Exposure levels of animal allergens, endotoxin, and ß-(1,3)-glucan on a university campus of veterinary medicine.

OBJECTIVES: The study aimed to determine the allergen, endotoxin and ß-(1,3)-glucan concentrations at various areas on a university campus of veterinary medicine. METHODS: Dust samples were collected four times a year for three years using electrostatic dust collectors (EDC) at 25 different locations on a campus of veterinary medicine and in laboratories of inorganic chemistry as a control area representing animal-free environment. Major animal allergens from dog, cat, horse, cattle and mouse, domestic mite (DM) allergens, and ß-(1,3)-glucan were measured using enzyme immunoassays and endotoxin using the limulus amoebocyte lysate (LAL) assay. Seasonal, annual and local influences on exposure levels were analyzed using Bayesian mixed models. RESULTS: With the exception of mouse allergens, all other determinants were found in almost all locations on the campus and in the control area, but in up to 10.000-fold variable concentrations. By far the highest levels of feline, canine, equine and bovine allergens were detected in buildings where the respective species were examined. The highest levels of mouse and DM allergens, ß-(1,3)-glucan and endotoxin occurred together and were associated with locations where large animals were present. In buildings without animals, allergen levels were considerably lower but still elevated at several locations compared to the control area, especially for dog and horse allergens, and ß-(1,3)-glucan. Significant seasonal effects were observed for dog, cat, horse and DM allergens, and ß-(1,3)-glucan. Variations between years were less apparent than between seasons (except for ß-(1,3)-glucan). CONCLUSIONS: The strongest influencing factor on the concentration of mammalian allergens was the presence of the corresponding animal at the collection site. Seasonal influence on allergen concentrations was observed, while the overall exposure remained constant over the years. At locations with horses, elevated levels of mite allergens, endotoxin, and ß-(1,3)-glucan can be expected, probably due to passive transfer from stable environment.

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.

What should be tested in patients with suspected mold exposure? Usefulness of serological markers for the diagnosis.

The associations of mold exposure, IgE-mediated sensitization, inflammatory markers, and respiratory symptoms were analyzed in 46 exposed and 23 non-exposed individuals. Both exposure and clinical symptoms were assessed by questionnaire. Specific (s)IgE to mold mixture (mx1) was significantly higher and found more frequently in exposed (41%) than non-exposed individuals (17%), which was not observed for sIgG to mold mix (Gmx6). Notably, exposed asthmatics were more frequently sensitized to molds (55%) compared to exposed non-asthmatics (18%). In addition, the serum concentrations of club cell protein (CC16) were significantly lower in exposed subjects, especially in asthmatics. Positive associations were observed among mold sensitization, asthma, and mold exposure, but not in subjects with predominantly environmental sensitizations without mold sensitization. Thus, sIgE to mx1 but not sIgG to Gmx6 is a useful diagnostic marker to verify mold-associated respiratory symptoms.

Comparing the concentration levels of allergens and endotoxins in employees' homes and offices.

OBJECTIVE: The aim of the study was to find out whether allergen and endotoxin concentrations in offices differ from those measured at the homes of employees, and identify the parameters that influence exposure. METHODS: Electrostatic dust collectors (EDCs) were placed in five office buildings (68 rooms, 436 EDCs), as well as the homes of the office workers (145 rooms, 405 EDCs) for 14 days, four times a year. In addition, surface samples were collected from the offices four times a year by vacuuming the carpeted floors. Domestic mite (DM), and the major cat and dog allergens (Fel d 1 and Can f 1) were quantified in all samples using fluorescence enzyme immunoassays. Endotoxin was measured in the EDC samples, using the Limulus amoebocyte lysate assay. The allergen and endotoxin concentrations were log transformed and analysed with multilevel models. RESULTS: Endotoxin concentrations were significantly higher in personal homes compared to levels measured in the offices, and depended on the number of persons living in each household, as well as the presence of a dog. DM allergens were significantly higher in households than in offices, and were significantly higher in bedrooms compared to living rooms. Offices occupied by cat owners had significantly higher Fel d 1 concentrations than offices or homes without. Additionally, Can f 1 concentrations were significantly higher in offices occupied by dog owners compared to those without. CONCLUSIONS: Pet owners appear to transfer cat and dog allergens to their offices. Therefore, in case of allergy complaints at the office, employers and physicians might consider possible contamination by cat and dog allergens.

Animal Allergens, Endotoxin, and ß-(1,3)-Glucan in Small Animal Practices: Exposure Levels at Work and in Homes of Veterinary Staff.

OBJECTIVES: In veterinary settings, high exposures to animal allergens and microbial agents can be expected. However, occupational exposure levels are largely unknown. The objective of this study was to estimate the allergen, endotoxin, and ß-(1,3)-glucan concentrations in small animal practices and in the homes of practice employees. METHODS: Dust samples were collected using electrostatic dust fall collectors in diverse rooms of 36 small animal practices, as well as in employees' homes. Major animal allergens (Fel d 1, Can f 1, Ory c 3, Cav p 1, Equ c 1, Bos d 2), domestic mite (DM) allergens, and ß-(1,3)-glucan levels were measured using enzyme immunoassays. Endotoxin was determined using the Limulus amoebocyte lysate assay. Influences on exposure levels were analyzed using multilevel models. RESULTS: The levels of Can f 1, Fel d 1, Ory c 3, and Cav p 1 were up to 30 times higher in practices compared with homes without animals, but significantly lower compared with the homes with the respective pet. Although horses were not treated in the practices, Equ c 1 was found in 87.5% of samples, with the highest concentrations measured in changing rooms. DM levels were significantly lower in practices than in all private homes, and endotoxin levels were similar to those in homes with pets. In the practice itself, exposure levels were significantly influenced by animal presence, type of the room, and area per employee; whereas, room volume and diverse cleaning measures had mostly no effect. CONCLUSIONS: Exposure to animal allergens is high in veterinary practices, but it does not reach levels of households with pets. Domestic mite allergen and endotoxin exposure seem to be low for workers in veterinary practices. The high Equ c 1 detection rate strongly indicates dispersal of allergens, most likely through clothing and hair.

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.

Analysis of inflammatory markers and metals in nasal lavage fluid of welders.

Welding fumes may produce adverse health effects in the respiratory tract. To assess the relationship between exposure to welding fumes and inflammation in the upper airways, 190 male welders were examined from the WELDOX study (median age 40 yr, 54.7% smokers, and 32.9% atopics). Inhalable welding fumes were collected in the breathing zone of welders during a single shift. Chromium (Cr), nickel (Ni), manganese (Mn), and iron (Fe) were measured in the welding-fume samples and in postshift nasal lavage fluid (NALF). In addition, the numbers of particles and inflammatory biomarkers, including total and differential cell counts, interleukin (IL)-8, leukotriene (LT) B4, 8-isoprostane (8-iso-PGF2α), tissue inhibitor of metalloproteinase-1 (TIMP-1), and immunoreactive matrix metalloproteinase (MMP)-9, were determined. Metal concentrations in NALF correlated with airborne concentrations. No significant association was found between airborne metal concentrations and biomarkers of inflammation in NALF, whereas increasing metal concentrations in NALF resulted in increased concentrations of total protein, IL-8, MMP-9, and TIMP-1. LTB4 and 8-iso PGF2α were elevated at higher concentrations of Cr or Ni in NALF. The same was true for Fe, although the effects were less pronounced and of borderline significance. In conclusion, our results showed a significant association between the concentrations of metals and soluble inflammatory markers in the NALF of welders. The noninvasive collection of NALF is applicable in field studies, where it may serve as a suitable matrix to simultaneously assess biomarkers of exposure and effect in the upper respiratory tract in workers who are occupationally exposed to airborne hazardous substances.

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.

Concentration of bioaerosols in composting plants using different quantification methods.

BACKGROUND: Bioaerosols (organic dusts) containing viable and non-viable microorganisms and their metabolic products can lead to adverse health effects in exposed workers. Standard quantification methods of airborne microorganisms are mainly based on cultivation, which often underestimates the microbial burden. The aim of the study was to determine the microbial load in German composting plants with different, mainly cultivation-independent, methods. Second purpose was to evaluate which working areas are associated with higher or lower bioaerosol concentrations. METHODS: A total of 124 inhalable dust samples were collected at different workplaces in 31 composting plants. Besides the determination of inhalable dust, particles, and total cell numbers, antigen quantification for moulds (Aspergillus fumigatus, Aspergillus versicolor, Penicillium chrysogenum, and Cladosporium spp.) and mites was performed. Concentrations of ß-glucans as well as endotoxin and pyrogenic activities were also measured. The number of colony forming units (cfu) was determined by cultivation of moulds and actinomycetes in 36 additional dust samples. RESULTS: With the exception of particle numbers, concentrations of all determined parameters showed significant correlations (P < 0.0001; r Spearman: 0.40-0.80), indicating a close association between these exposure markers. Colony numbers of mesophilic moulds and actinomycetes correlated also significantly with data of cultivation-independent methods. Exposure levels showed generally large variations. However, all parameters were measured highest in dusty working areas like next to the shredder and during processing with the exception of Cladosporium antigens that were found in the highest concentrations in the delivery area. The lowest concentrations of dust, particles, antigens, and pyrogenic activity were determined in wheel loader cabins (WLCs), which were equipped with an air filtration system. CONCLUSION: It was possible to assess the microbial load of air in composting plants with different quantification methods. Since allergic and toxic reactions may be also caused by nonliving microorganisms, cultivation-independent methods may provide additional information about bioaerosol composition. In general, air filtration reduced the bioaerosol exposure shown in WLCs. Due to the fact that the mechanical processing of compost material, e.g. by shredding or sieving is associated with the generation of high bioaerosol concentrations, there is still a need of improved risk assessment and state-of-the-art protective measures in composting plants.

Ex vivo cytokine release and pattern recognition receptor expression of subjects exposed to dampness: pilot study to assess the outcome of mould exposure to the innate immune system.

In rooms with moisture damage, the indoor air can be enriched with microorganisms causing a variety of symptoms. Due to the highly diverse composition of bioaerosols and the multiple effects on humans, an assessment of the health risk is not sufficiently possible. The aim of this study was to characterize the features of innate immunity using blood from subjects exposed to moisture damage compared to control subjects living in houses without visible moisture damage. We investigated the expression of TLR-2, TLR-4 and dectin-1 on the surface of monocytes from both fresh blood and after in vitro stimulation with the model substances E. coli endotoxin, zymosan A, Pam3Cys and Aspergillus versicolor in 25 exposed subjects and 25 control subjects. In vitro stimulation of whole blood with the same components was performed for 20 h and the release of inflammatory mediators IL-8 and IL-1ß were quantified. In addition to an enhanced number of blood leucocytes, the expression of the receptors TLR-2, TLR-4 and dectin-1 on blood monocytes was significantly enhanced in exposed subjects. In contrast, no different alteration in expression was detected between exposed and control group after in vitro stimulation with the model substances. The release of IL-8 and IL-1ß after stimulation of whole blood with A. versicolor was increased in subjects exposed to moisture damage. Furthermore, in the exposed subjects the IL-1ß release was significantly enhanced after in vitro stimulation with E. coli endotoxin (1000 pg/mL). In conclusion, features of the innate immune system (receptor expression and mediator release of monocytes) are altered in subjects exposed to moisture damage which may be a potential explanation for the increased incidence of respiratory health diseases observed in these populations.

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.

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.

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.

Occupational endotoxin-exposure and possible health effects on humans.

BACKGROUND: Endotoxins are commonly found at workplaces where large amounts of bioaerosols are generated. In Germany, especially since the Ordinance on Safety and Health Protection related to work involving biological agents (Biostoff-Verordnung) became effective (1999), threshold limit values are widely discussed. Up to the present, endotoxin values are measured with non-uniform methods and therefore values are of limited benefit for classification of exposure groups. In Germany there is no threshold limit value for endotoxin. METHODS: Relevant literature of the last 20 years was selected from Medline and discussed. RESULTS: In this review we focused on the impact of endotoxin exposure on human health with special respect to the measurements on workplace and methodological aspects of endotoxin determination. Methods for sampling and endotoxin determination have to be validated, optimized, and standardized first. CONCLUSION: The adverse health effects of endotoxins are known, standardization of measurements is a necessary goal and protection measures should be established immediately.