Or vehicle by oral gavage, and then mock-infected or infected with EW. Two dosing schedules were used: 1) GRA or vehicle alone was administered one day pre-infection and then one post-infection (pre/post), or 2) every two days through the course of infection. Nine days post-infection, cell populations isolated from the MLNs, PPs and LP were analyzed by flow cytometry for changes in B (CD19+) and T (CD4+ and CD8+) cells. *p,0.05, **p,0.01. Error bars are SEM. doi:10.1371/journal.pone.0049491.gGRA Induces ILF FormationFigure 4. GRA induces formation of B220+ aggregates in uninfected and rotavirus-infected mice. Mice (n = 3 mice per group) were administered GRA or vehicle by oral gavage, and infected or mock infected with EW. In these mice, GRA or vehicle was administered one day preinfection and then again one day post-infection. Ileal sections were prepared one day after the second GRA dose and then were stained for the detection of B cells (B220), DCs (CD11c), and T cells (CD3). Arrows indicate ILF-containing villi; arrowheads indicate adjacent ILF-absent villi. Magnification = 206. doi:10.1371/journal.pone.0049491.gnot as great (DMFI .1.5 fold, Figure 5). These data suggest rotavirus infection induces ILF which has not been observed before, and that GRA might augment the B cell response in the gut mucosa.Oral Administration of GRA Reduces the Duration of Rotavirus Antigen SheddingWe reported that GRA inhibits rotavirus replication in cell culture [23,30]. The ability of GRA to attenuate virus replication in vivo was tested. In the adult mouse model of rotavirus infection, the magnitude of replication is measured by fecal antigen shedding [20,31]. Mice were administered GRA or vehicle by oral gavage one day pre-infection with EW, and then again one day postinfection. The day of onset and magnitude of virus shedding was not different between GRA-treated and vehicle-treated animals (Figure 6A). However, virus 15755315 shedding in mice that received GRA was resolved one day earlier, and on the last positive day was reduced by ��-Sitosterol ��-D-glucoside web approximately 50 . Viral antigen was not detectable in either group at day nine post-infection. GRA treatment did not result in lower amounts of fecal antigen, but shortened theduration of virus shedding, suggesting an effect of GRA on the immune response to infection instead of a direct effect on virus replication. This idea is supported by the observation that antirotavirus serum antibody titers were statistically higher in GRAtreated animals relative to controls, although the difference was small (Figure 6B). Anti-rotavirus fecal IgA titers were not different between treated and untreated animals (data not shown).GRA MedChemExpress BIBS39 Enhances Accumulation of CD3+ T Cells in the PPs of Rotavirus-infected Mice Early Post-infectionPPs from infected mice treated with GRA or vehicle from the same experiment described above harvested at the early time point were evaluated for changes in B220, CD11c, and CD3 expression. There were marked increases in CD3+ T cells in the PPs of infected mice administered GRA compared to vehicle-treated or uninfected mice (Figure 7), suggesting GRA enhances T cell accumulation in mucosal inductive sites in response to infection, a correlation supported by flow cytometry (Figures 1 and 
3).GRA Induces ILF FormationFigure 5. GRA enhances formation of B220+ aggregates in rotavirus-infected mice at 9dpi. Mice (n = 3 mice per group) were administered GRA or vehicle by oral gavage, and infected or mock infected with EW. In.Or vehicle by oral gavage, and then mock-infected or infected with EW. Two dosing schedules were used: 1) GRA or vehicle alone was administered one day pre-infection and then one post-infection (pre/post), or 2) every two days through the course of infection. Nine days post-infection, cell populations isolated from the MLNs, PPs and LP were analyzed by flow cytometry for changes in B (CD19+) and T (CD4+ and CD8+) cells. *p,0.05, **p,0.01. Error bars are SEM. doi:10.1371/journal.pone.0049491.gGRA Induces ILF FormationFigure 4. GRA induces formation of B220+ aggregates in uninfected and rotavirus-infected mice. Mice (n = 3 mice per group) were administered GRA or vehicle by oral gavage, and infected or mock infected with EW. In these mice, GRA or vehicle was administered one day preinfection and then again one day post-infection. Ileal sections were prepared one day after the second GRA dose and then were stained for the detection of B cells (B220), DCs (CD11c), and T cells (CD3). Arrows indicate ILF-containing villi; arrowheads indicate adjacent ILF-absent villi. 
Magnification = 206. doi:10.1371/journal.pone.0049491.gnot as great (DMFI .1.5 fold, Figure 5). These data suggest rotavirus infection induces ILF which has not been observed before, and that GRA might augment the B cell response in the gut mucosa.Oral Administration of GRA Reduces the Duration of Rotavirus Antigen SheddingWe reported that GRA inhibits rotavirus replication in cell culture [23,30]. The ability of GRA to attenuate virus replication in vivo was tested. In the adult mouse model of rotavirus infection, the magnitude of replication is measured by fecal antigen shedding [20,31]. Mice were administered GRA or vehicle by oral gavage one day pre-infection with EW, and then again one day postinfection. The day of onset and magnitude of virus shedding was not different between GRA-treated and vehicle-treated animals (Figure 6A). However, virus 15755315 shedding in mice that received GRA was resolved one day earlier, and on the last positive day was reduced by approximately 50 . Viral antigen was not detectable in either group at day nine post-infection. GRA treatment did not result in lower amounts of fecal antigen, but shortened theduration of virus shedding, suggesting an effect of GRA on the immune response to infection instead of a direct effect on virus replication. This idea is supported by the observation that antirotavirus serum antibody titers were statistically higher in GRAtreated animals relative to controls, although the difference was small (Figure 6B). Anti-rotavirus fecal IgA titers were not different between treated and untreated animals (data not shown).GRA Enhances Accumulation of CD3+ T Cells in the PPs of Rotavirus-infected Mice Early Post-infectionPPs from infected mice treated with GRA or vehicle from the same experiment described above harvested at the early time point were evaluated for changes in B220, CD11c, and CD3 expression. There were marked increases in CD3+ T cells in the PPs of infected mice administered GRA compared to vehicle-treated or uninfected mice (Figure 7), suggesting GRA enhances T cell accumulation in mucosal inductive sites in response to infection, a correlation supported by flow cytometry (Figures 1 and 3).GRA Induces ILF FormationFigure 5. GRA enhances formation of B220+ aggregates in rotavirus-infected mice at 9dpi. Mice (n = 3 mice per group) were administered GRA or vehicle by oral gavage, and infected or mock infected with EW. In.
