterial exposure markers peptidoglycan (the main constituent of the cell walls of gram-positive bacteria, but also present in lesser amounts in gram-negative bacteria) and lipopolysaccharides (LPS; present only in gram-negative bacteria). LPS activity in airborne dust was measured with the Limulus amebocyte lysate assay (LPS(LAL)), and the total LPS was estimated from 3-hydroxy fatty acids, which were measured with gas chromatography-mass spectrometry (GC-MS) (LPS(GC-MS)). Peptidoglycan was estimated from muramic acid measured with GC-MS. The median (25th to 75th percentile) concentration of inhalable dust was 21 (16 to 25) mg/m3. LPS(LAL) was 1.2 (0.9 to 1.4) microg/m3; LPS(GC-MS) was 3.9 (2.5 to 4.9) microg/m3; and the peptidoglycan concentration in airborne dust was 6.5 (2.7 to 13) microg/m3. All exposure markers correlated significantly with an increase in serum interleukin-6. LPS(LAL) showed the highest correlation (r2 = 0.29) and total inhaled dust the lowest (r2 = 0.09). LPS(LAL) also correlated with symptoms and with an increase in bronchial responsiveness and decrease in vital capacity (VC). Peptidoglycan, but not LPS(LAL), correlated with an increase in the blood granulocyte concentration and in body temperature. The results suggest that several microbial agents in inhaled swine-house dust may contribute to acute systemic health effects.

Ergosterol and 3-hydroxy fatty acids, chemical markers for fungal biomass and the endotoxin of gramnegative bacteria, respectively, may be useful in studies of health effects of organic dusts, including domestic house dust. This paper reports a method for the combined determination of ergosterol and 3-hydroxy fatty acids in a single dust sample and a comparison of these chemical biomarkers determined by gas chromatography-mass spectrometry with results from fungal culture and Limulus assay. Analyses of replicate house dust samples resulted in correlations of 0.91 (ergosterol in six replicates; P < 0.01) and 0.94 (3-hydroxy fatty acids in nine replicates; P < 0.001). The amounts of ergosterol (range, 2 to 16.5 ng/mg of dust) correlated with those of total culturable fungi (range, 6 to 1,400 CFU/mg of dust) in 17 samples, (r ‫؍‬ 0.65; P < 0.005). The amounts of endotoxin (range, 11 to 243 endotoxin units/mg of dust) measured with a modified chromogenic Limulus assay correlated with those of lipopolysaccharide (LPS) determined from 3-hydroxy fatty acid analysis of 15 samples. The correlation coefficient depended on the chain lengths of 3-hydroxy acids used to compute the LPS content. The correlation was high (r ‫؍‬ 0.88 ؎ 0.01; P < 0.001) when fatty acid chains of 10 to 14 carbon atoms were included; the correlation was much lower when hydroxy acids of 16-or 18-carbon chains were included. In conclusion, the results of the described extraction and analysis procedure for ergosterol and 3-hydroxy fatty acids are reproducible, and the results can be correlated with fungal culture and endotoxin activity of organic dust samples.

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