Journal: Journal of Innate Immunity

Article Title: Intestinal Epithelium-Derived Galectin-9 Is Involved in the Immunomodulating Effects of Nondigestible Oligosaccharides

doi: 10.1159/000350515

Figure Lengend Snippet: Expression and secretion of galectin-9 in HT-29 cells is regulated by TLR9 ligation and scGOS/lcFOS. a-c Analysis of galectin (Gal) mRNA expression by HT-29 cells by quantitative PCR analysis. Evaluation of the expression profile of galectins by IEC (a). Apical TLR9 ligation of HT-29 cells, in the absence or presence of scGOS/lcFOS, specifically increases galectin-9 expression (b, c). Protein expression of galectin-4 and galectin-9 and its modulation upon TLR9 ligation and scGOS/lcFOS was confirmed by immunofluorescence microscopic staining of HT-29 monolayers (d). ELISA was performed in the basolateral supernatant of HT-29 monolayers (e). TLR9 ligation of HT-29 cells in the presence of scGOS/lcFOS enhanced galectin-9, but not galectin-4 secretion. n = 3 (a-d) or n = 6 (e) independent PBMC donors, means ± SEM, * p < 0.05, # p < 0.05, ** p < 0.01.

Article Snippet: IEC were fixed using 4% formalin in PBS for 10 min, permeabilized in 0.1% Triton X-100 (Sigma) and 1% BSA in PBS for 15 min and incubated with anti-human galectin-4 or galectin-9 antibodies or normal goat IgG as isotype control (all 0.75 μg/ml; R&D Systems) in 0.1% Triton X-100 and 1% BSA in PBS for 1 h. IEC were incubated with secondary Alexa Fluor 546 donkey anti-goat IgG (Invitrogen) in 0.1% Triton X-100 and 1% BSA for 30 min and embedded in Hoechst.

Techniques: Expressing, Ligation, Real-time Polymerase Chain Reaction, Immunofluorescence, Staining, Enzyme-linked Immunosorbent Assay

Journal: Journal of Innate Immunity

Article Title: Intestinal Epithelium-Derived Galectin-9 Is Involved in the Immunomodulating Effects of Nondigestible Oligosaccharides

doi: 10.1159/000350515

Figure Lengend Snippet: Galectin-9 neutralization in IEC/PBMC cocultures abrogates IFN-γ and IL-10 secretion by PBMC. HT-29 (unpolarized) and T84 (polarized) IEC were cocultured with CD3/CD28-activated PBMC for 24 h and apically exposed to TLR9 ligand in the presence or absence of scGOS/lcFOS. Basolateral galectin-9 was neutralized using TIM-3-Fc fusion protein. Galectin-9 neutralization abrogated the induction of IFN-γ (a, e) and IL-10 (b, f) by PBMC, but did not modulate IL-17A secretion by PBMC (c, g). Furthermore, the immunomodulating effects on IL-13 secretion induced by exposure of HT-29 or T84 cells to TLR9 ligand in the absence or presence of scGOS/lcFOS was abolished upon neutralization of galectin-9 (d, h). Means ± SEM of 3 independent PBMC donors. * p < 0.05, ** p < 0.01, *** p < 0.001. GF = scGOS/lcFOS.

Article Snippet: IEC were fixed using 4% formalin in PBS for 10 min, permeabilized in 0.1% Triton X-100 (Sigma) and 1% BSA in PBS for 15 min and incubated with anti-human galectin-4 or galectin-9 antibodies or normal goat IgG as isotype control (all 0.75 μg/ml; R&D Systems) in 0.1% Triton X-100 and 1% BSA in PBS for 1 h. IEC were incubated with secondary Alexa Fluor 546 donkey anti-goat IgG (Invitrogen) in 0.1% Triton X-100 and 1% BSA for 30 min and embedded in Hoechst.

Techniques: Neutralization

Journal: Journal of Innate Immunity

Article Title: Intestinal Epithelium-Derived Galectin-9 Is Involved in the Immunomodulating Effects of Nondigestible Oligosaccharides

doi: 10.1159/000350515

Figure Lengend Snippet: Functional responses of galectin-9-stimulated naïve CD4+CD45RA+ T cells and moDC. To study whether galectin-9 acts on moDC, T cells or via modulation of the interaction between moDC and T cells, galectin-9 was added during the differentiation of monocytes to moDC or during coculture of moDC with naïve CD4+ T cells (a). Naïve CD4+CD45RA+ T cells, moDC or moDC-T-cell cocultures were exposed to recombinant human galectin-9. Mixed lymphocyte cultures were maintained for 5 days. For Treg-cell differentiation, exogenous TGF-β was added to the culture. For intracellular cytokines, T cells were restimulated as described in the Materials and Methods. Stimulation of naïve CD4+ T cells with galectin-9 did not induce CD25+Foxp3+ Treg cells (b, d), IL-10 (b, e) or IFN-γ expression by CD4+ T cells (b, f). However, moDC generated in the presence of galectin-9 (Gal-9 DC) or exogenous galectin-9 added to mixed lymphocyte cultures with unconditioned (control) moDC resulted in increased differentiation of CD25+Foxp3+ Treg cells (c, d) and IL-10-expressing CD4+ cells (c, e). Only Gal-9 DC had the capacity to induce IFN-γ expression by CD4+ T cells (c, f). n = 3 independent PBMC donors. * p < 0.05, ** p < 0.01 vs. control DC.

Article Snippet: IEC were fixed using 4% formalin in PBS for 10 min, permeabilized in 0.1% Triton X-100 (Sigma) and 1% BSA in PBS for 15 min and incubated with anti-human galectin-4 or galectin-9 antibodies or normal goat IgG as isotype control (all 0.75 μg/ml; R&D Systems) in 0.1% Triton X-100 and 1% BSA in PBS for 1 h. IEC were incubated with secondary Alexa Fluor 546 donkey anti-goat IgG (Invitrogen) in 0.1% Triton X-100 and 1% BSA for 30 min and embedded in Hoechst.

Techniques: Functional Assay, Recombinant, Cell Differentiation, Expressing, Generated

Journal: Scientific Reports

Article Title: Tissue and plasma levels of galectins in patients with high grade serous ovarian carcinoma as new predictive biomarkers

doi: 10.1038/s41598-017-13802-5

Figure Lengend Snippet: Predictive value of circulating galectins on the OS and DFS. ( A) Gal-8 and ( B ) gal-9 concentration in the plasma of healthy donors and ovarian cancer patients. ( C ) Kaplan-Meier curves of the 5-years OS and 5-years DFS according to the presence of gal-8 and gal-9 in the plasma of HGSC patients. Blue bar: galectin negative samples, red bar: galectin positive samples.

Article Snippet: Rabbit anti-human gal-9 (1:100, Abcam), goat anti-human gal-9 (1:50, R&D Systems), rabbit anti-human LC3B (1:200, Sigma) and rabbit anti-human Cox IV (1:500, New England Biolabs, Ipswich, MA) primary antibodies were used.

Techniques: Concentration Assay

Journal: Scientific Reports

Article Title: Tissue and plasma levels of galectins in patients with high grade serous ovarian carcinoma as new predictive biomarkers

doi: 10.1038/s41598-017-13802-5

Figure Lengend Snippet: Predictive value of circulating galectin-8 and -9 according to CA-125 levels. Kaplan-Meier curves of 5-years OS and 5-years DFS according to the presence of circulating gal-8 and gal-9 in the plasma of ( A ) CA125 LOW or ( B ) CA125 HIGH patients. Blue bar: galectin negative samples, red bar: galectin positive samples.

Article Snippet: Rabbit anti-human gal-9 (1:100, Abcam), goat anti-human gal-9 (1:50, R&D Systems), rabbit anti-human LC3B (1:200, Sigma) and rabbit anti-human Cox IV (1:500, New England Biolabs, Ipswich, MA) primary antibodies were used.

Techniques:

Journal: Scientific Reports

Article Title: Tissue and plasma levels of galectins in patients with high grade serous ovarian carcinoma as new predictive biomarkers

doi: 10.1038/s41598-017-13802-5

Figure Lengend Snippet: Combining gal-8 and gal-9 increases the predictive value for OS and HFS. ( A ) Kaplan-Meier curves of the 5-years OS and 5-years DFS according to the presence of either or both gal-8 and gal-9 in the plasma of HGSC patients. Blue bar: Gal-8 LOW /Gal-9 LOW , green bar: Gal-8 High /Gal-9 LOW , Yellow bar: Gal-8 LOW /Gal-9 High , Magenta bar: Gal-8 High /Gal-9 HIGH . ( B ) Correlation between the concentration of gal-8 and gal-9 in the plasma of HGSC patients. ( C ) Kaplan-Meier curves of the 5-years OS and 5-years DFS according to the presence of both gal-8 and gal-9 in the plasma of HGSC patients. Blue bar: Gal-8 LOW /Gal-9 LOW , Gal-8 High /Gal-9 LOW or Gal-8 LOW /Gal-9 High sample; Red bar: Gal-8 High /Gal-9 HIGH samples. Patients with plasma gal-8 ≥ 2.7 ng/ml or plasma gal-9 ≥ 0.73 ng/ml were considered Gal-8 High and Gal-9 High , respectively.

Article Snippet: Rabbit anti-human gal-9 (1:100, Abcam), goat anti-human gal-9 (1:50, R&D Systems), rabbit anti-human LC3B (1:200, Sigma) and rabbit anti-human Cox IV (1:500, New England Biolabs, Ipswich, MA) primary antibodies were used.

Techniques: Concentration Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Activation of Human Basophils by A549 Lung Epithelial Cells Reveals a Novel IgE-dependent Response Independent of Allergen 1

doi: 10.4049/jimmunol.1700055

Figure Lengend Snippet: A, Flow cytometry demonstrating Gal-3 expression by A549 and BEAS-2B cell lines. B, Relative expression of Gal-3 and Gal-9 mRNA and C, protein for these galectins in the two epithelial cell lines.

Article Snippet: Special Reagents, buffers, and media The following reagents were purchased: crystallized human serum albumin (Calbiochem-Behring Corp, La Jolla, CA); PIPES, FCS, crystallized BSA, and n-acetyllactosamine (LacNAc) (Sigma-Aldrich, Allentown, PA); gentamicin, IMDM and nonessential amino acids (Life Technologies, Inc, Grand Island, NY); Percoll (Pharmacia Biotec, Inc, Piscataway, NJ); rhIL-3 (Biosource, Inc. Camarillo, CA); rhTSLP, rhGM-CSF, rhIL-5, Anti-galectin (Gal)-9 antibody and ELISAs (R&D Systems, Minneapolis, MN); anti-Gal-3 and IgG 1 isotype control (Santa Cruz, Dallas, TX); Gal-3 ELISAs (e-Bioscience, San Diego, CA).

Techniques: Flow Cytometry, Expressing