The chemical structure of the CCR4 antagonist.

<p>The CCR4 antagonist AF-399/42018025 is a small chemical molecule with a molecular weight of 565.93. It contains containing six 5 or 6 membered aromatic rings and 3 nitrogen, sulfur, and oxygen atoms. The chemical name of the molecule is 4-(1-benzofuran-2-ylcarbonyl)-1-{5-[4-chlorobenzyl)sulfanyl]-1,3,4-thiadiazol-2-yl}-3-hydroxy-5-(2-thienyl)-1,5-dihydro-2H-pyrrol-2-one [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0131364#pone.0131364.ref005" target="_blank">5</a>].</p

H&E and PAS staining of the fundus showing pronounced inflammation and severe pseudo-pyloric metaplasia.

<p>H&E (A) and PAS staining (B) of the fundus of an animal in the FC subcutaneously immunized and challenged group, showing pronounced inflammation and severe pseudo-pyloric metaplasia (original magnification: 100x).</p

Histopathological scoring of inflammation Study 1.

<p>Shown are the median scores (min-max) of pseudo-pyloric metaplasia of the fundus, infiltration of the mucosa of the fundus with granulocytes and mononuclear cells 3 weeks after challenge of the vaccinated mice. Scoring: 0 = normal, 1 = mild, 2 = moderate, 3 = severe. Significant differences between the positive control group and the immunized and challenged groups are noted by</p><p>* (p<0.05). (HBSS: Hank’s balanced salt solution FC: Freund’s complete, FIC: Freund’s incomplete, CT: Cholera toxin, Curd: Curdlan, CpG: CpG-DNA IN: intranasally, SC: subcutaneously, con: uninfected group, inf: infected group)</p><p>Histopathological scoring of inflammation Study 1.</p

H&E and PAS staining of the fundus, showing mild inflammation and pseudo-pyloric metaplasia.

<p>H&E (A) and PAS staining (B) of the fundus of an animal from the CCR4 antagonist subcutaneously immunized and challenged group, showing mild inflammation and pseudo-pyloric metaplasia (original magnification: 100x).</p

Histopathological analysis of fundus by H&E staining.

<p>Normal fundus from an uninfected BALB/c mouse (A). Metaplastic fundus of a Freund’s complete/lysate immunized and <i>H</i>. <i>suis</i>-infected BALB/c mouse, showing severe parietal cell loss, with replacement of the intestinal epithelium by a common glandular epithelium (B). Original magnification: 200x.</p

H&E and PAS staining of a normal fundus.

<p>H&E (A) and PAS staining (B) of a normal fundus of an animal from the negative control group (original magnification: 100x).</p

Experimental set-up Study 1.

<p>Shown is the</p><p>¹immunization protocol</p><p>²the administration route of the vaccine</p><p>³the amount of adjuvant used per mouse</p><p>⁴the volume of the vaccine</p><p>⁵the amount of lysate as shown by the protein concentration used per mouse</p><p>⁶whether the animals were challenged with <i>H</i>. <i>suis</i> or not and ⁶the number of animals in each group. (FC: Freund’s complete, FIC: Freund’s incomplete, CT: Cholera toxin, IN: intranasally, SC: subcutaneously, Pos. control.: sham-immunized/challenged, Neg. control: sham-immunized/not challenged)</p><p>Experimental set-up Study 1.</p

The protective efficacy of different adjuvants on the amount of colonizing <i>H</i>. <i>suis</i> bacteria, 3 weeks after challenge, in study 1.

<p>Subcutaneous immunization (SC) was done by mixing <i>H</i>. <i>suis</i> sonicate with Freund’s complete (FC), Freund’s incomplete (FIC) or Curdlan (Curd) and injecting this mixture at the lower back of the mice. Intranasal immunization (IN) was done by mixing <i>H</i>. <i>suis</i> sonicate with Cholera toxin (CT), CpG-DNA (CpG) or Curdlan (Curd) and applying this mixture on the external nares of the mice. Animals in the control groups were sham-immunized with Hank’s Balanced Salt Solution (HBSS). The bacterial load is illustrated as log(10) of <i>H</i>. <i>suis</i> copies/mg stomach tissue. Individual mice are illustrated as dots. Significant differences between immunized and non-immunized challenged animals are noted by * (p<0.05). (con: uninfected, inf: infected).</p

Relative gene expression of cytokines and chemokines in the stomach of challenged animals after immunization or after sham inoculation in study 1.

<p>The first bar represents the pooled data of the animals that were sham-immunized with HBSS (intranasally and subcutaneously) and that were not challenged with <i>H</i>. <i>suis</i> (Neg. con). The second bar represents the pooled data of the animals that were sham-immunized with HBSS (intranasally and subcutaneously) and that were challenged with <i>H</i>. <i>suis</i> (Pos. con). Bars 3, 4 and 5 represent the groups of animals that were immunized subcutaneously with Freund’s complete (FC/lysate/SC), Freund’s incomplete (FIC/lysate/SC) or Curdlan (Curd/lysate/SC) and challenged with <i>H</i>. <i>suis</i>. Bars 6, 7 and 8 represent the animals that were immunized intranasally with Cholera Toxin (CT/lysate/IN), CpG-DNA (CpG/lysate/IN) or Curdlan (Curd/lysate/IN) and challenged with <i>H</i>. <i>suis</i>. An * (p<0.05) indicates a significant modulation of mRNA expression levels compared to the sham-immunized/challenged groups. Cytokine expression between immunized and challenged groups was also compared with each other. When no differences could be found between the different immunized groups, the same letter designation was attributed.</p

Correlation between cytokine and chemokine expression and <i>H</i>. <i>suis</i> colonization in study 1.

<p>Shown are the correlation analyses between IL-17, IL-10, MIP-2, LIX, IFN-γ and IL-4 mRNA expression levels on the one hand and the number of <i>H</i>. <i>suis</i> bacteria colonizing stomach of mice in the immunized and challenged groups on the other hand. Correlation was measured by Spearman’s Rho (ρ). Statistical significance between the immunized and challenged groups and the positive control group is noted by the P-value.</p