All primer assays used for qRT-PCR were obtained commercially from Qiagen. PCRs were performed as follows: 2min at 50��C to activate uracil-N-glycosylase (UNG); 95��C for 10min to deactivate UNG; and 40 cycles at 94��C for 15s, 60��C for 35s, and 72��C for 30s. Reaction specificity was checked by performing dissociation curves after PCR. mRNA levels were Belinostat normalized to 18S rRNA. The fold induction of mRNA expression upon infection with E. papillata was determined using the 2?����CT method [9].2.8. Statistical AnalysisA two-tailed Student t-test was used for statistical analysis.3. ResultsThe course of E. papillata infections in mice was recently characterized in detail [10, 11]. The number of excreted oocysts varied among the individual mice between 200,000 and 260,000 per gram feces (mean value, 235,000 �� 24,000).

Light microscopical inspection of hematoxylin-and-eosin-stained sections revealed that the epithelial cells of the jejunum were infected by E. papillata. Concomitantly, there occurred some histological changes which were semiquantified by applying the scoring according to Dommels et al. [7]. Histological analysis revealed that mice infected with sporulated oocysts of E. papillata suffered a moderate inflammatory injury in jejunum (Figure 1).Figure 1E. papillata-induced jejunum injury on day 5. (a) Noninfected jejunum with normal architecture of the absorptive epithelium and lamina propria. (b) Infected jejunum with some pathological changes in lamina propria and absorptive epithelia. Developmental …Infection of mice with E.

papillata induced a significant increase in the number of TUNEL-positive cells of the jejunal crypts compared to the noninfected controls (Figure 2). In addition, there was a significant reduction of the goblet cell numbers seen at the site of the E. papillata infection in the jejunum (Figure 3).Figure 2Changes in apoptotic cells in mouse jejunum infected with E. papillata. (a) Noninfected jejunum (b) Infected jejunum with increased number of TUNEL-positive cells in lamina propria. Bar = 25��m. (c) Infection-induced changes in apoptotic …Figure 3Changes in goblet cell numbers in mouse jejunum infected with E. papillata. (a) Noninfected jejunum with more goblet cells. (b) Non-infected treated mouse jejunum. Sections are stained with Alcian blue. Bar = 25��m. (c) Number of goblet …

Quantitative real-time PCR was used to detect changes in the mRNA levels of inflammatory and goblet cell associated genes in the intestine. Upon infection GSK-3 with E. papillata, there was a significant increase in the mRNA expression of TNF-��, iNOS, IFN-��, and IL-1��. In contrast, the mRNA expression patterns of IL-6 in response to E. papillata infection did not differ significantly between control and infected mice (Figure 4). The mRNA expression of TLR4 is significantly upregulated, whereas the expression of MUC2 is significantly downregulated upon infection (Figure 5).