Effects of bacterial strains and gliadin on goblet cells.

<p>Histological staining of PAS-positive goblet cells in rat intestinal loops exposed to: <i>B. bifidum</i> IATA-ES2+gliadin (200 µg) (A), IFN-γ (225 U) (B), <i>Shigella</i> CBD8+gliadin (C), <i>Shigella</i> CBD8+gliadin +IFN-γ (D), <i>E. coli</i> CBL2+gliadin (E) and <i>E. coli</i> CBL2+gliadin+IFN-γ (F). Bacteria were applied at 10⁶/loop. Changes in goblet cells are expressed as medians and interquartile ranges (25% to 75%) of the number of PAS-positive goblet cells/100 epithelial cells (G). These values were for <i>B. bifidum</i> IATA-ES2 (39, 35–41), <i>E. coli</i> CBL2 (38, 35–41) and <i>Shigella</i> CBD8 (25, 20–27) when applied alone to the loops. Different letters (a–e) indicate statistically significant differences between medians as calculated by Mann-Whitney U test (P<0.05). Identical letters correspond to non-significant differences. The separate dots or asterisks indicate outliers. The pictures were obtained for the specimens viewed under an Olympus BX 40. Scale bar, 50 µm.</p

Cytokine array analysis of rat intestinal loops washes.

<p>Layout of the arrays (A), cytokine profiles from loops treated with PBS (control) (B), gliadin (C), gliadin+IFN-γ (D), <i>B. bifidum</i> IATA-ES2+gliadin+IFN-γ (E), <i>E. coli</i> CBL2+gliadin+IFN-γ (F), and <i>E. coli</i> CBL2+<i>B. bifidum</i> IATA-ES2+gliadin+IFN-γ (G). The data are expressed as relative levels of selected cytokines (percentage of positive controls). Cytokine-induced neutrophil chemoattractant (CINC)-2 and -3, monocyte chemoattractant protein (MCP)-1, macrophage inflammatory protein (MIP)-3α, nerve growth factor β-(NGF), tumor necrosis factor (TNF)-α, vascular endothelial growth factor (VEGF). The signal intensity was measured using the LAS-1000 luminescence detector (Fujifilm, Tokyo, Japan).</p

Adhesion of different bacterial strains to IEC-6 cells.

<p>The highest percentage of adhered bacteria was observed for <i>E. coli</i> CBL2 and <i>Shigella</i> CBD8. The differences between tested bacterial strains, as well as the effect of simultaneously added gliadin fragments were non-significant as established by applying the Mann-Whitney U-test. Data are expressed as medians and interquartile ranges (25% to 75%) of adhesion of four independent experiments. None of the differences was found to be statistically significant (P<0.05). The separate dot indicates an outlier.</p

Changes in intestinal permeability induced by gliadin and various bacterial strains.

<p>Intestinal loops were exposed to: gliadin fragments with IFN-γ and <i>B. bifidum</i> IATA-ES2 (A,D), gliadin+IFN-γ+<i>E. coli</i> CBL2 (B,E) and <i>Shigella</i> CBD8+gliadin+IFN-γ (C,F); The white arrows indicate gliadin fragments found by immunofluorescence (A–C) using mouse peroxidase-labeled monoclonal anti-gliadin antibody and TSA™ Plus Fluorescence systems and black arrows indicate goblet cells (D, E). Bacteria were applied at 10⁶/loop. The specimens were viewed under confocal microscope Olympus FV 1000 SIM using differential interference contrast (D–F). Scale bar, 20 µm.</p

Distribution of claudin-1 and ZO-1 in rat intestinal loops.

<p>Exposure of intestinal tissue to gliadin digest alone (A) or with IFN-γ (C) led to reduced ZO-1 expression at the periphery of the villi. The combination of gliadin+IFN-γ+<i>B. bifidum</i> IATA-ES2 (E) maintained the original level of ZO-1 as in PBS-treated loops (I). When gliadin+IFN-γ were applied with <i>E. coli</i> CBL2 (G) ZO-1 fluorescence was weaker. No changes in claudin-1 localization (B, D, F, H) were detected after any stimulus in comparison with PBS control (J). Representative pictures of three experiments are shown. The specimens were viewed under an Olympus BX 40 microscope. Scale bar, 20 µm. Western blot of tissue lysates (K) from intestinal loops stimulated with: 1. gliadin, 2. gliadin+IFN-γ, 3. <i>B. bifidum</i> IATA-ES2+gliadin, 4. <i>E. coli</i> CBL2+gliadin +IFN-γ, 5. <i>E. coli</i> CBL2+<i>B. bifidum</i> IATA-ES2+gliadin+IFN-γ, 6. <i>B. bifidum</i> IATA-ES2+gliadin+IFN-γ, and 7. PBS. The separated proteins on membranes were stained with anti ZO-1 or claudin-1 antibodies and re-probed with antibodies against β-actin to document the same protein concentration in all samples.</p

Decreased mast cell protease induction by heated-OVA.

<p>Heated OVA (h-OVA, black bar) induced significantly lower amounts of mast cell protease (MMCP-1), the marker of mast cell activation, compared to mice fed with native OVA (white bar). Data are represented as mean ± SEM (n = 10 mice/group), representative data from one out of three independent experiments. *P≤0.05, **P≤0.01, ***P≤0.001.</p

Numbers of Tregs in splenocytes and mesenteric lymph nodes of OVA treated mice.

<p>Typical plots depicting numbers of Tregs in mouse splenocytes (a) and mesenteric lymph node (b) in gated CD3+CD4+CD8– T helper cells after feeding with OVA, h-OVA or PBS, respectively. Numbers in upper quadrants shows proportions (mean ± SD) of either CD25–Foxp3+ or CD25+Foxp3+ Th cells out of all cells. Representative data from one out of three independent experiments. *P≤0.05.</p

Number of Tregs in spleen cell suspensions co-cultured <i>in vitro</i> with OVA digests.

<p>The percentage of Tregs in cell suspension isolated from spleens of non-stimulated (naïve) BALB/c mice cultured <i>in vitro</i> for 48 hours with undigested (0′) and after 20 (20′) and 40 minutes (40′) peptic digest of OVA (white bars), h-OVA (black bars) or b-OVA (grey bars). The data represent the percentage of CD4+Foxp3+ cells out of all live cells as measured by FACS. Representative data from one out of three independent experiments are shown. Data are represented as mean ± SEM.</p

Cytokine production after <i>in vitro</i> restimulation with OVA.

<p>The cytokine production from mesenteric lymph nodes (a) and splenocytes (b) of BALB/c mice fed with OVA (white bars) or h-OVA (black bars) and stimulated <i>in vitro</i> with appropriate allergen. Cytokine levels are expressed after subtraction of base line levels of unstimulated lymph node cells or splenocytes. Data shown are mean values ± SEM (n = 4–7 mice/group), representative data from one out of three independent experiments. *P≤0.05, **P≤0.01, ***P≤0.001, n.d. =  not detectable.</p

RP-HPLC separation profile of native-OVA and heated-OVA peptic digests.

<p>RP-HPLC separation profile monitored at 280 nm corresponds to OVA and OVA heated at 70°C (h)-OVA or boiled at 95°C (b)-OVA undigested (0′) and after 20 (20′) and 40 minutes (40′) of digestion by pepsin. RT – retention time.</p