Whole Blood Assay as a Tool to Describe the Effects of Zinc Oxide Exposure on Innate Immunity.

Inhalation of high concentrations of zinc oxide (ZnO) particles may cause metal fume fever. A useful tool to characterize the reactivity of innate immune cells of an individual, e.g., after in vivo exposure, is the whole blood assay (WBA). The measurable outcome of WBA is the release of cytokines, especially pro-inflammatory and pyrogenic cytokines induced by stimulation in vitro. The aim of the study was to evaluate whether inhalation of nano-sized zinc oxide particles modifies the results of WBA from healthy blood donors. Sixteen healthy subjects were exposed to filtered air and ZnO particles (0.5, 1.0, and 2.0 mg/m3) for 4 h on four different days. Blood was collected before and 24 h after exposure, and ex vivo stimulation of the whole blood was performed using different endotoxin concentrations. The release of interleukin (IL)-1ß and IL-8 after 22-h incubation was quantified with specific immunoassays. The dose-response relationship of ex vivo stimulation with different endotoxin concentrations was not affected by previous ZnO exposure. However, based on the previously established calculation models, changes due to ZnO exposure could be described. The range of cytokine release in WBA was calculated for the whole group of blood donors, for the subgroups of low and high responders (each n = 8), and on the individual level. Most changes were observed after 0.5 mg/m3 ZnO exposure. Higher ZnO exposure did not yield higher effects. We conclude that the effects of inhalation of nano-sized ZnO particles in blood of healthy donors using the WBA could be determined. However, it should be noted that cytokine release as outcome of WBA is not a marker of disease.

Cell Activation and Cytokine Release Ex Vivo: Estimation of Reproducibility of the Whole-Blood Assay with Fresh Human Blood.

The whole-blood assay (WBA) with human fresh blood may provide insight into the features of an individual's innate immunity. To assess this, ex vivo cytokine release is measured after stimulation of whole blood with various stimuli, for instance, endotoxin in vitro. The aim of the present study was to evaluate WBA reproducibility with fresh blood using different calculation models. The blood was collected from 16 healthy volunteers on 6 different days. Ex vivo stimulation was performed in each individual's blood sample for 22 h, using different endotoxin concentrations. Interleukin (IL)-1ß and IL-8 release were quantified using specific immunoassays in the cell-free supernatant. We found that a dose-response relationship between endotoxin and cytokine concentration could be verified for all blood donors in all tests. The median coefficient of variation of the repeated tests was 29% for IL-1ß and 52% for IL-8. Upon stimulation with 40 pg/mL endotoxin, a confidence interval of 60-140% was calculated for IL-1ß and 70-271% for IL-8 regarding test reproducibility. Furthermore, the classification into high or low responder was reproducible. We conclude that repeated blood collection offers an opportunity to evaluate the variability of WBA. Considering a high intragroup variability, an individual range assessment has been suggested to evaluate exposure effects.

Assessment of airborne exposure to endotoxin and pyrogenic active dust using electrostatic dustfall collectors (EDCs).

Passive airborne dust sampling using electrostatic dustfall collectors (EDCs) is one possibility especially for long sampling periods. In this study, EDCs were deposited in living rooms of private households and in social rooms of composting plants. The aim of the study was to determine whether endotoxin and pyrogenic activity are measurable using EDCs. In all extracts, endotoxin (via Limulus amebocyte lysate [LAL] assay) and pyrogenic activity (interleukin [IL]-1ß release via whole blood assay) were detectable. In addition, the monocyte chemotactic protein (MCP-1; CCL-2) as a secondary proinflammatory marker was measured with whole blood assay. Endotoxin activity and proinflammatory/pyrogenic activity of EDC extracts from social rooms in composting plants were higher compared to extracts obtained from EDCs in private household rooms. A significant correlation between LAL assay and whole blood assay was detectable. In conclusion, EDC sampling is an applicable method to evaluate settled dust from airborne bioaerosols displaying a longer period of exposure. The extraction of EDC without Tween enables one to measure endotoxin as well as proinflammatory/pyrogenic activity using the same sample for parallel detection and more reliable characterization of the airborne bioaerosol contamination.

Development and application of mold antigen-specific enzyme-linked immunosorbent assays (ELISA) to quantify airborne antigen exposure.

The aim of our study was to develop specific enzyme-linked immunosorbent assays (ELISAs) and apply these to assess mold antigen exposure in composting plants. Sandwich ELISAs based on polyclonal antibodies to Aspergillus fumigatus (Af), Penicillium chrysogenum (Pc), and Cladosporium herbarum (Ch) antigens were developed and validated. Reactivity to 18 different mold species was tested. To optimize extraction procedure, inhalable dust samples taken by a parallel sampler were extracted with or without homogenization. In 31 composting plants stationary pumps were installed at 4 sites to collect 124 inhalable dust samples. The newly developed ELISAs were used in addition to an anti ß-1,3-glucan ELISA to quantify mold antigens. The Cladosporium ELISA showed less than 0.04% reactivity to extracts from other fungal genera, while the Af ELISA demonstrated a reactivity of up to 3.6% and the Pc ELISA reacted up to 11% to other mold species. Extraction of parallel sampled filters gave higher antigen amounts with homogenization. The increase was highest for Pc-antigens, followed by Af-antigens, and lowest for Ch-antigens. Mean lower detection limits of homogenized inhalable dust samples were 5 ng/m(3) (Af), 0.6 ng/m(3) (Pc), 0.2 ng/m(3) (Ch), and 0.6 ng/m(3) (ß-1,3-glucan). The ELISAs were able to detect antigens in 43% (Af), 37% (Pc), 94% (Ch), or 100% (ß-1,3-glucan) of the 124 airborne dust samples. Inhalable dust, ß-1,3-glucan, and Af-, Pc-, and Ch-antigen concentrations were significantly correlated. The newly developed mold antigen ELISAs are thus able to measure airborne exposure levels in composting plants and differentiate between distinct fungi genera.

Is in vitro cytokine release a suitable marker to improve the diagnosis of suspected mold-related respiratory symptoms? A proof-of-concept study.

Indoor mold infestation can lead to a variety of adverse health effects, including allergic and non-allergic respiratory complaints. Especially if no evidence of an allergic reaction can be found for the complaints, diagnostic tools that might explain mold-associated health problems are missing. As a proof-of-concept, in the present study whole blood assay (WBA) was used to determine cellular response by measuring cytokine release (IL-1ß and IL-8) after in vitro stimulation. Blood was available from a total of 48 subjects. By questionnaire, complaints and possible mold exposure were documented. Specific in vitro blood stimulation was tested with Escherichia coli endotoxin and extracts of different molds (Aspergillus fumigatus, Penicillium chrysogenum, Aspergillus versicolor, and Cladosporium herbarum). To characterize the relevance of WBA in describing the mold-induced immune response, we compared the following groups: asthmatics vs. non-asthmatics, mx1-sensitized vs. non-mx1-sensitized, mold-exposed vs. non-mold-exposed. In response to endotoxin stimulation, a significantly higher IL-1ß release was found in mx1-sensitized than in non-mx1-sensitized subjects. Furthermore, the blood of asthmatics showed significantly higher IL-8 and IL-1ß release after stimulation with Penicillium chrysogenum and endotoxin, respectively, compared to non-asthmatics. However, no significant difference in the level of cytokine release was observed between the mold-exposed and non-exposed group, neither after endotoxin nor mold stimulation. In conclusion, the WBA used in this study is not a suitable tool for clinical routine diagnostic workup. Our data suggests that WBA reflects cellular differences that are disease-related but not directly attributable to mold exposure. However, in combination with further data, WBA will be a helpful und interesting tool in research, e.g., in description of the complex immune response to molds.

Determination of ATP-activity as a useful tool for monitoring microbial load in aqueous humidifier samples.

Air humidifier water tanks are potential sources of microbial contaminants. Aerosolization of these contaminants is associated with the development of airway and lung diseases; therefore, implementation of preventive strategies including monitoring of the microbial contamination is recommended. So far, culture-based methods that include measuring colony forming units (CFU) are widely used to monitor microbial load. However, these methods are time consuming and have considerable drawbacks. As a result, alternative methods are needed which provide not only clear and accurate results concerning microbial load in water samples, but are also rapid and easy to use in the field. This paper reports on a rapid test for ATP quantification as an alternative method for microbial monitoring, including its implementation, validation and application in the field. For this purpose, 186 water samples were characterized with different methods, which included ATP analysis, culture-based methods, endotoxin activity (common and rapid test), pyrogenic activity and number of particles. Half of the samples was measured directly in the field and the other half one day later in the laboratory. The results of both tests are highly correlated. Furthermore, to check how representative the result from one sample of a water source is, a second sample of the same water tank were collected and measured. Bioluminescence results of the undiluted samples covered a range between 20 and 25,000 relative light units (RLU) and correlated with the results obtained using the other methods. The highest correlation was found between bioluminescence and endotoxin activity (rs=0.79) as well as pyrogenic activity (rs=0.75). Overall, the results of this study indicate that ATP measurement using bioluminescence is a suitable tool to obtain rapid, reproducible and sensitive information on the microbial load of water samples, and is suitable to use in the field. However, to use ATP measurement as an indicator of water quality, criteria of assessment has to be discussed.

Determination of inflammatory responses to Aspergillus versicolor and endotoxin with human cryo-preserved blood as a suitable tool.

The aim of this study was to characterize the mediator release in the whole blood assay induced by stimulation with an extract of the indoor mold A. versicolor. In addition, the effect of concomitant stimulation with E. coli endotoxin and A. versicolor, and the involvement of the relevant Toll-like receptors were investigated. The stimulation of cryo-preserved whole blood with 400pg/ml E. coli endotoxin induced a significant IL-1ß release (976±133pg/ml), whereas, none of the tested A. versicolor concentrations (10-1000mg/ml) caused IL-1ß release. However, stimulation with A. versicolor resulted in a maximum IL-8 release of 6200pg/ml. The simultaneous stimulation with 5pg/ml E. coli endotoxin and A. versicolor induced a synergistic effect on both IL-1ß and IL-8 release. Addition of anti-TLR-4 reduced the release of IL-1ß and IL-8 up to 56% and 43%, respectively. In summary, the whole blood assay is useful to characterize the inflammatory potential of A. versicolor, especially when the release of the pro-inflammatory chemokine IL-8 is included. Additionally, it is also possible to study the influence of cell-surface receptors of the innate immunity involved in the effects of specific microbial stimuli.

Standardization of whole blood assay for determination of pyrogenic activity in organic dust samples.

To characterize bioaerosol exposure at workplaces standardized methods are necessary. Activity of endotoxin, one component of organic dust, can be quantified with the Limulus-Amoebocyte Lysat test (LAL test). Further information with respect to pyrogenic activity of the organic dust can be achieved by measuring cytokine release of human blood after stimulation with the dust or its extract (whole blood assay). The aim of our study was the standardization of the whole blood assay (WBA) while using cryo-preserved human blood (Qualis Laboratorium) and to compare the outcome of the different cytokines determined by incubation of the blood cells with extracts from dust samples collected at various workplaces. Cytokine release (IL-1 beta, IL-6, IL-8, TNF-alpha, MCP-1) was measured by ELISA after stimulation of fresh blood from ten donors as well as cryo-preserved human blood. In both cases blood was stimulated with E. coli endotoxin as well as with 30 dust filter extracts from various workplaces. All dust filter extracts were investigated in the WBA using cryo-preserved blood as well as with LAL test. E. coli endotoxin stimulated the release of IL-1 beta, IL-6, IL-8, TNF-alpha and MCP-1 in a dose-dependent manner in fresh as well as cryo-preserved human whole blood. 200 pg/ml E. coli endotoxin induced maximal cytokine release in cryo-preserved blood (mean value for IL-1 beta 2509+/-418 pg/ml; n=13 experiments) whereas fresh blood of single donors reached a maximum release when stimulated with 50 ng/ml endotoxin (mean value of ten donors 1125+/-553 pg/ml IL-1beta). Using cryo-preserved blood the coefficient of variation (CV) regarding the interassay variability was below 21% for all cytokines measured. Regarding 26 dust sample extracts correlation coefficient r2 for LAL test and WBA was between 0.90 and 0.93 (Pearson) for IL-1 beta, IL-6, IL-8 and TNF-alpha whereas correlation for MCP-1 was lower (r(2)=0.59). Two dust sample extracts which showed similar reactivity patterns in LAL test as well as in WBA with respect to IL-1 beta, IL-6, IL-8 and TNF-alpha could be differentiated by measuring MCP-1. In conclusion, cryo-preserved blood pools are suitable to standardize WBA. Combination of different outcome variables like IL-1 beta and MCP-1 improve the characterization from the inflammatory potency of workplace related dust samples.