Important roles of multiple Sp1 binding sites and epigenetic modifications in the regulation of the methionine sulfoxide reductase B1 (MsrB1) promoter-5

<p><b>Copyright information:</b></p><p>Taken from "Important roles of multiple Sp1 binding sites and epigenetic modifications in the regulation of the methionine sulfoxide reductase B1 (MsrB1) promoter"</p><p>http://www.biomedcentral.com/1471-2199/8/39</p><p>BMC Molecular Biology 2007;8():39-39.</p><p>Published online 22 May 2007</p><p>PMCID:PMC1885803.</p><p></p>showing MsrA (26 kDa) and Sp1 (105 kDa) protein levels

Important roles of multiple Sp1 binding sites and epigenetic modifications in the regulation of the methionine sulfoxide reductase B1 (MsrB1) promoter-1

<p><b>Copyright information:</b></p><p>Taken from "Important roles of multiple Sp1 binding sites and epigenetic modifications in the regulation of the methionine sulfoxide reductase B1 (MsrB1) promoter"</p><p>http://www.biomedcentral.com/1471-2199/8/39</p><p>BMC Molecular Biology 2007;8():39-39.</p><p>Published online 22 May 2007</p><p>PMCID:PMC1885803.</p><p></p>uence of the band from the RACE -2 primer reaction is reported. The upstream sequence from the fragment cloned from the band is in upper case and the arrow indicates the transcription start site (+ 1). The open reading frame codifying for MsrB1 is in lower case and the start codon is in boldface type

Important roles of multiple Sp1 binding sites and epigenetic modifications in the regulation of the methionine sulfoxide reductase B1 (MsrB1) promoter-7

<p><b>Copyright information:</b></p><p>Taken from "Important roles of multiple Sp1 binding sites and epigenetic modifications in the regulation of the methionine sulfoxide reductase B1 (MsrB1) promoter"</p><p>http://www.biomedcentral.com/1471-2199/8/39</p><p>BMC Molecular Biology 2007;8():39-39.</p><p>Published online 22 May 2007</p><p>PMCID:PMC1885803.</p><p></p>g region of with respect to the start site of transcription; the y axis indicates the percentage of the subclones that were methylated

Important roles of multiple Sp1 binding sites and epigenetic modifications in the regulation of the methionine sulfoxide reductase B1 (MsrB1) promoter-0

<p><b>Copyright information:</b></p><p>Taken from "Important roles of multiple Sp1 binding sites and epigenetic modifications in the regulation of the methionine sulfoxide reductase B1 (MsrB1) promoter"</p><p>http://www.biomedcentral.com/1471-2199/8/39</p><p>BMC Molecular Biology 2007;8():39-39.</p><p>Published online 22 May 2007</p><p>PMCID:PMC1885803.</p><p></p>t levels in MCF7 and MDA-MB231 cells were analysed using Chemi Doc System (Bio-Rad) and normalized by GAPDH signal intensities of the corresponding lanes. The data are reported as mean ± S.D. of three independent experiments; * P < 0.001. Immunoblotting of MCF7 and MDA-MB231 extracts with a mouse monoclonal anti-MsrB1. The MsrB1 protein (13 kDa) levels in MCF7 and MDA-MB231 cells were analysed using Chemi Doc System (Bio-Rad) and normalized by β-actin (42 kDa) signal intensities of the corresponding lanes. Values are reported as mean ± S.D. of at least five different immunoblotting experiments; ** P < 0.005

Important roles of multiple Sp1 binding sites and epigenetic modifications in the regulation of the methionine sulfoxide reductase B1 (MsrB1) promoter-4

<p><b>Copyright information:</b></p><p>Taken from "Important roles of multiple Sp1 binding sites and epigenetic modifications in the regulation of the methionine sulfoxide reductase B1 (MsrB1) promoter"</p><p>http://www.biomedcentral.com/1471-2199/8/39</p><p>BMC Molecular Biology 2007;8():39-39.</p><p>Published online 22 May 2007</p><p>PMCID:PMC1885803.</p><p></p>complexes. The control + lane was DNA that had been sonicated and pre-cleared with protein G beads. The control-lanes were processed according to the protocol, but did not have any Ab added to the samples. PCR were performed on isolated DNA using primers encompassing MsrB1 promoter region -169 to +76. A schematic map of the amplified DNA fragment (245 bp) containing Sp1 binding motifs and TSS position is illustrated as well

Important roles of multiple Sp1 binding sites and epigenetic modifications in the regulation of the methionine sulfoxide reductase B1 (MsrB1) promoter-3

<p><b>Copyright information:</b></p><p>Taken from "Important roles of multiple Sp1 binding sites and epigenetic modifications in the regulation of the methionine sulfoxide reductase B1 (MsrB1) promoter"</p><p>http://www.biomedcentral.com/1471-2199/8/39</p><p>BMC Molecular Biology 2007;8():39-39.</p><p>Published online 22 May 2007</p><p>PMCID:PMC1885803.</p><p></p> was designed P-203 Sp1EMut. Luciferase reporter activity was assessed following transfection into MCF7 cells. Luciferase activity was inhibited by approximately 80% by mutation of Sp1E site. () the sequences spanning Sp1-binding sites (Sp1C and Sp1E) contained in the P-296 WT construct were subjected to site-directed mutagenesis. The constructs obtained were designated as P-296 Sp1CMut and P-296 Sp1EMut. In addition a double mutant containing both mutations was indicated as P-296 Sp1C/EMut. The mutated constructs were transiently expressed in MCF7 cells for luciferase assays. Luciferase activity was inhibited by approximately 30% by mutation of Sp1C site; 24% by mutation of Sp1E site and by approximately 76% by mutation of both Sp1 sites (Sp1C/Sp1E). The results are expressed as mean ± S.D. of at least five different experiments, in duplicate for each construct. Statistically significant differences compared to the appropriate WT construct are indicated by *P < 0.005 and **P < 0.002

IL-22 and IDO1 Affect Immunity and Tolerance to Murine and Human Vaginal Candidiasis

<div><p>The ability to tolerate <i>Candida albicans</i>, a human commensal of the gastrointestinal tract and vagina, implicates that host defense mechanisms of resistance and tolerance cooperate to limit fungal burden and inflammation at the different body sites. We evaluated resistance and tolerance to the fungus in experimental and human vulvovaginal candidiasis (VVC) as well as in recurrent VVC (RVVC). Resistance and tolerance mechanisms were both activated in murine VVC, involving IL-22 and IL-10-producing regulatory T cells, respectively, with a major contribution by the enzyme indoleamine 2,3-dioxygenase 1 (IDO1). IDO1 was responsible for the production of tolerogenic kynurenines, such that replacement therapy with kynurenines restored immunoprotection to VVC. In humans, two functional genetic variants in <i>IL22</i> and <i>IDO1</i> genes were found to be associated with heightened resistance to RVVC, and they correlated with increased local expression of IL-22, IDO1 and kynurenines. Thus, IL-22 and IDO1 are crucial in balancing resistance with tolerance to <i>Candida</i>, their deficiencies are risk factors for RVVC, and targeting tolerance via therapeutic kynurenines may benefit patients with RVVC.</p></div

Vaginal candidiasis in IL-22- or IDO1-deficient mice.

<p>C57BL/6, IL-22- or IDO1-deficient mice (<i>n</i> = 6) were intravaginally inoculated with 5×10⁶<i>C. albicans</i> blastoconidia. (<b>A</b>) Periodic acid-Schiff-staining of vaginal sections and inflammatory cell recruitment in vaginal fluids (May–Grünwald Giemsa staining in the insets) at different days post-infection (dpi). Representative images of histology sections and vaginal fluids were acquired with a 40× and 100× objective respectively. Scale bars, 100 µm. (<b>B</b>) Polymorphonuclear cells (PMNs) quantification in the vaginal fluids. PMNs were identified by nuclear morphology and enumerated per field at ×100 magnification. Each point represents an individual mouse, and horizontal bar indicates the means. **<i>P</i><0.01 and ***<i>P</i><0.001, IDO1- or IL-22-deficient <i>vs.</i> wild-type mice at the indicated days. (<b>C</b>) <i>S100a8</i> and <i>S100a9</i> mRNA expression in vaginal tissue by real-time RT-PCR. The mRNA-normalized data were expressed as relative mRNA in IDO1- or IL-22-deficient <i>vs.</i> wild-type mice. *<i>P</i><0.05 and **<i>P</i><0.01, IDO1- or IL-22-deficient <i>vs.</i> wild-type mice at the indicated days. (<b>D</b>) Levels of calprotectin during vaginal candidiasis. **<i>P</i><0.01, IDO1- or IL-22-deficient <i>vs.</i> wild-type mice. (<b>E</b>) Vaginal fungal burden in mice at different dpi. CFU were quantified by culturing serial dilutions of vaginal fluids (VF) from each mouse and expressed as Log₁₀ CFU/100 µl VF ± s.e.m. *<i>P</i><0.05, IDO1- or IL-22-deficient <i>vs.</i> wild-type mice at the days indicated. Data are pooled or representative (histology) from four independent experiments. (<b>F</b>) Cytokine levels (pg/mg, cytokine/total proteins for each sample) in the vaginal fluids (at 3 dpi for IL-17F and IL-17A). Results represent mean cytokine levels (± s.e.m.) from samples pooled from three experiments (<i>n</i> = 4–6 total samples per group). *<i>P</i><0.05, IDO1- or IL-22-deficient <i>vs.</i> wild-type mice. (<b>G</b>) Levels of IL-22 (pg/mg, cytokine/total proteins) and (<b>H</b>) fungal growth (at 3 dpi) in mice treated with 300 µg of mAb neutralizing IL-22 or isotype control mAb (None) given intraperitoneally the day of and 1 day after the primary infection. (<b>I</b>) Fungal growth (at 3 dpi) in mice treated intravaginally with rIL-22 or PBS (None) the day of and 1 and 2 days after the infection. Pooled data from two experiments (<i>n</i> = 6). *<i>P</i><0.05 and ***<i>P</i><0.001, treated <i>vs.</i> untreated mice. N.S., not significant.</p

IDO1 and kynurenines mediate tolerance in murine VVC.

<p>(<b>A</b>) IDO1 protein and (<b>B</b>) gene expression in the vagina of C57BL/6 mice (<i>n</i> = 4) intravaginally infected with <i>C. albicans</i>. Proteins in vaginal cell lysates (3 dpi) were visualized by western blotting with rabbit polyclonal IDO1 specific antibody. Scanning densitometry was done on a Scion Image apparatus. Western blots out of 2 independent experiments and corresponding pixel density ratio normalized against β-tubulin. <i>Ido1</i> mRNA expression [normalized to mRNA of naïve (dpi 0) mice] in vaginal tissue (RT-PCR) at different dpi. (<b>C</b>) Relative concentrations of kynurenines (Kyn) and (<b>D</b>) kynurenine-to-tryptophan (Kyn/Trp) ratio in vaginal fluids at different dpi. Pooled results from 3 different experiments. *<i>P</i><0.05, IDO1-deficient <i>vs.</i> C57BL/6 mice at the days indicated. N.S., not significant. (<b>E</b>) Vaginal fungal growth (Log₁₀ CFU/100 µl VF ± s.e.m.) at different dpi in mice (<i>n</i> = 6) treated intraperitoneally with a mixture of l-kynurenine, 3-hydroxykynurenine and 3-hydroxyanthranilic acid or PBS (None). Pooled data from 3 different experiments.*<i>P</i><0.05, treated <i>vs.</i> untreated mice at the days indicated. N.S., not significant. (<b>F</b>) Periodic acid-Schiff-stained vaginal sections and inflammatory cell recruitment in vaginal fluids (May–Grünwald Giemsa staining in the insets) acquired with a 40× and 100× objective, respectively, at 21 dpi. Scale bars, 100 µm. Representative image from 3 experiments. (<b>G</b>) Cytokine levels (pg/mg, cytokine/total proteins for each sample, at 21 dpi) in the vaginal fluids of mice treated as above. Pooled data from 3 different experiments. *<i>P</i><0.05, treated <i>vs.</i> untreated (None) mice. (<b>H</b>) Vaginal immunohistochemistry of naïve or infected mice at 3 days after re-challenge. Double staining was done with anti-IL-10-FITC and polyclonal rabbit to FoxP3 followed by anti-rabbit TRITC. Cell nuclei were stained with DAPI (blue). Representative pictures (out of 2 experiments) were taken with a 20× objective. Scale bars, 50 µm.</p

Vaginal candidiasis in IL-17A- or IL-17F-deficient mice.

<p>(<b>A</b>) Vaginal fungal burden in C57BL/6, IL-17A- or IL-17F-deficient mice (<i>n</i> = 6) intravaginally inoculated with 5×10⁶<i>C. albicans</i> blastospores. CFU were quantified by culturing serial dilutions of vaginal fluid (VF) from each mouse at different days post-infection (dpi) and expressed as Log₁₀ CFU/100 µl VF ± s.e.m. Pooled data from 3 experiments. (<b>B</b>) Histological analysis of periodic acid-Schiff-stained vaginal sections and inflammatory cell recruitment in vaginal fluids (May–Grünwald Giemsa staining in the insets) at 3 dpi. Representative images (out of 3 experiments) of histology sections and vaginal fluids were acquired with a 40× and 100× objective respectively. Scale bars, 100 µm. (<b>C</b>) Polymorphonuclear cells (PMNs) quantification in the vaginal fluids at different dpi. PMNs were identified by nuclear morphology and enumerated per field at ×100 magnification. Each point represents an individual mouse, and horizontal bar indicates the means. (<b>D</b>) Cytokine levels (pg/mg, cytokine/total proteins for each sample) in the vaginal fluids of naïve or infected (3 dpi) mice. Data are pooled or representative (histology) from 3 independent experiments. * <i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001, knockout <i>vs.</i> wild-type mice. N.S., not significant.</p