Immunohistochemistry. Immunohistochemical analysis at the peak AZD-5438 of phosphorylation revealed the presence of extensive pJNK in rat ME mucosae 48 h after bacterial inoculation. Immunoreactivity to pJNK was observed in the nuclei of both stromal and epithelial cells. No immunostaining was observed when the primary Ab was omitted. Inhibition of bacterially induced ME mucosal hyperplasia by C. difficile toxin B, CEP11004, or SP600125. Figure 2 shows the effects of C. difficile toxin B, CEP11004, or SP600125 on the surface area of cultured ME mucosal explants from bacterially inoculated rats. We have previously shown that ME mucosal explants from the MEs of rats infected 48 h before dissection with NTHI display permanently enhanced tissue proliferation , which can be used as a model for bacterial enhancement of ME mucosal growth.
All explants maintained a healthy appearance and remained firmly attached to the plate surface throughout the duration of the study. C. difficile toxin B significantly inhibited the growth of mucosal 2-Methoxyestradiol epithelial cells, decreasing the surface area by 53.8 . C. difficile toxin B treatment at 0.1 ng ml or 1 ng ml showed no effect on explant outgrowth. Similarly, 1,000 nM CEP11004 significantly inhibited explant outgrowth, decreasing the surface area by 56.4 . No inhibition was observed for CEP11004 at 10 or 100 nM. Both 2 M and 20 M SP600125 significantly inhibited explant outgrowth, decreasing the surface area by 34.6 and 92.2 , respectively. No inhibition was observed for SP600125 at 0.2 M. Figure 3 shows the typical microscopic appearances of treated and untreated mucosal explants from infected rat ME epithelia on day 7 of culture.
As determined with trypan blue daily between days 4 and 10, the viability of individual cells from trypsinized and dissociated explants, with and without the highest dose of C. difficile toxin B, CEP11004, or SP600125, was 95 for all cultures. There were no statistical differences between control explants and those treated with any inhibitor. Inhibition of uninfected ME mucosae by C. difficile toxin B, CEP11004, or SP600125. The growth of uninfected ME mu cosae was also affected by JNK pathway inhibition, although to a lesser extent than that of infected mucosae. C. difficile toxin B at 10 ng ml significantly inhibited the growth of explant cultures, decreasing the surface area by 60.4 , compared with that of control specimens.
As with infected mucosal explants, C. difficile toxin B treatment at 0.1 ng ml or 1 ng ml did not inhibit explant growth. CEP11004 at 1,000 nM significantly inhibited explant outgrowth , decreasing the surface area by 73.0 . No inhibition was observed at 10 or 100 nM. In contrast to the case for infected mucosal explants, only 20 M SP600125 significantly inhibited explant outgrowth, decreasing the surface area by 56.4 . No inhibition was observed for SP600125 at 0.2 M or 2 M. As determined with trypan blue daily between days 4 and 10, the viability of cells from trypsinized explants, with and without the highest dose of C. difficile toxin B, CEP11004, or SP600125, was 95 . Inhibition of bacterially infected ME mucosae by SP600125 in vivo.