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The measurement of airborne endotoxins is thus far not standardized. Earlier studies reported higher endotoxin yields when Tween 20 was added to the media used for filter extraction and in the Limulus amebocyte lysate (LAL) assay. This study compared four common media and assessed the effects of Tween during extraction and analysis separately. Parallel airborne dust samples from five work environments (n = 250) were used to compare the four media (pyrogen-free water [PFW], PFW-Tween 20, PFW-Tris, and PFW-triethylamine-phosphate [TAP]) and an extraction time of 10 or 60 min. A subset of the extracts in PFW or PFW-Tween (n = 40) were analyzed in parallel LAL assays with PFW or PFW-Tween as the assay medium. The results produced by a shorter extraction time or the presence of Tris were similar to the results for the reference procedure (PFW and 60 min of shaking). The use of PFW-TAP showed overall lower yields and a deviant calibration curve. The presence of Tween in the extraction medium resulted in significantly (P <0.05) higher endotoxin yields from all dust types, independent of the effect of Tween in the assay. Tween in the LAL assay, however, also strongly inhibited the reactivity of the lipopolysaccharide (LPS) standard, thus shifting the calibration curve to higher values. The inhibition of LPS in test samples was less pronounced and varied between dust sources, resulting in enhanced calculated concentrations. This assay effect could be circumvented by diluting extracts at least 50-fold before the LAL assay. In conclusion, of the media tested, only Tween enhances the efficiency of endotoxin extraction from airborne dust samples in a consistent manner. We recommend extraction in PFW-Tween combined with dilution and LAL analysis in PFW.

BACKGROUND: Several studies have consistently reported inverse associations between exposure to endotoxin in house dust and atopy. With regard to the association between house dust endotoxin and asthma, the results are inconsistent. OBJECTIVES: To study the association between house dust endotoxin levels and respiratory symptoms and atopy in populations from largely different countries. METHODS: Data were collected within the International Study on Asthma and Allergies in Childhood Phase Two, a multi-centre cross-sectional study of 840 children aged 9-12 years from six centres in the five countries of Albania, Italy, New Zealand, Sweden and the United Kingdom. Living room floor dust was collected and analysed for endotoxin. Health end-points and demographics were assessed by standardized questionnaires. Atopy was assessed by measurements of allergen-specific IgE against a panel of inhalant allergens. Associations between house dust endotoxin and health outcomes were analysed by logistic regression. Odds ratios (ORs) were presented for an overall interquartile range increase in exposure. RESULTS: Many associations between house dust endotoxin in living room floor dust and health outcomes varied between countries. Combined across countries, endotoxin levels were inversely associated with asthma ever [adjusted OR (95% confidence interval (CI)) 0.53 (0.29-0.96) for endotoxin levels per m(2) of living room floor] and current wheeze [adjusted OR (95% CI) 0.77 (0.64-0.93) for endotoxin levels per gram of living room floor dust]. There were inverse associations between endotoxin concentrations and atopy, which were statistically significant in unadjusted analyses, but not after adjustment for gender, parental allergies, cat and house dust mite allergens. No associations were found with dust quantity and between endotoxin exposure and hayfever. CONCLUSION: These findings suggest an inverse association between endotoxin levels in living room floor dust and asthma in children.

Neuropeptide S receptor 1 (NPSR1, GPRA 154, GPRA) has been verified as a susceptibility gene for asthma and related phenotypes. The ligand for NPSR1, Neuropeptide S (NPS), activates signalling through NPSR1 and microarray analysis has identified Tenascin C (TNC) as a target gene of NPS-NPSR1 signalling. TNC has previously been implicated as a risk gene for asthma. We aimed therefore to study the genetic association of TNC in asthma- and allergy-related disorders as well as the biological and genetic interactions between NPSR1 and TNC. Regulation of TNC was investigated using NPS stimulated NPSR1 transfected cells. We genotyped 12 TNC SNPs in the cross-sectional PARSIFAL study (3113 children) and performed single SNP association, haplotype association and TNC and NPSR1 gene-gene interaction analyses. Our experimental results show NPS-dependent upregulation of TNC-mRNA. The genotyping results indicate single SNP and haplotype associations for several SNPs in TNC with the most significant association to rhinoconjunctivitis for a haplotype, with a frequency of 29% in cases (P = 0.0005). In asthma and atopic sensitization significant gene-gene interactions were found between TNC and NPSR1 SNPs, indicating that depending on the NPSR1 genotype, TNC can be associated with either an increased or a decreased risk of disease. We conclude that variations in TNC modifies, not only risk for asthma, but also for rhinoconjunctivitis. Furthermore, we show epistasis based on both a direct suggested regulatory effect and a genetic interaction between NPSR1 and TNC. These results suggest merging of previously independent pathways of importance in the development of asthma- and allergy-related traits.

BACKGROUND: Occupational allergen exposure assessment usually requires airborne dust sampling at the worksite followed by dust extraction and enzyme immunoassay (EIA) analysis at the laboratory. Use of semiquantitative lateral flow immunoassays (LFIAs) may allow a more rapid detection procedure with direct on-site demonstration of a bioallergen exposure hazard. OBJECTIVE: In a field study, we evaluated a recently developed LFIA for fungal alpha-amylase, an important bakery allergen. METHODS: Airborne and surface dust (wipe) samples and samples from flours and baking additives used at the workplace were collected in 5 industrial bakeries and tested in the LFIA for fungal amylase. For comparison, amylase was measured in sample eluates with the reference EIA method. RESULTS: Sensitivity of the LFIA was 1 to 10 ng/mL, and of EIA, approximately 25 pg/mL. In LFIA, most flour samples, 84% of wipe samples, 26% of personal airborne dust, and none of the 26 ambient air dust samples produced a visible reaction. Wipe samples from dough-making areas and flour samples gave the strongest reactions. All extracts with >5 ng allergen per milliliter showed a positive LFIA reaction. CONCLUSION: The LFIA for fungal amylase is an easy and rapid method to demonstrate the allergen directly at the worksite in less than 10 to 20 minutes. Similar LFIA methods may be used for other occupational allergens in other work environments. CLINICAL IMPLICATIONS: Lateral flow immunoassays for occupational allergens may be of great value in occupational hygiene surveys to demonstrate directly to workers and supervisors the hazards of work-related bioallergen exposure.

AIMS: Episodes of serious work related health problems resembling organic dust toxic syndrome (ODTS) in workers of a grass seed quality inspection laboratory prompted the authors to study personal endotoxin exposure levels in this facility and in the agricultural seed processing industry. In addition, microbial and inflammatory characteristics of agricultural seeds were studied. METHODS: The authors assessed inhalable dust and endotoxin levels in 101 samples from 57 workers in grass, cereal, and vegetable seed plants who were handling mainly grass seeds as bulk product, and horticulture seeds in smaller quantities. Additionally, real-time dust exposure was measured using a DataRAM monitor in 12 grass seed workers to obtain more information on exposure patterns during specific tasks. Endotoxin concentrations in seed extracts were determined by LAL assay and seed samples were analysed by scanning electron microscopy. Release of inflammatory cytokines was measured in supernatants of whole blood samples stimulated with lipopolysaccharide (LPS) or agricultural seed extracts in a human whole blood assay (WBA). RESULTS: Endotoxin concentrations in personal samples were high (geometric mean 1800 EU/m3), particularly in the grass seed quality inspection lab where endotoxin levels up to 274 000 EU/m3 were measured. The recommended health based endotoxin exposure limit of 50 EU/m3 was amply exceeded in almost all personal samples. Job tasks dumping and mixing were associated with highest dust and endotoxin exposures, which was confirmed by real-time measurements. Microbial infestation was found in almost all seed samples. WBA results showed that most seed extracts were capable of inducing a pronounced dose dependent cytokine release. CONCLUSIONS: Workers handling grass, cereal, or vegetable seeds are at risk of exposure to high levels of endotoxin containing seed dust. Occupational exposure to inhalable agricultural seed dust can induce inflammatory responses, and is a potenti

BACKGROUND: Exposure to airborne wheat allergen is a well-known cause of bakers' allergy and asthma. Airborne wheat allergen can be measured by enzyme immunoassays (EIAs) in extracts of inhalable dust samples, but only limited knowledge is available on the size distribution of wheat allergen-carrying particles. Recently, a new sampling medium, porous polyurethane foam, has been introduced for the size-selective sampling of airborne dust in various occupational settings. We investigated the applicability of these foams for size-selective wheat allergen measurements. METHODS: Personal and stationary measurements were performed in a flour mill, using respirable and thoracic foams inserted into the conventional IOM inhalable sampler, together with PTFE (Teflon) filters. Foams and filters were eluted and wheat allergen levels determined by human IgG4 inhibition EIA. RESULTS: Wheat allergen levels could be determined in both filter and foam eluates. Inhalable dust levels from filters and foams ranged from 1.4 to 53 mg m(-3), and wheat allergen levels from 15 to 580 microg m(-3). The allergen was mainly borne on particles with D(ae) (particle aerodynamic diameter) > 10 microm and particles with 4 microm

Well-validated methods for measuring airborne occupational allergens are essential for effective control and reduction of allergen exposures. For wheat. our allergens, specific immunoassays are available, but there is a need for optimisation and standardization of sample processing procedures. Wheat. our allergen elution and storage were studied using airborne dust samples collected in bakeries with a new parallel sampler. Forty-eight series of 9 parallel filters were subjected to extraction procedures varying in elution medium, shaking method, extraction vial, and centrifugation speed. Wheat allergens were measured with enzyme immunoassays, and the effect of various procedures evaluated by mixed regression analyses. The stability of the eluted allergens was assessed after storage for 20 days and 4 months at -20 degrees C, in the presence or absence of casein in the medium. Only the type of elution medium had significant effects on allergen recovery: addition of Tween-20 resulted in 3- to 100-fold increased levels, an effect that was most pronounced at low concentrations. Allergen levels in extracts were stable for at least 4 months at -20 degrees C, irrespective of the presence of casein in the medium. Addition of Tween-20 to the elution medium is essential for optimal extraction of wheat allergen. The recommended procedure further includes the use of conventional polystyrene tubes, simple shaking methods, and centrifugation after extraction. Wheat dust extracts in PBS-Tween can be stored frozen for at least 4 months, and addition of a stabilising protein is not required.

BACKGROUND: Exposure to airborne wheat allergens in the bakery trade is associated with a high risk of occupational allergy and asthma. Control and reduction of allergen exposure require relatively simple but reliable monitoring techniques. We developed new rabbit IgG-based enzyme immunoassays (EIA) for wheat allergens, which might be a convenient alternative for the thus far used human IgG4 inhibition assay. METHODS: The reactivity and specificity of rabbit antibodies were assessed by EIA and immunoblotting, and compared with those of IgE from wheat-sensitized bakers, and with the antibodies used in the IgG4 inhibition EIA. An IgG inhibition and a sandwich EIA were developed for analysis of airborne dust samples. RESULTS: Human IgG4 and rabbit IgG inhibition EIAs had comparable sensitivities, with limits of detection (LOD) between 18 and 88 ng/mL, while the sandwich EIA was much more sensitive (LOD0.95) and their absolute values showed less than 10-20% differences. CONCLUSION: The rabbit IgG EIAs are valid substitutes for the human IgG4 inhibition EIA, with important practical advantages. The inhibition EIA is recommended for routine wheat allergen measurements. The sandwich EIA may be used to measure low allergen levels, as in short task-related exposure measurements or in subfractions of airborne dust samples.

Background and objectives We have previously reported that high rat urinary allergen (RUA) exposure was not associated with increased risk of rat allergy in long‐term‐exposed laboratory animal (LA) workers. We aimed to assess whether strong allergen‐specific IgG4 responses could explain the absence of a dose response in these subjects. We investigated whether IgG4 was associated with allergen exposure and prevalence of sensitization or respiratory symptoms to rats. The longitudinal relation between IgG4 and rat allergy was studied using data obtained during 2 years of follow‐up. Methods Five hundred and twenty‐nine LA workers answered a questionnaire on respiratory symptoms and occupational history and participated in skin prick testing. Blood samples were analysed for specific IgG4 and IgE to RUA. Exposure to RUA was estimated based on personal air samples. The relation between IgG4 and newly occurring sensitization or rat allergy was studied in workers who were not sensitized or did not report respiratory symptoms to rats. Results IgG4 titres were higher in atopic than in non‐atopic subjects, and increased with higher allergen exposure. Titres were highest in subjects who were sensitized and reported respiratory symptoms to rats when compared with those who were not (geometric mean [geometric standard deviation]=202 [5.7] vs. 8.4 [18.3] AU). The association between IgG4 and sensitization or symptomatic rat allergy was independent of estimated allergen exposure. IgG4 was a strong predictor of newly occurring sensitization and symptomatic rat allergy during follow‐up in atopic and rat‐sensitized subjects. Conclusion High exposure to RUA is associated with a strong allergen‐specific IgG4 antibody response. High anti‐RUA IgG4 is a strong predictor of prevalent and incident sensitization and symptomatic rat allergy in atopic and rat‐sensitized subjects. IgG4 can therefore not explain the absence of a dose response between allergen exposure an