After calculations, the number of eggs eliminated by each infected mouse was expressed as eggs/g of faeces. Given the fact that S. venezuelensis filiform larvae develop only into female worms in the small intestine of the host, fecundity rate was estimated by dividing number of eggs per number of worms recovered from the intestine of each animal. The eosinophil peroxidase (EPO) assay was used to measure eosinophil activity in the skin and lung as previously described by Strath et al.(28) and modified by Silveira et al.(16).
Briefly, 100 mg of tissue (skin or lung) was homogenized in 1·9 mL of PBS using a tissue homogenizer (Power Gen 125; Fisher Scientific, Pittsburgh, PA, USA). The homogenate was centrifuged (3000g for 10 min), red blood cells in the pellet underwent hypotonic lysis (1·5 mL of 0·2% NaCl) and the molarity was restored
with 1·5 mL of 1·6% NaCl solution containing 5% glucose. After a further centrifugation MLN0128 (3000g selleck for 10 min), the pellet was resuspended in PBS (pH 7·4) containing 0·5% hexadecyltrimethylammonium bromide (PBS-HTAB). The cell solution was homogenized again and the homogenates were then freeze-thawed three times in liquid nitrogen, centrifuged for 15 min at 3000 g and the supernatant was used to measure EPO activity. For this assay, 75 μL of each experimental sample obtained from different tissues was incubated with 75 μL of substrate [1·5 mm o-phenylenediamine (OPD) in 0·075 mm Tris–HCl buffer, pH 8·0, containing 6·6 mm
of hydrogen peroxide] for 30 min at room temperature (RT) in the dark. Reaction was stopped by adding 50 μL of 1 m H2SO4. Reaction intensity was read at 492 nm on a micro-plate reader (Emax; Molecular Devices, Sunnyvale, CA, USA) and results are shown as absorbance units. The extent of neutrophil activity was indirectly estimated by myeloperoxidase (MPO) assay as previously described by Ivey et al. (29) and modified by Matos et al. (30). Tissue samples (100 mg of skin else and lung) were homogenized in extraction buffer (0·1 m NaCl, 0·02 m NaPO4, 0·015 m NaEDTA; pH 4·7) and the pellet underwent hypotonic/hypertonic lysis as described in EPO assay. After further centrifugation (3000g for 10 min), the pellet was resuspended and rehomogenized in 0·05 m NaPO4 buffer, pH 5·4, containing 0·5% HTAB, followed by three freeze-thaw cycles using liquid nitrogen. The resulting solution was centrifuged for 15 min at 10 000g and the supernatant was used for the colorimetric assay. For this, 75 μL of supernatant was incubated with 75 μL of substrate (1·6 mm tetramethylbenzidine and 0·5 mm H2O2 in 0·05 m NaPO4 buffer, pH 5·4) for 30 min at room temperature in the dark. Reaction was stopped by adding 50 μL of 1 m H2SO4. Myeloperoxidase activity present in the sample was measured at 450 nm on a micro-plate reader (Emax; Molecular Devices).