GRA enhances formation of B220⁺ aggregates in rotavirus-infected mice at 9dpi.

<p>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 pre-infection and then again one day post-infection. Ileal sections were prepared nine days post-infection, 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 = 20×.</p

CD138⁺ cells are increased in MLNs and PPs 48 hours post-treatment (hpt) with GRA.

<p>Mice (n = 3 mice per group) were administered GRA or vehicle by oral gavage, and cell populations in the MLNs and PPs were analyzed with antibodies to CD11c, CD11b, and CD138. **p<0.01. Error bars are SEM.</p

GRA induces CD19⁺ cell accumulation in the lamina propria in uninfected and rotavirus infected mice.

<p>Mice (n = 5 mice per group) were administered GRA 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.</p

GRA reduces the duration of rotavirus antigen shedding.

<p>C57Bl/6 mice (n = 5 mice per group) were administered 50 mg/kg GRA or vehicle alone by oral gavage one day pre-infection and then one day post-infection. A) Fecal samples from individual mice were collected and rotavirus antigen was detected by ELISA. B) Total endpoint anti-rotavirus serum antibody titers were measured by ELISA. Error bars are SEM; p = 0.03.</p

GRA-induced changes in cytokine expression in intestinal tissue.

<p>Representative data are shown for RNA isolated from duodenal or ileal tissue. Data shown for duodenal tissue are from the initial full array. Data from ileal sections from uninfected and EW infected mice were obtained with the custom array. Data are presented as fold-increase over mock-treated controls. ND – not done.</p

GRA induces formation of B220⁺ aggregates in uninfected and rotavirus-infected mice.

<p>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 pre-infection 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 = 20×.</p

Immune cell populations modulated by GRA in uninfected and rotavirus -infected mice.

<p>C57Bl/6 mice (n = 5 per group) were administered GRA or vehicle alone orally one day pre-infection with 10⁵ SD₅₀ of murine rotavirus strain EW, and then one day post-infection. Cells isolated from the MLNs and PPs were analyzed for changes in B cells (CD19), T cells (CD4 and CD8), their activation (CD69); and dendritic cells (CD11c^high and CD11c^low), macrophages (CD11b), and plasma cells (CD138). *p<0.05, **p<0.01. Error bars are SEM.</p

<i>L</i>. <i>lactis</i>-CFA/I confers protection against CIA via reduction of neutrophil influx into joints with concomitant reduction in TNF-α, but only slightly reduces anti-CII Ab titers.

<p>Reduced tissue destruction in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117825#pone.0117825.g002" target="_blank">Fig. 2</a> is attributed to diminished neutrophil infiltration of the joints <b>(A, B)</b> in <i>L</i>. <i>lactis</i>-CFA/I-treated mice (as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117825#pone.0117825.g002" target="_blank">Fig. 2B</a>). Cell suspensions were analyzed by flow cytometry for Ly-6G⁺Ly-6C⁺CD11b⁺ neutrophils; * <i>p</i> < 0.001 compared with control groups (n = 5/group). <b>(C)</b><i>L</i>. <i>lactis</i>-CFA/I fails to elicit serum IgG and IgG subclass anti-CFA/I fimbriae and <b>(D)</b> modestly reduces IgG1 and IgG2a anti-CII antibody titers (day 35 post-CIA induction) in orally treated mice from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117825#pone.0117825.g002" target="_blank">Fig. 2</a>; n = 5 mice/group. <b>(E)</b> LN lymphocytes from <i>L</i>. <i>lactis</i>-CFA/I-treated mice from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117825#pone.0117825.g002" target="_blank">Fig. 2B</a> (n = 5 mice/group) were CII-restimulated in vitro and showed reduced TNF-α production; * <i>p</i> < 0.001 compared to each control group.</p

Expression of <i>E</i>. <i>coli cfaI</i> fimbrial operon in <i>Lactococcus lactis</i>.

<p><b>(A)</b> Schematic map for expression of <i>E</i>. <i>coli cfaI</i> in <i>L</i>. <i>lactis</i> (pBzMM153). The elements of the composite promoter are boxed. Each structural gene is fused in-frame to a different lactococcal secretion signal peptide, and each gene is followed by its own STOP codon. No other functional elements are interspersed except for one Shine-Dalgarno (SD) sequence upstream of a fusion between extracellular (Exp4) protein and <i>cfaA</i>. All (SD) sequences are marked. <b>(B)</b> CFA/I fimbriae expression in <i>L</i>. <i>lactis</i>. Lane1, molecular weight (MW) markers; lane 2, <i>Salmonella</i>-CFA/I (H696) strain; lane 3, <i>L</i>. <i>lactis</i> bearing the empty pMSP3535H3 vector; lanes 4,5, pBzMM156 (nisin-inducible promoter) clones 1 and 2; lanes 6,7, pBzMM155 (p23 promoter) clones 1 and 2; and lanes 8–10, pBzMM153 (synthetic composite promoter) clones 1–3. <b>(C, D)</b> Immunogold staining of <b>(C)</b><i>L</i>. <i>lactis</i> vector and <b>(D)</b><i>L</i>. <i>lactis</i>-CFA/I with anti-CFA/I antibody. Arrows point to gold particles on fimbrial structures projecting from the cell wall.</p

<i>L</i>. <i>lactis</i>-CFA/I, not <i>L</i>. <i>lactis</i> vector, is a potent therapeutic for collagen-induced arthritis (CIA).

<p>C57BL/6 males (n = 5/group) were CII-challenged on day 0, and treated orally with 5×10⁸ CFUs <i>L</i>. <i>lactis</i>-CFA/I (<i>L</i>. <i>lactis</i>-pBzMM153) or vector control (<i>L</i>. <i>lactis</i>-pMSP3535H3) in sterile PBS or with vehicle alone on days <b>(A)</b> 14, 21, 28 or <b>(B)</b> on days 18 and 25 post-CIA induction as diagramed. Bacteria were grown in synthetic M17 medium supplemented with 0.5% glucose. A representative example of 6 experiments (n = 5/group) is depicted; * <i>p</i> < 0.01; ^✢<i>p</i> < 0.05 as compared to each control group. <b>(C)</b> Joint pathology was evaluated from decalcified knees from mice in each treatment group (n = 5/group). Representative examples of mid-sagittal knee joint sections are stained with H&E (top row) or toluidine blue (bottom row) from mice treated with two doses of <i>L</i>. <i>lactis</i>-CFA/I <b>(B)</b> upon termination of the study.</p