Implementation of non-invasive methods in the diagnosis of diisocyanate-induced asthma.

Diisocyanate-induced asthma is difficult to diagnose since the immunopathological mechanisms and exposure determinants at the workplace are not well defined. The aim of this study was to evaluate the non-invasive methods of nasal lavage fluid (NALF) and induced sputum (IS) to enhance the diagnostic efficiency. Sixty-three diisocyanate-exposed workers with work-related shortness of breath underwent a standardized 4-steps-1-day-whole body exposure test with diisocyanates used at work up to 30 ppb. NALF and IS were collected before, 0.5, and 19 h after the end of exposure. Cellular composition and soluble inflammatory biomarkers were studied in the samples. In addition, ten controls with bronchial hyperresponsiveness, but without prior occupational diisocyanate exposure, were also examined. Twelve out of the 63 subjects (19 %) showed a significant asthmatic reaction (pulmonary responders) after challenge (FEV1 decrease >20 %). NALF samples did not demonstrate significant effects either on cellular composition or on mediator concentrations in the responders, non-responders, or controls at any time point. In contrast, in the IS samples of the pulmonary responders collected 19 h after challenge, the percentage of eosinophils was higher (p = 0.001) compared with baseline before challenge. Eosinophils were also increased 30 min and 19 h after challenge in IS samples of the responders compared with the non-responders or controls. In addition, 19 h after challenge the eosinophilic cationic protein (ECP) concentration was significantly higher in the responders than non-responders (p < 0.04) or controls (p < 0.002). In conclusion, positive asthmatic reactions to diisocyanates are accompanied by an influx of eosinophils into lower airways. Analysis of induced sputum should be implemented in the diagnostic procedure of diisocyanate-related airway diseases.

Comparison of different non-invasive methods for detection of allergic asthma.

Non-invasive methods to assess inflammation of lower airways are induced sputum (IS), exhaled nitric oxide (eNO), and exhaled breath condensate (EBC). Here we focused on the assessment of airway inflammation with a panel of non-invasive methods in health care workers (HCWs) with suspected latex allergy with and without current allergic respiratory symptoms about 10 years after the latex ban in German health care facilities. Seventy-seven non-smoking subjects were examined by skin prick test and specific IgE measurements, eNO, IS, and EBC. Sensitivity, specificity, and positive and negative predicted values for relevant biomarkers were calculated using current asthma symptoms as the gold standard. Twenty-nine subjects (38%) reported ongoing asthmatic symptoms (AS). In these subjects the EBC concentrations of nitrogen oxides (NO(x); p=0.027) and leukotriene B(4) (p=0.025) were significantly higher than in subjects without AS. In addition, in the subjects with AS the numbers of eosinophils (p=0.015) and the concentrations of IL-5 (p= 0.021) in IS samples were significantly higher than in the subjects without AS. A good correlation between several inflammatory markers in IS was detected. The maximum Youden Index was reached for IS total eosinophils ≥3.5·10(4) with a test efficiency of 0.72. In conclusion, non-invasive inflammatory monitoring with EBC and IS may assist the diagnosis of allergic asthma. Self-reported current asthmatic symptoms were reflected by eosinophilic inflammation and the best parameter to support the asthma diagnosis is a total number of eosinophils ≥3.5·10(4) in IS.

Pulmonary lesions and serum levels of soluble Fas (sCD95) in former hard coal miners.

OBJECTIVE: Fas/APO-1 (CD95) and Fas Ligand (FasL) is a major mediator system that activates programmed cell death (apoptosis) and is most important for pulmonary cellular homeostasis. Another form of Fas, circulating soluble Fas (sCD95), produced by alternative mRNA splicing antagonizes the cell-surface Fas function. It was the aim of the study to test the hypothesis that the Fas/FasL system is implicated in the development of silica-induced pulmonary nodular lesions. MATERIAL AND METHODS: We investigated the serum levels of sCD95 in 55 former hard coal miners. Coal workers' pneumoconiosis (CWP) was assumed when the profusion of small round opacities according to the ILO 2000 classification system was 1/1 or greater. Analyses of sCD95 were performed by a sandwich ELISA. RESULTS: Radiologic CWP was found in 34 of the 55 individuals. The age of subjects with and without CWP was similar (73.5 (SD 7.2) years vs. 73.5 (7.1) years; P = 0.924). sCD95 could be quantified in all samples; significantly higher levels were observed in subjects with radiologic signs of CWP (914 (752-1251) pg/ml vs. 632 (509-804) pg/ml, P < 0.001). However, there was no relationship between sCD95 serum concentrations and the quantity of profusion according to ILO. CONCLUSIONS: The hypothesis of elevated sCD95 concentrations in CWP was corroborated. The usefulness of sCD95 for prevention and diagnosis of CWP and other forms of silica-induced fibrosis needs to be established by epidemiological studies.

Detection of DNA strand breaks by comet assay in sputum leucocytes of bitumen-exposed workers: a pilot study.

DNA strand breaks were determined in leucocytes of induced sputum (IS) and compared with DNA strand breaks in blood lymphocytes from 42 bitumen-exposed workers pre and post shift. Comet assay results were expressed in arbitrary units based on visual scoring (sputum leucocytes) and Olive tail moment (OTM, blood lymphocytes). DNA damage in IS leucocytes was overall high but did not change during shift. Level of DNA strand breaks in IS samples correlated with total cell count and neutrophil content (Spearman rank correlation coefficient r(s) = 0.47, p = 0.001, r(s)= 0.48, p = 0.001, respectively) and with IL-8 concentration before and after shift (r(s) = 0.31, P = 0.048, and r(s) = 0.43, P = 0.005). DNA damage in IS was not associated with DNA strand breaks in blood lymphocytes (r(s) = -0.04, p = 0.802 before shift, r(s) = 0.27, p = 0.088 after shift). A higher level of DNA strand breaks was measured in blood lymphocytes before shift (median OTM 1.7 before and 1.3 after shift, p = 0.023). A strong correlation was found between the number of neutrophils and IL-8 concentration in IS before and after shift (r(s) = 0.77 and r(s)= 0.75, p < 0.001). This study showed an association between genotoxic and inflammatory effects in the lower airways and compared simultaneously DNA strand breaks in IS and blood of bitumen-exposed workers.

Development of a monoclonal antibody-based sandwich ELISA for detection of the latex allergen Hev b 1.

BACKGROUND: Natural rubber latex (NRL) products are complex mixtures consisting of different allergenic components. Among them, Hev b 1 belongs to the important and well-characterized ones. To quantify the relevant allergen Hev b 1 in NRL products, a two-site monoclonal antibody (mAb)-based assay was developed. METHODS: Two Hev b 1-specific mAbs with different epitope recognition and ability to bind simultaneously to an Hev b 1 molecule were used in the study. Both mAbs (II4F9 and II4G9) were enriched by in vitro production in a modular minifermenter and affinity purified. Wells of micro-ELISA plates coated with captured mAb II4G9 were incubated with samples containing Hev b 1. Bound Hev b 1 was detected by a combination of biotinylated mAb II4F9 as detection antibody and peroxidase-labeled avidin. RESULTS: The optimized sandwich ELISA was highly reproducible in the linear range of the standard curve and Hev b 1 concentrations ranging from 12.5 to 400 ng/100 microl could be detected. The assay was suitable for the detection of Hev b 1 concentrations in latex sap and latex products, e.g. gloves, with a detection limit of 1.25 microg of Hev b 1/g of rubber. In a preliminary study with five different brands of latex gloves, Hev b 1 concentrations were found to be in the range of 18-40 microg per gram of rubber material, corresponding to 2-4% of the total extractable protein content in latex glove extracts. CONCLUSIONS: A sensitive sandwich assay was developed to quantify the latex allergen Hev b 1. This assay can be used to standardize latex extracts with regard to the content of the major allergen Hev b 1.

Development of a two-site enzyme-linked immunosorbent assay for alpha-amylase from Aspergillus oryzae based on monoclonal antibodies.

A two-site monoclonal antibody ELISA was developed to quantify the allergen Asp o 2 (alpha-amylase from Aspergillus oryzae). Two mAbs recognizing distinct epitopes were selected, enriched by in vitro production in a modular minifermenter and affinity-purified. The first antibody was bound to microtiter plates which were then incubated with samples containing the allergen. Bound allergen was detected using a biotinylated second antibody and peroxidase-polymer-labelled streptavidin. The assay had a sensitivity of 0.6 ng/ml and did not react to high concentrations of wheat and rye flour or yeast proteins. The mAb ELISA will be useful in individual or epidemiological studies of baker's asthma to assess workplace allergen concentrations and the efficacy of allergen exposure prevention. It can be used as a standard assay for the quantification of alpha-amylase and the establishment and control of threshold limits in European bakeries.