Michael Timm

Seasonal Variations of Indoor Microbial Exposures and Their Relation to Temperature, Relative Humidity, and Air Exchange Rate

ABSTRACT Indoor microbial exposure has been related to adverse pulmonary health effects. Exposure assessment is not standardized, and various factors may affect the measured exposure. The aim of this study was to investigate the seasonal variation of selected microbial exposures and their associations with temperature, relative humidity, and air exchange rates in Danish homes. Airborne inhalable dust was sampled in five Danish homes throughout the four seasons of 1 year (indoors, n = 127; outdoors, n = 37). Measurements included culturable fungi and bacteria, endotoxin, N-acetyl-beta-d-glucosaminidase, total inflammatory potential, particles (0.75 to 15 μm), temperature, relative humidity, and air exchange rates. Significant seasonal variation was found for all indoor microbial exposures, excluding endotoxin. Indoor fungi peaked in summer (median, 235 CFU/m3) and were lowest in winter (median, 26 CFU/m3). Indoor bacteria peaked in spring (median, 2,165 CFU/m3) and were lowest in summer (median, 240 CFU/m3). Concentrations of fungi were predominately higher outdoors than indoors, whereas bacteria, endotoxin, and inhalable dust concentrations were highest indoors. Bacteria and endotoxin correlated with the mass of inhalable dust and number of particles. Temperature and air exchange rates were positively associated with fungi and N-acetyl-beta-d-glucosaminidase and negatively with bacteria and the total inflammatory potential. Although temperature, relative humidity, and air exchange rates were significantly associated with several indoor microbial exposures, they could not fully explain the observed seasonal variations when tested in a mixed statistical model. In conclusion, the season significantly affects indoor microbial exposures, which are influenced by temperature, relative humidity, and air exchange rates.

Comparison of sampling methods for the assessment of indoor microbial exposure.

UNLABELLED Indoor microbial exposure has been related to allergy and respiratory disorders. However, the lack of standardized sampling methodology is problematic when investigating dose-response relationships between exposure and health effects. In this study, different sampling methods were compared regarding their assessment of microbial exposures, including culturable fungi and bacteria, endotoxin, as well as the total inflammatory potential (TIP) of dust samples from Danish homes. The Gesamtstaubprobenahme (GSP) filter sampler and BioSampler were used for sampling of airborne dust, whereas the dust fall collector (DFC), the electrostatic dust fall collector (EDC), and vacuum cleaner were used for sampling of settled dust. The GSP assessed significantly higher microbial levels than the BioSampler, yet measurements from both samplers correlated significantly. Considerably higher levels of fungi, endotoxin, and TIP were found in the EDC compared with the DFC, and regarding fungi, the EDC correlated more strongly and significantly with vacuumed dust than the DFC. Fungi in EDC and vacuum dust correlated most strongly with airborne dust, and in particular, the measurements from the EDC associated well with those from GSP. Settled dust from the EDC was most representative of airborne dust and may thus be considered as a surrogate for the assessment of indoor airborne microbial exposure. PRACTICAL IMPLICATIONS Significant discrepancies between sampling methods regarding indoor microbial exposures have been revealed. This study thus facilitates comparison between methods and may therefore be used as a frame of reference when studying the literature or when conducting further studies on indoor microbial exposure. Results also imply that the relatively simple EDC method for the collection of settled dust may be used as an alternative to otherwise tedious and time-consuming airborne dust sampling.

Sampling, extraction and measurement of bacteria, endotoxin, fungi and inflammatory potential of settling indoor dust

Selection of sampling device, sampling location and period are important first steps in the measurement of exposure to bioaerosols in indoor air. The steps following the sampling include treatment of samples and laboratory analysis. In this study, settling bacteria, endotoxin, fungi and serine protease have been measured in Danish homes using Electrostatic Dust Fall Collectors (EDCs). The effects of the presence of occupants, sampling on open surfaces versus in bookcases and treatment of samples have been studied. Concentrations of bacteria and endotoxin were significantly higher when occupants were at home than when they were absent. Across homes, higher concentrations of fungi were found in spring than in winter, as was the total inflammatory potential, while higher concentrations of protease were found in winter than in spring. The placement of the EDCs in bookcases versus on an open surface significantly affected the measured concentrations of bacteria and endotoxin. Direct extraction of EDC cloths caused a higher measured concentration of bacteria, fungi and serine protease than if EDC cloths were extracted post-storage at -20 °C. Extraction of EDC cloths caused an average of 51% and 58% extraction of bacteria and fungi respectively. In conclusion, EDCs should be placed on open surfaces during the sampling, how much occupants are present in their home during sampling and sampling season should be considered, EDC cloths should not be stored in a freezer before extraction of microorganisms, but extraction suspensions can be stored at -80 °C without affecting the number of microorganisms significantly.

Immunomodulatory effects of honey cannot be distinguished from endotoxin

In recent years, the use of honey has re-emerged as a remedy for wound treatment. Effects of honey have been related to the presence of an unidentified component that induces release of inflammatory cytokines from monocytic cells. The present study was intended to further characterize the reported in vitro effects of honey. Our results show that natural honeys induce interleukin-6 release from Mono Mac 6 cells as well as release of reactive oxygen species from all-trans retinoic acid (ATRA) differentiated HL-60 cells. The natural honeys contained substantial amounts of endotoxin, and the responses observed in the cell based assays were similar to the responses induced by endotoxin alone. In addition, we determined that the immunomodulatory component present in the natural honeys was retained in the ultra filtrated fraction with a molecular weight greater than 20 kDa. The component was resistant to boiling and its immunomodulatory activity could be abrogated by the addition of polymyxin B. We speculate that the observed in vitro immunomodulatory effects of honey might solely be explained by the endotoxin content in the natural honeys.

Assessment of the Total Inflammatory Potential of Bioaerosols by Using a Granulocyte Assay

ABSTRACT Occupational health symptoms related to bioaerosol exposure have been observed in a variety of working environments. Bioaerosols contain microorganisms and microbial components. The aim of this study was to estimate the total inflammatory potential (TIP) of bioaerosols using an in vitro assay based on granulocyte-like cells. A total of 129 bioaerosol samples were collected in the breathing zone of workers during their daily working routine at 22 biofuel plants. The samples were analyzed by traditional assays for dust, endotoxin, fungal spores, (1→3)-β-d-glucan, total number of bacteria, the enzyme N-acetyl-β-d-glucosaminidase (NAGase; primarily originating from fungi), Aspergillus fumigatus, and mesophilic and thermophilic actinomycetes; the samples were also assayed for TIP. In a multilinear regression four factors were significant for the TIP values obtained: endotoxin (P < 0.0001), fungal spores (P < 0.0001), (1→3)-β-d-glucan (P = 0.0005), and mesophilic actinomycetes (P = 0.0063). Using this model to estimate TIP values on the basis of microbial composition, the correlation to the measured values was r = 0.91. When TIP values obtained in the granulocyte assay were related to the primary working area, we found that bioaerosol samples from personnel working in straw storage facilities showed high TIP values (≈50 times the TIP of unstimulated controls). In contrast, bioaerosol samples from personnel with work functions in offices or laboratories showed low TIP values (≈5 times the TIP of the unstimulated control). This indicates, as expected, that these areas were less contaminated. In conclusion, the granulocyte assay reacts to multiple contaminants in the environmental samples and can be used to obtain a measurement of TIP. Therefore, potential occupational health effects related to inflammation of the airways in a working environment can be estimated using this assay.

Cryopreservation of differentiated HL-60 cells for pyrogen testing.

All-trans retinoic-acid (ATRA) differentiated HL-60 cells can be used to detect pyrogens such as bacteria, bacterial components, yeasts and fungi. Differentiated HL-60 cells obtain neutrophil like characteristics and if stimulated the differentiated HL-60 cells produce reactive oxygen species in a dose dependent manner. Culturing and differentiation of cell lines are time consuming activities and require suitable facilities; cryopreservation of pre-differentiated cells could provide the basis for an easily distributable pyrogen testing kit. Cryopreservation of granulocytes has proven to be very complicated and neutrophils are especially difficult to cryopreserve, most likely due to their large degree of granulation. Here we present evidence that HL-60 cells can be differentiated with ATRA and subsequently cryopreserved. Upon thawing the cells retain their ROS producing capabilities and reactivity towards pyrogens. Further, the cells retain their ability to react dose dependently towards lipopolysaccharide (LPS), lipoteichoic acid (LTA) and zymosan. At pathophysiologically relevant concentrations of LPS, LTA and zymosan the cells retain full reactivity for at least two months when stored in liquid nitrogen. In conclusion, ATRA differentiated HL-60 cells are cryopreservable and retain reactivity upon thawing. It is therefore possible to produce an in-vitro in-house pyrogen test kit for medicines and related products.

Effect of moist heat sterilisation on the pyrogenicity of cell wall components from Staphylococcus aureus.

As opposed to endotoxins very little is known about the heat resistance of Gram positive pyrogens. The aim of this study is to examine the pyrogenic activity of the cell wall components lipoteichoic acid and peptidoglycan from Staphylococcus aureus after moist heat sterilisation. The pyrogenic activity is determined as the ability of the substances to induce interleukin-6 secretion in Mono Mac 6 cells. The standard terminal moist heat sterilisation procedures (121 degrees C for 15min and 134 degrees C for 3min) are not able to inactivate the pyrogenic activity of S. aureus lipoteichoic acid and peptidoglycan. However after longer treatment times the pyrogenic activity of lipoteichoic acid is removed at both 121 degrees C and 134 degrees C. In contrast the activity of peptidoglycan is not removed after 160min at neither 121 degrees C nor 134 degrees C where only a 2-log reduction is obtained. In conclusion the terminal moist heat sterilisation procedures described by the European Pharmacopoeia are not able to inactivate the interleukin-6 inducing activity of S. aureus lipoteichoic acid and peptidoglycan.

The effect of dietary fish oil-supplementation to healthy young men on oxidative burst measured by whole blood chemiluminescence

Dietary long-chain n-3 PUFA (n-3 LCPUFA) are thought to have immune-modulating effects, but the specific effects and mechanisms are not fully elucidated. The aim of this study was to determine whether dietary n-3 LCPUFA could affect ex vivo oxidative burst in healthy young men. The study had a randomised 2 x 2-factorial design in which subjects were randomly assigned to 8-week supplementation with capsules containing fish oil (about 2.9 g n-3 LCPUFA/d) or olive oil (control). Subjects were also randomly assigned to household use of oils and fat spreads with a high or a low 18:2n-6 content. At baseline and at the end of the intervention, the fatty acid composition of peripheral blood mononuclear cells (PBMC) was analysed by GLC and oxidative burst was studied in whole blood stimulated with zymosan using luminol-enhanced chemiluminescence. The PBMC content of n-3 LCPUFA was markedly increased by the fish oil-supplementation (P < 0.001, compared to the olive oil groups). No effect of the intervention was observed on neutrophil count, but one measure of the zymosan-induced oxidative burst was higher in the fish oil groups (P = 0.03) compared to the olive oil groups. The fat intervention did not in itself affect oxidative burst neither did it change the effect of the fish-oil intervention. The measures of oxidative burst at the end of the intervention period were found to be associated with the DHA content of PBMC (r 0.44, P = 0.016), suggesting a dose-response relationship. These results indicate that n-3 LCPUFA may have immuno-stimulating effects.

Immunomodulatory effects of testosterone evaluated in all-trans retinoic acid differentiated HL-60 cells, granulocytes, and monocytes

The sex hormones are known to affect innate immunity in humans. In this study we evaluated the immunomodulatory effects of testosterone in a model system comprising of all-trans retinoic acid differentiated HL-60 cells, and confirmed the results in human granulocytes and monocytes. Results showed that testosterone at pharmacological doses reduced the production of interleukin-8 and reactive oxygen species from differentiated HL-60 cells in a concentration dependent manner without affecting phagocytosis. The cells were stimulated with zymosan, lipopolysaccharide, or Bacillus subtilis. At the highest concentration of testosterone (120 μM), interleukin-8 secretion was reduced 42-80%, and production of reactive oxygen species was reduced 32-46%. Flutamide, an antagonist of the classical intracellular androgen receptor, was unable to antagonize the immunosuppressive effect of testosterone. We further demonstrated that the suppressive effect of testosterone has a short onset time. Our results suggest that testosterone affects the fast operating membrane bound androgen receptor or a rapid acting enzyme system. Testosterone, at pharmacological doses, was also shown to suppress generation of reactive oxygen species and interleukin-8 in human granulocytes and monocytes, respectively, to a similar extent as observed in differentiated HL-60 cells.

Comment on: Harte et al. High Fat Intake Leads to Acute Postprandial Exposure to Circulating Endotoxin in Type 2 Diabetic Subjects. Diabetes Care 2012;35:375–382

We read with great interest the article by Harte et al. (1), which reports that high intake of dietary fat results in increased levels of endotoxin in the blood of both type 2 diabetic patients and control subjects, thereby supporting an earlier article by Erridge et al. (2) who studied healthy subjects. Low-grade inflammation is associated with obesity, and much evidence suggests that such chronic tissue inflammation is a key factor contributing to development of type 2 diabetes. Some of this inflammation may be caused by endotoxin from the gut microbiota, from where endotoxin may be absorbed together with dietary fat. In such a case, endotoxin should increase …