Specificity and dilution of monoclonal antibodies.

a<p>Sialyl-T antigen was detected using the anti-T antibody after neuraminidase treatment as described in Material and Methods.</p

Histochemical characterization of mice gastric mucosa treated with <i>Pteridium aquilinum</i> and/or infected with <i>Helicobacter pylori.</i>

<p>Histochemical staining with hematoxylin and eosin (<b>Panel A</b>) and Alcian Blue (pH = 2,5) with nuclear red staining (<b>Panel B</b>) of mice gastric mucosa of: Control (Group 1) (a,b,i,j), <i>Pteridium aquilinum</i> treated (Group 2) (c,d,k,l), <i>Helicobacter pylori</i> infected (Group 3) (e,f,m,n) and both <i>Pteridium aquilinum</i> treated and <i>Helicobacter pylori</i> infected (Group 4) (g,h,o,p). Since the results observed at 4 and 7 weeks time points were identical, only a representative picture is included for each experimental condition. Bar = 40 µm (a,c,e,g and i-p) and bar = 20 µm (b,d,f,h). Arrows indicate inflammatory cells.</p

Primer sequences and Real-time PCR conditions for the analysis of gene expression in mice gastric mucosa.

<p>Primer sequences and Real-time PCR conditions for the analysis of gene expression in mice gastric mucosa.</p

DHA effect on <i>H. pylori</i> growth.

<p>Growth of <i>H. pylori</i> strains A) 26695, B) SS1 and C) B128 during 48 hours in the presence of increasing concentrations of DHA from 50 to 1000 µM. Ethanol 0.06% v/v was used as a vehicle in DHA original stock solution, and therefore the presence of ethanol at the same concentration was also analyzed on <i>H. pylori</i> control culture with no effect on bacterial growth for the three strains. Data are expressed as the mean ± Standard Deviation and are representative of three independent experiments. * Refers to significant differences in <i>H. pylori</i> growth between controls and DHA-treated conditions (50 µM to 1000 µM of DHA).</p

Glycosylation alterations in mice gastric mucosa upon <i>Pteridium aquilinum</i> treatment and concomitant <i>Helicobacter pylori</i> infection.

<p><b>Panel A –</b> Heatmap of the Glyco-gene Chip array analysis obtained when comparing group 1 (control) with group 4 (treated with <i>Pteridium aquilinum</i> and <i>Helicobacter pylori</i> infected). Red indicates increased and blue indicates decreased expression relative to the mean transcript expression value. The transcripts identified as differentially expressed were those with adjusted p-value <0.1 and fold change >1.3. <b>Panel B</b> – Schematic representation of the biosynthesis of sialylated terminal structures. The enzymes or family of enzymes responsible for each step of the biosynthesis are indicated. The enzymes identified as showing different expression levels in <i>Pteridium aquilinum</i> treated and <i>Helicobacter pylori</i> infected mice (Group 4) are highlighted in red (increased expression) and blue (decreased expression). <b>Panel C</b> – Immunohistochemistry for evaluation of expression of Sialyl-Lewis a (SLe^a) antigen (a–d) and for Sialyl-Lewis x (SLe^x) (e–h). Additional immunofluorescence evaluation performed for SLe^x (i–l). Bar = 20 µm.</p

Expression of carbohydrate antigens ^a in control, <i>Helicobacter pylori</i> infected and <i>Pteridium aquilinum</i> treated with or without infection mice.

a<p>Staining was graded in three categories: (-) negative, (+) positive, and (+ +) strongly positive.</p>b<p>Fisher’s exact test (p-value); n.a. – not applicable.</p

Glycosyltransferases expression analysis of gastric mucosa from <i>Pteridium aquilinum</i> exposed mice. Panel A –

<p>Heatmap of the Glyco-gene Chip array analysis obtained when comparing group 1 (control mice) with group 2 (mice treated with <i>Pteridium aquilinum</i>). Red and blue indicate increased and decreased expression relative to the mean transcript expression value, respectively. The transcripts identified as differentially expressed were those with adjusted p-value <0.1 and fold change >1.3. <b>Panel B –</b> Real-time PCR analysis for expression of <i>St6galnac6</i>, <i>Galntl4</i>, <i>St3gal2</i> and <i>C1galt1</i> in gastric mucosa from control (Group 1), <i>Pteridium aquilinum</i> treated (Group 2) and concomitantly <i>Pteridium aquilinum</i> treated and <i>Helicobacter pylori</i> infected (Group 4) mice. The gene used as reference was <i>Hprt1</i>. Significance was evaluated using the Shapiro-Wilk test. n.s. not significant p<0.05(*); p<0.01(**); p<0.005 (***).</p

Characterization of cystically dilated gland of gastric mucosa of mouse exposed to <i>Pteridium aquilinum.</i>

<p><i> Pteridium aquilinum</i> treated mice (Group 2) displaying a cystically dilated gland stained with hematoxylin and eosin (a), Alcian Blue (pH = 2,5) (b), and immunostained for Sialyl-Lewis x antigen (c). Bar = 20 µm.</p

Anti-inflammatory effects of DHA in the infected gastric mucosa of mice.

<p><b>A</b>) Histological analysis of inflammatory lesions of the gastric mucosa of <i>H. pylori</i> infected or non-infected mice treated or not with DHA 50 µM after 6 and 9 months. No differences were observed between non-infected mice DHA-treated or non-treated. In infected mice, the DHA treatment in infected mice leads to a decrease of the gastric mucosa thickness compared to <i>H. pylori</i> infected mice but non-DHA treated at both time-points. Infiltrates of polymorphonuclear (PMN) cells and plasmocytes as well as number of lymphoid aggregates (arrows) were lower in <i>H. pylori</i> infected mice treated with DHA compared to infected mice non-treated. The infection leads to the formation of lymphoid aggregate (arrows) not observed in mice treated with DHA. Scale bars correspond to 100 µm. <b>B</b>) Semi-quantification of inflammation score grading in the gastric mucosa. At each time-point, 6 and 9 months, mean score grading for each group of mice are higher in the <i>antrum</i> than <i>fundus</i> part. The presence of DHA leads to a significant decrease of the <i>antrum</i> inflammation of the <i>H. pylori</i> infected gastric mucosa. PMN: Polymorphonuclear cells; Linf: Lymphocytes aggregates; Submuc: Submucosa. Comparison was made between infected mice non-DHA supplemented and infected mice DHA supplemented. C) Measurement of PGE2 in the mice serum. Lower PGE2 values were observed in infected mice that received DHA 50 µM compared to infected mice non-DHA treated (adjusted <i>P</i> values 0.016 at 1 month; 0.008 at 3 months; 0.054 at 6 months; 0.012 at 9 months). PGE2 levels were lower at 6 months in DHA-treated mice compared to untreated mice, even though the difference cannot be considered statistically significant. Control group corresponds to non-infected and non-DHA treated mice. Bars represent means with standard deviation.</p

Alterations in simple mucin-type carbohydrate antigens in gastric mucosa from mice exposed to <i>Pteridium aquilinum.</i> Panel A –

<p>Schematic representation of the biosynthesis of simple mucin-type carbohydrate antigens. The enzymes or family of enzymes responsible for each step of the biosynthesis are indicated. The enzymes identified as showing different expression levels in <i>Pteridium aquilinum</i> treated mice (Group 2) are highlighted in red (increased expression) and blue (decreased expression). <b>Panel B</b> – Immunohistochemistry of simple mucin-type carbohydrate antigens, Tn (a,b), STn (c,d), T (e,f) and ST (g,h) in gastric mucosa of control (Group 1) (a,c,e,g) and <i>Pteridium aquilinum</i> treated (Group 2) (b,d,f,h) mice. Bar = 20 µm.</p