11,655 research outputs found
Gal-9 binds CD44 on T cells and enhances T-cell activation.
No full text(A) Images showing the expression of CD44 and Gal-9 on the surface of T cells. (B) Cumulative data showing percent colocalization of Gal-9 with CD44 and CD137. (C) Percent of Gal-9+ T cells in the presence or absence of recombinant Gal-9 following in vitro incubation. (D) Percent CD44 clustering in the presence and absence of Gal-9. (E) Representative plots and (F) cumulative data showing percentages of CD4+ T cells expressing CD38 or (G) HLA-DR following stimulation with anti-CD3/CD28 antibodies in the presence of exogenous Gal-9 (1,000 pg/ml) and/or Gal-9 and anti-CD44 and anti-CD137 antibodies. (H) Representative plots and (I) cumulative data showing percentages of CD8+ T cells expressing CD38 or (J) HLA-DR following stimulation with anti-CD3/CD28 antibodies in the presence of exogenous Gal-9 (1,000 pg/ml) and/or Gal-9 and anti-CD44 and anti-CD137 antibodies. (K) Representative plots and (L) cumulative data showing phospho-LCK (Y505) in CD4+ and (M) CD8+ T cells in the absence and presence of Gal-9 (1,000 pg/ml). The underlying data can be found in S1 Data. Gal-9, Galectin-9.</p
Gal-9 epitope recognition by Gal-Nab1 and Gal-Nab2.
No full textA. Apoptotic cell death assessed by flow cytometry in CD3+ T cells treated for 36h with gal-9 (gal-9S; 40 nM) alone or in combination with mAbs (ctrl IgG1, Gal-Nab1, Gal-Nab2; 67 nM) pre-incubated or not with scramble peptide (Scr.) or gal-9 CTB-peptide (6.7 μM). B. Human recombinant gal-9 was immobilized in 96-wells plates and binding of Gal-Nab1 and Gal-Nab2 mAbs was measured after pre-incubation with overlapping peptides representative of human gal-9, as described under “Material and Methods”. Gal-Nab1 (left) or Gal-Nab2 (right) were then detected using secondary HRP-conjugated anti-mouse antibodies. Percentages of inhibition induced by each peptide were calculated from the absorbance data as described under “Material and Methods”.</p
Anti-gal-9 mAbs efficiently neutralize gal-9-induced apoptosis in primary T cells.
No full textCD3+ T-cells were isolated from healthy donors, activated by a combination of CD3/CD28 antibodies and treated or not with gal-9 (gal-9S; 40 nM) alone or in combination with lactose (5 mM), control isotype mAbs (ctrl IgG1) or anti-gal-9 mAbs (Gal-Nab1 and Gal-Nab2) at 67 nM (i.e. 10 μg/mL). After 36 h, they were subjected to annexin-V/PI staining and flow cytometry analysis. A. Examples of flow cytometry plots for purified CD3+ cells from one donor. B. Synthesis of data from 3 similar experiments made with CD3+ cells from 3 donors. C and D. Dose-response curves for apoptotic cell death (annexin-V+ PI+) (C) or PS translocation (annexin-V+ PI-) (D) in activated CD3+ cells treated for 36h with gal-9 combined with increasing concentrations of Gal-Nab1 and Gal-Nab2 (0.3 to 100 nM). Empty squares indicate the percentages obtained in conditions without gal-9. Black crosses indicate the percentages obtained with isotype control IgG1 mAbs used at maximal concentration (100 nM). Data are presented as means ± SEM of three independent experiments made with three distinct donors.</p
Activation of Silent gal Genes in the lac-gal Regulon of Streptococcus thermophilus
No full textStreptococcus thermophilus strain CNRZ 302 is unable to ferment galactose, neither that generated intracellularly by lactose hydrolysis nor the free sugar. Nevertheless, sequence analysis and complementation studies with Escherichia coli demonstrated that strain CNRZ 302 contained structurally intact genes for the Leloir pathway enzymes. These were organized into an operon in the order galKTE, which was preceded by a divergently transcribed regulator gene, galR, and followed by a galM gene and the lactose operon lacSZ. Results of Northern blot analysis showed that the structural gal genes were transcribed weakly, and only in medium containing lactose, by strain CNRZ 302. However, in a spontaneous galactose-fermenting mutant, designated NZ302G, the galKTE genes were well expressed in cells grown on lactose or galactose. In both CNRZ 302 and the Gal+ mutant NZ302G, the transcription of the galR gene was induced by growth on lactose. Disruption of galR indicated that it functioned as a transcriptional activator of both the gal and lac operons while negatively regulating its own expression. Sequence analysis of the gal promoter regions of NZ302G and nine other independently isolated Gal+ mutants of CNRZ 302 revealed mutations at three positions in the galK promoter region, which included substitutions at positions -9 and -15 as well as a single-base-pair insertion at position -37 with respect to the main transcription initiation point. Galactokinase activity measurements and analysis of gusA reporter gene fusions in strains containing the mutated promoters suggested that they were gal promoter-up mutations. We propose that poor expression of the gal genes in the galactose-negative S. thermophilus CNRZ 302 is caused by naturally occurring mutations in the galK promoter.
Effect of anti-gal-9 mAbs on the “T<sub>CM</sub>-like” phenotype shifting induced by gal-9 in resting PBMCs.
No full textA-B. PBMCs freshly isolated from healthy donors were treated for one week with human recombinant gal-9 (Gal-9S; 40 nM) with or without combination with lactose (5 mM), control isotype antibody (ctrl IgG1), Gal-Nab1 or Gal-Nab2 (67 nM i.e. 10 μg/mL). A. Flow cytometry analysis was performed to determine the level of naive (CCR7+ CD45RO-), central memory (CCR7+ CD45RO+) effector memory (CCR7- CD45RO+) and effector (CCR7- CD45RO-) T cells within the CD3+ population. At least 50% of the cells were alive. Dead cells were gated out. B. For each treatment condition, percentages of cells with an apparent central memory phenotype were normalized with the basal level of this subpopulation in untreated cells. Data are represented as means ± SEM of four independent experiments with different donors. **p<0.01; ns: not significant; compared with gal-9 treatment (one-way ANOVA/Dunnet post-test).</p
X-gal-negative Newly Formed Cardiomyocytes Increase Following Cardiac Injury.
No full text(A) X-gal staining and immunofluorescent images of sections comparing sham and myocardial infarction (MI) CreLacZ mice: left panel, sham; middle panel, MI remote area; and right panel, MI area. The upper panels show X-gal-stained images. The bottom panels show the corresponding immunofluorescent images (SA-actinin, green; laminin, red). Asterisks show X-gal-negative cardiomyocytes. Scale bar, 20 μm. The X-gal-negative cardiomyocytes are further enlarged in the inset. Scale bar, 10 μm. (B) Number of X-gal-negative cardiomyocytes per area 3 months after MI: sham (n = 5) and MI (n = 5). *p < 0.05. (C) Comparison of the number of X-gal-negative cardiomyocytes in the MI remote and MI area 3 months after MI (n = 5 per area). *p < 0.05. (D) Left: comparison of the cross-sectional area between X-gal-negative (MI2w neg) and -positive (MI2w pos) cardiomyocytes at 2 weeks after MI. Right: comparison of the cross-sectional area of X-gal-negative cardiomyocytes at 2 weeks (MI2w neg) and 6 months (MI6m neg) after MI. n = 50–66 cardiomyocytes pooled from two MI mice per group. *p < 0.05. The Mann–Whitney U-test was used for statistical analysis.</p
Anti-gal-9 mAbs inhibit the “Th1-like” phenotype shifting induced by gal-9 in resting or stimulated PBMCs.
No full textA-C. PBMCs from healthy donors were stimulated or not with anti-CD3/CD28 antibodies and treated for one week with human recombinant gal-9 (Gal-9S; 40 nM) with or without combination with lactose (5 mM), control isotype antibody (ctrl IgG1), Gal-Nab1 or Gal-Nab2 (67 nM i.e. 10 μg/mL). Intracellular cytokine expression was assessed by flow cytometry as explained under “Materials and Methods”. At least 50% of the cells were alive. Dead cells were gated out. A. Examples of flow cytometry plots obtained with stimulated PBMCs for one donor after gating on the CD3+ T cell population: IFN-γ (upper plots) and IL-2 (lower plots) expression were analyzed. B-C. Percentages of CD3+ IFNγ+ (upper histogram) and CD3+ IL-2+ (lower histogram) cells were normalized with the basal percentages obtained in untreated cells using either unstimulated (B) or stimulated (C) PBMCs. Data are represented as means ± SEM of four independent experiments with different donors. All statistical differences displayed are compared with gal-9 treatment; ***p<0.001; **p<0.01; *p<0.05; ns: not significant (one-way ANOVA/Dunnet post-test).</p
Gal-4 protein levels in PaTu-S and PaTu-T cells, and localization of Gal-4 in PaTu-T/Gal-4.
No full text<p><b>A)</b> Proteins from whole-cell extracts (75 ug total protein) and culture medium (4 days culture, 25 ul) of PaTu-S (P-S), PaTu-T (P-T), PaTu-T/Gal-4 (P-T/Gal-4) and PaTu-T/mock (P-T/M) were separated by SDS-PAGE. After transfer of the proteins to a nitrocellulose membrane, the blots were stained using goat anti-hGal-4 for detection of Gal-4, and mouse anti-tubulin as control for the presence of intracellular protein. <b>B)</b> Photographs of representative ICC analysis of the cellular localization of Gal-4 in PaTu-T/Gal-4 and PaTu-T/mock cells<b>.</b> Gal-4 was detected using Alexa-labeled anti-Gal-4 Abs (green), Actin was stained using Phalloidin (red) and nucleus staining obtained using HOESCHS (blue); the third panel shows the merging of the different stainings. Bar = 25 µm.</p
LIF Increases the Number of X-gal-negative Newly Formed Cardiomyocytes and the Frequency of BrdU Incorporation.
No full text(A) Representative images of X-gal-negative cardiomyocytes in PBS- and LIF-treated CreLacZ mice. X-gal staining (left) and immunofluorescence images (right; SA-actinin, green; laminin, red; nuclei were stained with DAPI, blue) are shown. Arrows indicate X-gal-negative cardiomyocytes. Scale bar, 20 μm. (B) Number of X-gal-negative cardiomyocytes in the MI remote area (closed bar) and the MI area (open bar) in the PBS- and LIF-treated mice after MI. Asterisks indicate significant differences between two groups. *p < 0.05. (C) Frequencies of BrdU-positive X-gal-negative cardiomyocytes among all X-gal-negative cardiomyocytes in the MI remote area (closed bar) and the MI area (open bar) in the PBS- and LIF-treated mice after MI. Asterisks indicate significant differences between two groups. *p < 0.05. Three pairs of adjacent heart sections were examined per mouse. Data indicate the mean of five mice. (D) Representative images of a pair of adjacent sections. Left panels were stained with SA-actinin (green), laminin (red), DAPI (blue), and X-gal. Right panels were stained with BrdU (green), DAPI (blue) and X-gal. Top panels represent a group of X-gal-negative cardiomyocytes in the MI area of a LIF-treated mouse. The enlarged images of a region of interest (white square) are shown. Two X-gal-negative cardiomyocytes (arrowheads in the left panels) and the corresponding BrdU-positive nuclei (arrowheads in the right panels) are shown. Scale bar, 20 μm.</p
Gal-4 and Gal-4 binding sites in PaTu-S and PaTu-T cells.
No full text<p>Detection of endogenous Gal-4, and Gal-4 ligands, in PaTu-S and PaTu-T cells by flow cytometry. A histogram of one representative experiment is depicted for each condition of least two independent experiments. <b>A)</b> Dot plots of Gal-4 staining of permeabilized PaTu-S and PaTu-T cells. Gal-4 was detected at 4°C with anti-hGal-4 Abs in fixed permeabilized cells. Secondary Abs staining without anti-hGal-4 Abs was used as background autofluorescence control. <b>B)</b> Presence of endogenous bound Gal-4 to the surface of PaTu-S and PaTu-T after washing the cells with 500 mM lactose prior to Gal-4 staining. The presence of Gal-4 was established by FACS analysis using anti-hGal-4 Abs at 4°C. Endogenous Gal-4 bound to the surface is shown by a black line. <b>C)</b> The presence of Gal-4 binding sites on PaTu-S and PaTu-T cells was determined after washing the cells with 500 mM lactose prior to Gal-4 staining. The binding of externally added recombinant (rec) hGal-4 (5 µg/ml, black line) was investigated. Binding of rec hGal-4 to the surface could be inhibited by adding lactose (dark field). Background staining with secondary Abs is depicted as light grey fields in B and C.</p
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