Intestinal immune cell responses following ileitis induction in PACAP-treated mice.

<p>Ileitis was induced by peroral infection of mice with <i>T. gondii</i> at day 0. The average number of cells positive for (A) CD3 (T lymphocytes), (B) FOXP3 (regulatory T cells, Tregs), (C) MPO7 (myeloperoxidase-7, neutrophils and monocytes), and (D) F4/80 (macrophages) from at least six high power fields (HPF, 400× magnification) per animal were determined microscopically in immunohistochemically stained ileum sections derived from mice following PACAP prophylactic treatment (Proph, white bars), PACAP therapy (Therap, gray bars), or placebo application (black bar) at day 7 post infection as compared to naïve controls (lined bars). Numbers of analyzed animals are given in parentheses. Mean values, standard errors of the mean, and significance levels as determined by the Mann-Whitney-U test are indicated. Data are pooled from three independent experiments.</p

Less severe extra-intestinal histopathology following PACAP prophylaxis.

<p>Paraffin section of (A) lung and (B) kidney samples were obtained from naïve animals (right panel) and from mice following PACAP prophylactic treatment (PACAP, middle panel) and compared to placebo controls (PLC, left panel) at day 7 post infection. Representative photomicrographs of H&E stained paraffin sections (upper panel: 100× magnification; scale bar 100 µm; lower panel: 400× magnification; scale bar 20 µm) from three independent experiments are shown. White arrow head points towards hemosiderin deposit (in A), black arrows indicate an inflammatory peri-bronchial cuff (in A) and a pyknotic, degenerated, apoptotic glomerulum (in B).</p

Amelioration of acute ileitis in PACAP-treated mice.

<p>Ileitis was induced by peroral infection of mice with <i>T. gondii</i> at day 0. (A) Relative body weight loss of mice (in %), (B) relative small intestinal shortening (in %), and (C) histopathological scores of the terminal ileum following PACAP prophylactic treatment (Proph, white bars), PACAP therapy (Therap, gray bars) or placebo application (black bar) at day 7 post infection. Numbers of analyzed animals are given in parentheses. Mean values, standard errors of the mean, and significance levels as determined by the Mann-Whitney-U test are indicated. Data are pooled from three independent experiments. (D) Representative photomicrographs (100× magnification; scale bar 100 µm) of histopathological changes in ileal paraffin sections from three independent experiments are shown. Naïve, uninfected mice served as controls.</p

Pro-inflammatory cytokine responses in ilea of PACAP-treated mice.

<p>Ileitis was induced by peroral infection of mice with <i>T. gondii</i> at day 0. (A) IL-23p19 and (B) IL-22 mRNA expression levels (by RT-PCR) as well as (C) IFN-γ and (D) MCP-1 protein concentrations (by CBA) were determined in <i>ex vivo</i> ileum biopsies obtained from mice following PACAP prophylactic treatment (Proph, white bars), PACAP therapy (Therap, gray bars), or placebo application (black bars) at day 7 post infection as compared to naïve animals (lined bars). mRNA expression levels are expressed as fold changes relative to HPRT mRNA expression (Arbitrary Units). Numbers of analyzed animals are given in parentheses. Mean values, standard errors of the mean, and significance levels as determined by the Mann-Whitney-U test are indicated. Data are pooled from three independent experiments.</p

Survival rates of PACAP treated mice.

<p>Ileitis was induced by peroral infection of mice with <i>T. gondii</i> at day 0 as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108389#s2" target="_blank">methods</a>. Survival of animals following prophylactic (Proph, black line; n = 10) or therapeutic (Therap, gray line; n = 10) PACAP treatment as compared to placebo controls (PLC, dotted line; n = 16) was monitored twice daily until day 28 post infection (p.i.). Significance levels (as compared to respective groups) were determined by Kaplan-Meier analysis. Data are pooled from three independent experiments.</p

Pro-inflammatory cytokine responses in extra-intestinal compartments of PACAP-treated mice.

<p>Ileitis was induced by peroral infection of mice with <i>T. gondii</i> at day 0. Hepatic (A) IFN-γ, (B) TNF-α, (C) MCP-1, (D) IL-6, and (E) splenic TNF-α secretion were determined in overnight cultures of <i>ex vivo</i> biopsies derived from respective organs of mice following PACAP PACAP prophylactic treatment (Proph, white bars), PACAP therapy (Therap, gray bars), or placebo application (black bars) at day 7 post infection as compared to naïve animals (lined bars). Numbers of analyzed animals are given in parentheses. Mean values, standard errors of the mean, and significance levels as determined by the Mann-Whitney-U test are indicated. Data are pooled from three independent experiments.</p

Pro-inflammatory cytokine responses in mesenteric lymph nodes of PACAP-treated mice.

<p>Ileitis was induced by peroral infection of mice with <i>T. gondii</i> at day 0. (A) IFN-γ and (B) nitric oxide secretion were determined in overnight cultures of <i>ex vivo</i> biopsies derived from mesenteric lymphnodes (MLNs) of mice following PACAP PACAP prophylactic treatment (Proph, white bars), PACAP therapy (Therap, gray bars), or placebo application (black bars) at day 7 post infection as compared to naïve animals (lined bars). Numbers of analyzed animals are given in parentheses. Mean values, standard errors of the mean, and significance levels as determined by the Mann-Whitney-U test are indicated. Data are pooled from three independent experiments.</p