Деякі проблеми використання тимчасово зайнятих земель

<div><p>Glucocorticoid induced-leucine zipper (GILZ) has been shown to be induced in cells by different stimuli such as glucocorticoids, IL-10 or deprivation of IL-2. GILZ has anti-inflammatory properties and may be involved in signalling modulating apoptosis. Herein we demonstrate that wildtype <em>Yersinia enterocolitica</em> which carry the pYV plasmid upregulated GILZ mRNA levels and protein expression in epithelial cells. Infection of HeLa cells with different <em>Yersinia</em> mutant strains revealed that the protease activity of YopT, which cleaves the membrane-bound form of Rho GTPases was sufficient to induce GILZ expression. Similarly, <em>Clostridium difficile</em> toxin B, another bacterial inhibitor of Rho GTPases induced GILZ expression. YopT and toxin B both increased transcriptional activity of the GILZ promoter in HeLa cells. GILZ expression could not be linked to the inactivation of an individual Rho GTPase by these toxins. However, forced expression of RhoA and RhoB decreased basal <em>GILZ</em> promoter activity. Furthermore, MAPK activation proved necessary for profound GILZ induction by toxin B. Promoter studies and gel shift analyses defined binding of upstream stimulatory factor (USF) 1 and 2 to a canonical c-Myc binding site (E-box) in the <em>GILZ</em> promoter as a crucial step of its trans-activation. In addition we could show that USF-1 and USF-2 are essential for basal as well as toxin B induced GILZ expression. These findings define a novel way of <em>GILZ</em> promoter trans-activation mediated by bacterial toxins and differentiate it from those mediated by dexamethasone or deprivation of IL-2.</p> </div

Depletion of pDCs Abrogates <i>B. adolescentis</i>-Mediated Prevention of <i>Yersinia</i> Dissemination.

<p>Lamina propria dendritic cells (lpDC) were analyzed by gating on linage negative (CD3ε, CD19, DX5) and CD11c-intermediate cells, as demonstrated in exemplarily depicted dot plots (<b>A</b>) Mean percentage ± SD of B220⁺ lamina propria plasmacytoid DCs (solid black line) and fluorescence minus one control (gray filled histograms) (<b>B</b>) and splenic CFU of <i>Yersinia</i> in log₁₀ per gram of <i>B. adolescentis</i> fed and <i>Yersinia</i> infected mice, either injected with anti-mouse PDCA-1 (n = 5) or respective isotype control (n = 3).</p

Cell Counts of Intraepithelial Plasmacytoid DCs, and Lamina Propria FoxP3⁺ T_reg Cells.

<p>Numbers indicate mean cell counts±SD of untreated mock (M) and streptomycin (S) treated mice. Data represent at least four mice per group.</p

<i>B. adolescentis</i> Depletion Results in Comparable Frequencies of Intraepithelial pDCs and Lamina Propria T_reg Cells.

<p>Mean percentage ± SD of (<b>A</b>) intraepithelial B220⁺PDCA-1⁺ plasmacytoid DCs and (<b>B</b>) lamina propria FoxP3-expressing regulatory T cells (solid black line) and fluorescence minus one control (gray filled histograms) of vancomycin and metronidazole treated mock (M+ antibiotics) and <i>B. adolescentis</i> (B+ antibiotics) (B) fed mice. Intraepithelial plasmacytoid cells and lamina propria CD4+T cells were gated as indicated in Fig. 2 and Fig. 5, respectively. Data represent five mice per group.</p

<i>B. adolescentis</i> Feeding Prevents From Weight Loss and Dissemination of <i>Yersinia</i> to the Spleen.

<p>(<b>A</b>) Graph represents percent modulation of initial body weight over four days of <i>Yersinia</i> infected mice (Y, black line and squares) and <i>B. adolescentis</i> fed and <i>Yersinia</i> infected mice (BY, gray line and diamonds). Colony forming units (CFU) of <i>Yersinia</i> in log₁₀ per gram Peyer's patches (PP) (<b>B</b>), spleen (<b>C</b>), or feces (<b>D</b>) of <i>Yersinia</i> infected mice (Y) and <i>B. adolescentis</i> fed and <i>Yersinia</i> infected mice (BY). Data represent mean and SEM of at least 8 mice.</p

Streptomycin Treatment does Neither Induce Intraepithelial pDCs nor Lamina Propria T_reg Cells.

<p>Mean percentage ± SD of (<b>A</b>) intraepithelial B220⁺PDCA-1⁺ plasmacytoid DCs and (<b>B</b>) lamina propria FoxP3-expressing regulatory T cells (solid black line) and fluorescence minus one control (gray filled histograms) of untreated mock (M) and streptomycin (S) treated mice. Intraepithelial plasmacytoid cells and lamina propria CD4⁺ T cells were gated as indicated in Fig. 2 and Fig. 5, respectively. Data represent at least four mice per group.</p

Cell Counts of Total Intraepithelial DCs, Conventional, and Plasmacytoid DCs.

<p>Numbers indicate mean cell counts ± SD of untreated mock (M), <i>B. adolescentis</i> fed (B), <i>Yersinia</i> infected (Y), as well as <i>B. adolescentis</i> fed and <i>Yersinia</i> infected mice (BY) mice. ^a M vs. Y p<0.001, B vs. Y p<0.01, BY vs. Y p<0.001; ^b M vs. Y p<0.05, B vs. Y p<0.05, BY vs. Y p<0.01; ^c Mock vs. B p<0.01, and B vs. BY p<0.05. Data represent five mice per group.</p

B. adolescentis Feeding Results in Increased Plasmacytoid DC Frequency.

<p>(<b>A</b>) Intraepithelial dendritic cells (ieDC) were analyzed by gating on linage negative (CD3ε, CD19, DX5) and CD11c-positive cells, as demonstrated in exemplarily depicted dot plots. Graph represents mean percentage ± SEM of CD11c⁺ intraepithelial leukocytes of untreated mock (M, dark gray bar), <i>B. adolescentis</i> fed (B, white bar), <i>Yersinia</i> infected (Y, black bar), as well as <i>B. adolescentis</i> fed and <i>Yersinia</i> infected mice (BY, gray bar). (<b>B</b>) Histograms represent mean percentage ± SD of CD11b-expressing conventional ieDCs (solid black line) and respective fluorescence minus one control (gray filled histogram). (<b>C</b>) Dot plots indicate mean percentage ± SD of B220 and PDCA-1-expressing plasmacytoid ieDCs of mock (M), <i>B. adolescentis</i> fed (B), <i>Yersinia</i> infected (Y), as well as <i>B. adolescentis</i> fed and <i>Yersinia</i> infected mice (BY). Data represent five mice per group.</p

Cell Counts of Intraepithelial Plasmacytoid DCs, and Lamina Propria FoxP3⁺ T_reg Cells.

<p>Numbers indicate mean cell counts±SD of vancomycin and metronidazole treated mock (M)and <i>B. adolescentis</i> (B) fed mice. Data represent five mice per group. ^a p = 0.00494.</p

Refolding of invasin membrane anchor (InvMA).

<p><b>(A)</b>Heat-modifiability of refolded InvMA. Guanidine-denatured, recombinant InvMA (expected molecular weight  = 29 kDa) was refolded by rapid dilution into 1% LDAO. The majority of the protein remains in the supernatant (SN) after centrifugation. Heating refolded InvMA at 95°C in the presence of urea leads to slower migration in SDS-PAGE compared to an unheated sample (RT  =  room temperature). The faster migration of the unheated sample is typical of folded β-barrel membrane proteins. (<b>B</b>) CD spectrum of refolded InvMA in LDAO buffer. The spectrum is typical of a β-structured protein with the characteristic broad minimum at 215 nm, showing that InvMA is folded.</p