The polyethylene tube was connected to a syringe

The see more polyethylene tube was connected to a syringe find more containing 4% buffered paraformaldehyde, and the lungs were inflated in situ with the fixative to normal size. After 5 minutes the lungs were removed in toto

and further fixated for at least 24 hours. Tissues were embedded in paraffin in a standardized way (horizontal cut through the hilum regions) and subsequently 7 μm thick slices were cut and stained with haematoxylin/periodic acid Schiff (PAS). The degree of inflammation and morphological changes in the lungs were evaluated blindly by microscopy by two experienced researchers and revaluated in case of discrepancy as described previously [24]. Statistics The numbers of inflammatory cells in biopesticide-exposed mice were compared to the control group by means of non-parametric analysis of variance (Kruskall-Wallis). In case of significant difference in the Kruskall-Wallis test, pair-wise comparisons between the water control group and the biopesticide-exposed animals were further analysed using the Mann-Whitney’s U-test. Statistical significant

difference was accepted at p < 0.05. Results Validation of actual deposited dose after inhalation Comparing the theoretically inhaled dose of Vectobac® (3.5 × 104 CFU) and actual deposited dose (2.9 × 104 CFU) revealed that 83% of the theoretically inhaled dose was deposited. For the 10 × higher concentration, the mean theoretically inhaled dose was 5.6 × 105 CFU and actual deposited dose was 5.1 × 105 CFU, i.e. 91% was deposited.

The particle counts from APS and LHPC particle counters were stable throughout the exposure (Figure 1). Figure 1 Aerosol characteristics and validation of actual deposited dose (ADD) per mouse. Particles (counts min-1) of the Vectobac® × 10 exposure aerosol were measured by APS (n= 21) and LHPC (n = 24) for different particle sizes. The theoretically Sitaxentan inhaled dose (TID) per mouse based on CFU measurements from a GSP filter sampler were compared to the ADD per mouse (n = 5 per group) for the two different exposure concentrations. Values are means with SEM. CFU recovery from BAL fluid and from total lung homogenate Comparison of the CFU present in total lung homogenate to the CFU recovered from BAL fluid revealed that an average of 13% (range 10-20%) of the total CFU was recovered by the BAL procedure. The remaining 80-90% of the CFUs were recovered from the lung homogenate of the flushed lungs. Acute inflammatory response to biopesticide instillation A clear dose-dependent increase in number of neutrophils was apparent 24 hours post i.t. instillation of the biopesticide Vectobac®. Statistically significant increased numbers of neutrophils were seen after instillation of 2 × 105 CFU or more. Furthermore, at the 1.2 × 106 CFU Vectobac®dose a significant increased number of lymphocytes and eosinophils were seen (Figure 2). Figure 2 Cells in BAL fluid after instillation of different doses of biopesticide.

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