No stomach or spleen tissues were sampled

at 7 days post challenge in single infection against G. strigosum or B. bronchiseptica, respectively; mesenteric lymph nodes were collected only during infections of the small intestine. In the dual bacteria–helminth infections we also collected the uninfected gastrointestinal organs, for example, in the B. bronchiseptica–G. strigosum infection the uninfected duodenum was sampled and cytokines estimated. Our previous work showed that 7 DPI is when we observe the strongest cytokine response during the course of these infections, thus we focused on this time point for our analysis [18], [19], [22], [25], [26] and [27] (unpublished data). In the current study we chose not to re-stimulate any organ but quantified cytokine gene learn more expression in organs preserved in situ. The lungs were blended for 10 s and 3 g of tissue is diluted into 30 ml of RNAlater (Ambion, TX, USA) for storage at −80 °C. The remaining lung tissue was transferred CAL-101 mouse to ice-cold PBS and bacterial counts obtained by plating 10-fold serial

dilutions of the homogenate on Bordet–Gengou blood agar plates supplemented with streptomycin (VWR Intl., West Chester, PA) [18]. The small intestine was divided into four equal sections, SI-1 to SI-4, from the duodenum to the ileum, respectively. Each section was further divided into four equal segments. Segments 1 and 3 from the SI-1 and SI-4 sections were transferred to PBS (pH 7.4) for T. retortaeformis

counts [22]. Five pieces of tissue (0.5 cm×0.5 cm) were collected from segments 2 and 4, transferred to 5 ml RNAlater and stored at −80 °C. The stomach was divided into two parts, fundus and antrum (i.e. Bay 11-7085 top and bottom). Each section was then divided longitudinally and the right sections with the food contents stored in PBS for G. strigosum counts. Five pieces of tissue were collected from the top and bottom of the left sections and stored in RNAlater at −80 °C for subsequent cytokine gene expression analyses [22]. The spleen and mesenteric lymph nodes were cut into 0.5 cm×0.5 cm sized pieces, transferred to 5 ml of RNAlater and stored at −80 °C. It should be noted that the titration of the whole lungs precluded the quantification of cytokine expression in regions of high and low infection intensity, which was scattered within the organ and not consistent among individuals, in contrast to the gastrointestinal sections. Indeed, histopathological analyses confirmed the patchy inflammation in the lungs (unpublished data). Except for the lung where the entire tissue was processed, 2 pieces of small intestine and stomach (approx. 0.