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  • 99
    Millipore fingolimod
    Potentiation of dasatinib cytotoxicity by <t>FTY‐720.</t> (A) Cell proliferation of dasatinib‐resistant pancreatic cancer cell lines (MiaPaCa2, SU8686) treated with increasing concentrations of dasatinib either with or without FTY720 for 48 h.
    Fingolimod, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 27 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Tocris fty 720
    Effect of S1P receptor agonist pretreatment with <t>FTY720</t> and SEW2871 on contractile responses at 1 day following recovery from Sham or CPB. Left Panels : responses to PE in mesenteric arteries. Right panels : responses to SE in coronary arteries. Abbreviations: CPB, cardiopulmonary bypass; PE, phenylephrine; SE, serotonin. Data are mean±SEM. a - indicates P
    Fty 720, supplied by Tocris, used in various techniques. Bioz Stars score: 93/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Cayman Chemical fingolimod
    Effect of <t>fingolimod,</t> fingolimod-phosphate and the serums of patients under fingolimod therapy on CD56 expression in vitro . ( a ) Kinetic change of CD56 + T cells co-cultured with fingolimod (F) or fingolimod-phosphate (F-p) for 5 days in healthy subjects (HS) and multiple sclerosis patients without disease-modifying drug (MS without DMD). ( b ) CD56 expression on T cells stimulated by PHA in the presence or absence of fingolimod or fingolimod phosphate at a concentration of 0.1 and 1 μM. Representative graphs of CD56 expression on T cells cultured in the presence of PHA, PHA with 0.1 μM fingolimod and PHA with 0.1 μM fingolimod-phosphate (left panel) ( c ) CD56 expression on T cells of MS without DMD co-cultured with serum of the same patient after fingolimod therapy. Each column represents mean ± SEM. p-values: *p
    Fingolimod, supplied by Cayman Chemical, used in various techniques. Bioz Stars score: 92/100, based on 49 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Selleck Chemicals ao fty 720
    Effect of <t>FTY720</t> on inflammatory cytokines, including monocyte chemoattractant protein-1 (MCP-1), macrophage-related TNF-α and IL-6, arginase-1 and IL-10, in the three groups. (A) Western blot analysis of MCP-1 and M1 and M2 macrophage-related cytokines; β-actin was used as a control. (B) Quantitative analysis of protein expression of MCP-1 and macrophage-related cytokines. (C) Expression of MCP-1 and macrophage-related cytokines mRNA; β-actin was used as a control. Values are presented as the mean±SEM. n =6. b P
    Ao Fty 720, supplied by Selleck Chemicals, used in various techniques. Bioz Stars score: 94/100, based on 33 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Novartis fingolimod
    Comparison of NEDA status in the core study and the first extension year by treatment group (A) IFN-switch group (B) <t>Continuous-fingolimod</t> group. Data presented here are for the pooled fingolimod 0.5 and 1.25 mg groups. N, total number of patients in the group; n, number of patients achieving NEDA; IFN, interferon; NEDA, no evidence of disease activity.
    Fingolimod, supplied by Novartis, used in various techniques. Bioz Stars score: 94/100, based on 467 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    BioVision fty 720
    Dynamics of H60-specific CD8 + T cells under different conditions. ( a ) J15 CD8 + T cells were harvested from the spleen and peritoneum of adoptive hosts at the indicated times after exposure to different priming conditions, ( b ) quantified and ( c ) analysed by flow cytometry to compare CFSE-dilution profiles. Numbers and CFSE profiles of J15 CD8 + T cells from <t>FTY-720-treated</t> mice were obtained on day 5 post priming and were incorporated in plots designated as day 5+F in ( b , c ). ( d ) Contraction degrees (peak numbers/contraction numbers) of splenic J15 CD8 + T cells obtained from adoptive hosts are plotted. Data shown were obtained from adoptive hosts transferred in high numbers ( b–d ). ( e ) Dynamics of J15-LucTg CD8 + T cells in B6-albino-adoptive hosts (transferred with 1 × 10 5 cells) were monitored periodically after priming under different conditions through secondary expansion. Those in B6-albino-adoptive hosts without priming are also shown as control (naive). Photon flux values as the primary responses progressed were plotted after designating the whole body as the ROI. Fold changes in peak values in 1 o peak relative to the values of 1 o day 0 (before priming) are shown. The mean relative ratios of 2 o peak: 2 o day 0 (before boosting) photon flux values are presented in the table. Data shown represent at least three ( n =2 per group; b–d ) or two ( n =3 per group; e ) independent experiments. A.T., Adoptive Transfer. Data are presented as means±s.e.m. Not Significant (NS) P > 0.05, * P
    Fty 720, supplied by BioVision, used in various techniques. Bioz Stars score: 88/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Brinkmann Instruments v fty 720 mechanism
    A diagram shows differentially expressed genes (in a category of ECM organization/inflammation response) by Affymetrix gene array. Gene expression profiling in sham hearts was normalized to yellow, which is defined as unchanged. Orange and red represent increased expression, and grey and blue represent decreased expression. Fold change of expression is shown for <t>TAC/FTY-720</t> versus TAC only (n=2 for each group). All genes identified as significantly altered (P
    V Fty 720 Mechanism, supplied by Brinkmann Instruments, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Novartis fty720 fingolimod gilenya
    A diagram shows differentially expressed genes (in a category of ECM organization/inflammation response) by Affymetrix gene array. Gene expression profiling in sham hearts was normalized to yellow, which is defined as unchanged. Orange and red represent increased expression, and grey and blue represent decreased expression. Fold change of expression is shown for <t>TAC/FTY-720</t> versus TAC only (n=2 for each group). All genes identified as significantly altered (P
    Fty720 Fingolimod Gilenya, supplied by Novartis, used in various techniques. Bioz Stars score: 88/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Selleck Chemicals pump fingolimod
    Electrophysiological characteristics of <t>fingolimod</t> treatment in CD86 −/− NOD mice. ( A ) The compound motor action potential amplitude (CMAP) of the sciatic nerve declined similarly in both fingolimod and vehicle treated CD86 −/− NOD mice. ( B ) Motor fibers showed electrophysiological signs of demyelination indicated by the decrease of motor nerve conduction velocity (MCV), which was more pronounced in the fingolimod treated mice compared to vehicle treatment. ( C ) F-wave latency increased in both groups with disease progression. Again, fingolimod treated mice were more affected than mice in the vehicle group. Similar to motor fibers, the ( D ) sensory nerve action potential amplitude (SNAP) and ( E ) sensory nerve conduction velocity (SCV) declined in both treatment groups. Statistical analysis: ( A ) Kruskall-Wallis test with Dunn’s method, ( B – E ) 2-way ANOVA with Sidak post hoc, group sizes: n = 10 (vehicle), n = 9–10 (fingolimod). *p ≤ 0.05, ns: not significant.
    Pump Fingolimod, supplied by Selleck Chemicals, used in various techniques. Bioz Stars score: 93/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Selleck Chemicals fty720
    Effect of <t>FTY720</t> on inflammatory cytokines, including monocyte chemoattractant protein-1 (MCP-1), macrophage-related TNF-α and IL-6, arginase-1 and IL-10, in the three groups. (A) Western blot analysis of MCP-1 and M1 and M2 macrophage-related cytokines; β-actin was used as a control. (B) Quantitative analysis of protein expression of MCP-1 and macrophage-related cytokines. (C) Expression of MCP-1 and macrophage-related cytokines mRNA; β-actin was used as a control. Values are presented as the mean±SEM. n =6. b P
    Fty720, supplied by Selleck Chemicals, used in various techniques. Bioz Stars score: 92/100, based on 667 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore fty 720 propranolol propranolol hydrochloride
    Ischemic lesion size in propranolol-treated (a) FTY720-treated (b) and the respective vehicle-treated control mice with tMCAO of 1 h occlusion time after an observation period of 24 h after tMCAO (propranolol: n = 7, vehicle: n = 9; FTY720: n = 8, vehicle: n = 10). The box boundaries mark the 25th and 75th percentile, the line within the box indicates the mean. Whiskers above and below the box mark the minimum and maximum. Statistical significance was assessed with Student’s t test, * p = 0.01. FTY720, <t>fingolimod;</t> tMCAO, transient middle cerebral artery occlusion.
    Fty 720 Propranolol Propranolol Hydrochloride, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Potentiation of dasatinib cytotoxicity by FTY‐720. (A) Cell proliferation of dasatinib‐resistant pancreatic cancer cell lines (MiaPaCa2, SU8686) treated with increasing concentrations of dasatinib either with or without FTY720 for 48 h.

    Journal: Molecular Oncology

    Article Title: Activation of protein phosphatase 2A tumor suppressor as potential treatment of pancreatic cancer

    doi: 10.1016/j.molonc.2015.01.002

    Figure Lengend Snippet: Potentiation of dasatinib cytotoxicity by FTY‐720. (A) Cell proliferation of dasatinib‐resistant pancreatic cancer cell lines (MiaPaCa2, SU8686) treated with increasing concentrations of dasatinib either with or without FTY720 for 48 h.

    Article Snippet: Thioridazine, chlorpromazine, perphenazine, penfluridol, pimethixene, and fingolimod (FTY‐720) were from Sigma–Aldrich (St. Louis, MO).

    Techniques:

    Scheme representing inhibition of cell proliferation in pancreatic cancer cells by penfluridol and FTY‐720. Modulation of PI3K/AKT and MEK/ERK signaling pathways mediated through PP2A. Phosphorylation of AKT, p70S6K, and GSK3β are suppressed

    Journal: Molecular Oncology

    Article Title: Activation of protein phosphatase 2A tumor suppressor as potential treatment of pancreatic cancer

    doi: 10.1016/j.molonc.2015.01.002

    Figure Lengend Snippet: Scheme representing inhibition of cell proliferation in pancreatic cancer cells by penfluridol and FTY‐720. Modulation of PI3K/AKT and MEK/ERK signaling pathways mediated through PP2A. Phosphorylation of AKT, p70S6K, and GSK3β are suppressed

    Article Snippet: Thioridazine, chlorpromazine, perphenazine, penfluridol, pimethixene, and fingolimod (FTY‐720) were from Sigma–Aldrich (St. Louis, MO).

    Techniques: Inhibition

    Anti‐proliferative activity of FTY‐720 in pancreatic cancer cells. (A) Colony formation assay. Two pancreatic cancer cell lines (MiaPaCa2, Panc0403) were grown in soft agar and treated with FTY‐720 for 14 days. Representative

    Journal: Molecular Oncology

    Article Title: Activation of protein phosphatase 2A tumor suppressor as potential treatment of pancreatic cancer

    doi: 10.1016/j.molonc.2015.01.002

    Figure Lengend Snippet: Anti‐proliferative activity of FTY‐720 in pancreatic cancer cells. (A) Colony formation assay. Two pancreatic cancer cell lines (MiaPaCa2, Panc0403) were grown in soft agar and treated with FTY‐720 for 14 days. Representative

    Article Snippet: Thioridazine, chlorpromazine, perphenazine, penfluridol, pimethixene, and fingolimod (FTY‐720) were from Sigma–Aldrich (St. Louis, MO).

    Techniques: Activity Assay, Colony Assay

    Electron microscopy shows organelle transformation mediated by FTY-720 induced fusions. ( A,B ) EM micrographs of representative cultured control chromaffin cells ( A ), and after incubation with FTY-720 for 15 min ( B ). ( C ) Mean ± SEM vesicle number (white bars) and area (black bars) per condition (N control = 6 cells, 1235 vesicles; N FTY720 15min = 6 cells, 542 vesicles). ( D ) Mean ± SEM values of mitochondrial roundness (white bars) and area (black bars) per condition (N control = 6 cells, 932 mitochondria; N FTY720 15min = 6 cells, 406 mitochondria). (E ) EM micrographs from cultured control chromaffin cells and after FTY-720 (15 min) showing different fusion levels (I to III) after treatment. ( F–H ) Amplified images of representative cytoplasmic sections including examples of all types of fusion (rows) and levels (columns; I to III) between vesicles (V), and mitochondria (M). Red stars and boxes indicate interaction between elements. Statistical significance was assessed with a Mann-Whitney U-Test: **P ≤ 0.01, ***P ≤ 0.001. Scale bars represent 2 μm ( A,B,E ) and 1 μm ( F–H ).

    Journal: Scientific Reports

    Article Title: Multiple sclerosis drug FTY-720 toxicity is mediated by the heterotypic fusion of organelles in neuroendocrine cells

    doi: 10.1038/s41598-019-55106-w

    Figure Lengend Snippet: Electron microscopy shows organelle transformation mediated by FTY-720 induced fusions. ( A,B ) EM micrographs of representative cultured control chromaffin cells ( A ), and after incubation with FTY-720 for 15 min ( B ). ( C ) Mean ± SEM vesicle number (white bars) and area (black bars) per condition (N control = 6 cells, 1235 vesicles; N FTY720 15min = 6 cells, 542 vesicles). ( D ) Mean ± SEM values of mitochondrial roundness (white bars) and area (black bars) per condition (N control = 6 cells, 932 mitochondria; N FTY720 15min = 6 cells, 406 mitochondria). (E ) EM micrographs from cultured control chromaffin cells and after FTY-720 (15 min) showing different fusion levels (I to III) after treatment. ( F–H ) Amplified images of representative cytoplasmic sections including examples of all types of fusion (rows) and levels (columns; I to III) between vesicles (V), and mitochondria (M). Red stars and boxes indicate interaction between elements. Statistical significance was assessed with a Mann-Whitney U-Test: **P ≤ 0.01, ***P ≤ 0.001. Scale bars represent 2 μm ( A,B,E ) and 1 μm ( F–H ).

    Article Snippet: FTY-720 toxicity on cultured chromaffin cells We used cell monolayer cultures incubated with supplemented DMEM (see above) and trypan blue solution (1:5: Sigma Aldrich, T6146) cultured for 24 h and evaluated at: t0 , 5 min, 10 min, 15 min, 1 h, 2 h, 4 h, 8 h, 12 h and 24 h. We prepared control cultures and others that were treated with FTY-270 (20 μM; Sigma Aldrich, SML0700, Lot#056M4724V) for 5, 10 and 15 min.

    Techniques: Electron Microscopy, Transformation Assay, Cell Culture, Incubation, Amplification, MANN-WHITNEY

    Morphometric changes of vesicles and mitochondria in control and FTY-720 treated cells. ( A ) Time-lapse confocal fluorescence microscopy of a representative cultured chromaffin cell expressing RFP-NPY (red) to show vesicles from control and FTY-720 treated cells (5, 10 and 15 min). ( B ) Vesicular size frequency distribution comparing all conditions ( C ; FTY720 5,10 and 15 min) by size. ( C,D ) Box and whiskers and scatter plot vertical graphs of vesicle area ( C ) and number ( D ) by condition (N = 10 cells; N control = 686, N FTY720 5min = 554, N FTY720 10min = 419, N FTY720 15min = 393 vesicles). (E ) Time-lapse confocal fluorescence microscopy images from a representative live cultured chromaffin cell labeled with MitoTracker green to identify the mitochondria in control and FTY-720 treated cells (5, 10 and 15 min). ( F–I ) Box and whiskers vertical graphs of the mitochondrial area ( F ), number ( G ), roundness ( H ) and aspect ratio ( I ) per condition (N = 5 cells: N control = 804, N FTY720 5min = 621, N FTY720 10min = 424, N FTY720 15min = 350 mitochondria). Box and whiskers graph shows the mean as the central box line, the SEM as the box limits (top and bottom) and the amplitude variability through the whiskers (box emergent lines). Statistical significance was assessed by a Kruskal-Wallis Test: n.s., non-significant (P ≥ 0.05), *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. Scale bars represent 1 μm.

    Journal: Scientific Reports

    Article Title: Multiple sclerosis drug FTY-720 toxicity is mediated by the heterotypic fusion of organelles in neuroendocrine cells

    doi: 10.1038/s41598-019-55106-w

    Figure Lengend Snippet: Morphometric changes of vesicles and mitochondria in control and FTY-720 treated cells. ( A ) Time-lapse confocal fluorescence microscopy of a representative cultured chromaffin cell expressing RFP-NPY (red) to show vesicles from control and FTY-720 treated cells (5, 10 and 15 min). ( B ) Vesicular size frequency distribution comparing all conditions ( C ; FTY720 5,10 and 15 min) by size. ( C,D ) Box and whiskers and scatter plot vertical graphs of vesicle area ( C ) and number ( D ) by condition (N = 10 cells; N control = 686, N FTY720 5min = 554, N FTY720 10min = 419, N FTY720 15min = 393 vesicles). (E ) Time-lapse confocal fluorescence microscopy images from a representative live cultured chromaffin cell labeled with MitoTracker green to identify the mitochondria in control and FTY-720 treated cells (5, 10 and 15 min). ( F–I ) Box and whiskers vertical graphs of the mitochondrial area ( F ), number ( G ), roundness ( H ) and aspect ratio ( I ) per condition (N = 5 cells: N control = 804, N FTY720 5min = 621, N FTY720 10min = 424, N FTY720 15min = 350 mitochondria). Box and whiskers graph shows the mean as the central box line, the SEM as the box limits (top and bottom) and the amplitude variability through the whiskers (box emergent lines). Statistical significance was assessed by a Kruskal-Wallis Test: n.s., non-significant (P ≥ 0.05), *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. Scale bars represent 1 μm.

    Article Snippet: FTY-720 toxicity on cultured chromaffin cells We used cell monolayer cultures incubated with supplemented DMEM (see above) and trypan blue solution (1:5: Sigma Aldrich, T6146) cultured for 24 h and evaluated at: t0 , 5 min, 10 min, 15 min, 1 h, 2 h, 4 h, 8 h, 12 h and 24 h. We prepared control cultures and others that were treated with FTY-270 (20 μM; Sigma Aldrich, SML0700, Lot#056M4724V) for 5, 10 and 15 min.

    Techniques: Fluorescence, Microscopy, Cell Culture, Expressing, Labeling

    A new mechanism of action for FTY-720 affecting neuroendocrine function and cell death. FTY-720 accumulates in the cell cytoplasm where it can promote SNARE complex assembly and reduce F-actin mobility, thereby limiting vesicle transport to the plasma membrane for secretion. SNARE complex enhancement mediates intracellular vesicle fission. Vesicles could fuse among themselves (homotypic fusion) or with mitochondria (heterotypic fusion). Mixed organelles cannot maintain their membrane potential, leading to cell death.+Indicates potentiation and − reduction.

    Journal: Scientific Reports

    Article Title: Multiple sclerosis drug FTY-720 toxicity is mediated by the heterotypic fusion of organelles in neuroendocrine cells

    doi: 10.1038/s41598-019-55106-w

    Figure Lengend Snippet: A new mechanism of action for FTY-720 affecting neuroendocrine function and cell death. FTY-720 accumulates in the cell cytoplasm where it can promote SNARE complex assembly and reduce F-actin mobility, thereby limiting vesicle transport to the plasma membrane for secretion. SNARE complex enhancement mediates intracellular vesicle fission. Vesicles could fuse among themselves (homotypic fusion) or with mitochondria (heterotypic fusion). Mixed organelles cannot maintain their membrane potential, leading to cell death.+Indicates potentiation and − reduction.

    Article Snippet: FTY-720 toxicity on cultured chromaffin cells We used cell monolayer cultures incubated with supplemented DMEM (see above) and trypan blue solution (1:5: Sigma Aldrich, T6146) cultured for 24 h and evaluated at: t0 , 5 min, 10 min, 15 min, 1 h, 2 h, 4 h, 8 h, 12 h and 24 h. We prepared control cultures and others that were treated with FTY-270 (20 μM; Sigma Aldrich, SML0700, Lot#056M4724V) for 5, 10 and 15 min.

    Techniques:

    Colocalization of organelles during FTY-720 treatment. ( A ) Time-lapse TIRF microscopy images from a representative cultured chromaffin cell expressing RFP-NPY (red) to detect vesicles at the plasma membrane of control and FTY-720 treated cells (5, 10 and 15 min). ( B ) Vesicular masks at plasma membrane in each condition. ( C ) Time-lapse confocal fluorescence images from a representative live cultured chromaffin cell labelled with MitoTracker green to visualize mitochondria, and expressing RFP-NPY (red) to detect vesicles in control and FTY-720 treated cells (5, 10 and 15 min). ( D ) Representative cytofluorograms per condition (Y axis for red fluorescence and X axis for green fluorescence). The black lines indicate the fluorescence colocalization as a direct correlation of the line’s slopes. ( E ) Mean ± SEM values of the cortical vesicular mask area per condition (N = 5 cells: N control = 106, N FTY720 5min = 59, N FTY720 10min = 37, N FTY720 15min = 19 masks). ( F ) Mean ± SEM values of the Pearson’s Correlation Coefficient per condition (N = 5 cells: N = 10 points/cell). Statistical significance was assessed with a Kruskal-Wallis Test: ***P ≤ 0.001. Scale bars represent 1 μm.

    Journal: Scientific Reports

    Article Title: Multiple sclerosis drug FTY-720 toxicity is mediated by the heterotypic fusion of organelles in neuroendocrine cells

    doi: 10.1038/s41598-019-55106-w

    Figure Lengend Snippet: Colocalization of organelles during FTY-720 treatment. ( A ) Time-lapse TIRF microscopy images from a representative cultured chromaffin cell expressing RFP-NPY (red) to detect vesicles at the plasma membrane of control and FTY-720 treated cells (5, 10 and 15 min). ( B ) Vesicular masks at plasma membrane in each condition. ( C ) Time-lapse confocal fluorescence images from a representative live cultured chromaffin cell labelled with MitoTracker green to visualize mitochondria, and expressing RFP-NPY (red) to detect vesicles in control and FTY-720 treated cells (5, 10 and 15 min). ( D ) Representative cytofluorograms per condition (Y axis for red fluorescence and X axis for green fluorescence). The black lines indicate the fluorescence colocalization as a direct correlation of the line’s slopes. ( E ) Mean ± SEM values of the cortical vesicular mask area per condition (N = 5 cells: N control = 106, N FTY720 5min = 59, N FTY720 10min = 37, N FTY720 15min = 19 masks). ( F ) Mean ± SEM values of the Pearson’s Correlation Coefficient per condition (N = 5 cells: N = 10 points/cell). Statistical significance was assessed with a Kruskal-Wallis Test: ***P ≤ 0.001. Scale bars represent 1 μm.

    Article Snippet: FTY-720 toxicity on cultured chromaffin cells We used cell monolayer cultures incubated with supplemented DMEM (see above) and trypan blue solution (1:5: Sigma Aldrich, T6146) cultured for 24 h and evaluated at: t0 , 5 min, 10 min, 15 min, 1 h, 2 h, 4 h, 8 h, 12 h and 24 h. We prepared control cultures and others that were treated with FTY-270 (20 μM; Sigma Aldrich, SML0700, Lot#056M4724V) for 5, 10 and 15 min.

    Techniques: Microscopy, Cell Culture, Expressing, Fluorescence

    SNAP-25 is involved in organelle fusion induced by FTY-720. ( A,B ) Time-lapse confocal fluorescence microscopy images from representative cultured chromaffin cells expressing WT and the truncated form GFP-SNAP-25 ∆9 (green), combined with RFP-NPY (red) expression to show vesicles ( A ) and MitoTracker Red CMXRos labeling to detect mitochondria ( B ), in control cells and after FTY-720 treatment (5, 10 and 15 min). ( C,D ) Mean ± SEM values of vesicular area ( C ) and number ( D ) for WT SNAP-25 cells (N = 5 cells: N control = 686, N FTY720 5min = 507, N FTY720 10min = 419, N FTY720 15min = 263 vesicles). ( E-F ) Mean ± SEM values of vesicular area ( E ) and number ( F ) for ∆9 SNAP-25 cells (N = 5 cells: N control = 601, N FTY720 5min = 587, N FTY720 10min = 584, N FTY720 15min = 490 vesicles). ( G,H ) Mean ± SEM values of mitochondrial area ( G ) and roundness ( H ) for WT SNAP-25 cells (N = 5 cells: N control = 665, N FTY720 5min = 612, N FTY720 10min = 420, N FTY720 15min = 341 mitochondria). ( I,J ) Mean ± SEM values of mitochondria area ( I ) and roundness ( J ) for ∆9 SNAP-25 cells (N = 5 cells: N control = 579, N FTY720 5min = 582, N FTY720 10min = 557, N FTY720 15min = 497 mitochondria). Statistical significance was assessed with a Two-way ANOVA Test: n.s. non-significant (P ≥ 0.05), *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. Scale bars represent 1 μm.

    Journal: Scientific Reports

    Article Title: Multiple sclerosis drug FTY-720 toxicity is mediated by the heterotypic fusion of organelles in neuroendocrine cells

    doi: 10.1038/s41598-019-55106-w

    Figure Lengend Snippet: SNAP-25 is involved in organelle fusion induced by FTY-720. ( A,B ) Time-lapse confocal fluorescence microscopy images from representative cultured chromaffin cells expressing WT and the truncated form GFP-SNAP-25 ∆9 (green), combined with RFP-NPY (red) expression to show vesicles ( A ) and MitoTracker Red CMXRos labeling to detect mitochondria ( B ), in control cells and after FTY-720 treatment (5, 10 and 15 min). ( C,D ) Mean ± SEM values of vesicular area ( C ) and number ( D ) for WT SNAP-25 cells (N = 5 cells: N control = 686, N FTY720 5min = 507, N FTY720 10min = 419, N FTY720 15min = 263 vesicles). ( E-F ) Mean ± SEM values of vesicular area ( E ) and number ( F ) for ∆9 SNAP-25 cells (N = 5 cells: N control = 601, N FTY720 5min = 587, N FTY720 10min = 584, N FTY720 15min = 490 vesicles). ( G,H ) Mean ± SEM values of mitochondrial area ( G ) and roundness ( H ) for WT SNAP-25 cells (N = 5 cells: N control = 665, N FTY720 5min = 612, N FTY720 10min = 420, N FTY720 15min = 341 mitochondria). ( I,J ) Mean ± SEM values of mitochondria area ( I ) and roundness ( J ) for ∆9 SNAP-25 cells (N = 5 cells: N control = 579, N FTY720 5min = 582, N FTY720 10min = 557, N FTY720 15min = 497 mitochondria). Statistical significance was assessed with a Two-way ANOVA Test: n.s. non-significant (P ≥ 0.05), *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. Scale bars represent 1 μm.

    Article Snippet: FTY-720 toxicity on cultured chromaffin cells We used cell monolayer cultures incubated with supplemented DMEM (see above) and trypan blue solution (1:5: Sigma Aldrich, T6146) cultured for 24 h and evaluated at: t0 , 5 min, 10 min, 15 min, 1 h, 2 h, 4 h, 8 h, 12 h and 24 h. We prepared control cultures and others that were treated with FTY-270 (20 μM; Sigma Aldrich, SML0700, Lot#056M4724V) for 5, 10 and 15 min.

    Techniques: Fluorescence, Microscopy, Cell Culture, Expressing, Labeling

    Secretory responses of chromaffin cells exposed to FTY-720. ( A,B) Amperometric measurements from cultured bovine chromaffin cells stimulated by depolarization with a 59 mM KCl solution: control cells ( A ) and cells treated with a 20 µM FTY-720 for 15 mins ( B ). ( C ) Averaged amperometric fusion events and parameters from control (N = 1262 events) and FTY-720-treated cells (N = 901 events). ( D ) FTY-720 enhances the quantal amount of catecholamines released per single event. ( E ) Distribution of the quantal events in control and FTY-720 treated cells. ( F ) Example of amperometrical responses to cell depolarization in repetitive 10 s pulses. ( G ) Mean secretory responses obtained by amperometry trace integration during each pulse (charge) in both control and FTY-treated cells.Statistical significance was assessed with a Mann-Whitney U-Test: **P ≤ 0.005. ***P ≤ 0.0001.

    Journal: Scientific Reports

    Article Title: Multiple sclerosis drug FTY-720 toxicity is mediated by the heterotypic fusion of organelles in neuroendocrine cells

    doi: 10.1038/s41598-019-55106-w

    Figure Lengend Snippet: Secretory responses of chromaffin cells exposed to FTY-720. ( A,B) Amperometric measurements from cultured bovine chromaffin cells stimulated by depolarization with a 59 mM KCl solution: control cells ( A ) and cells treated with a 20 µM FTY-720 for 15 mins ( B ). ( C ) Averaged amperometric fusion events and parameters from control (N = 1262 events) and FTY-720-treated cells (N = 901 events). ( D ) FTY-720 enhances the quantal amount of catecholamines released per single event. ( E ) Distribution of the quantal events in control and FTY-720 treated cells. ( F ) Example of amperometrical responses to cell depolarization in repetitive 10 s pulses. ( G ) Mean secretory responses obtained by amperometry trace integration during each pulse (charge) in both control and FTY-treated cells.Statistical significance was assessed with a Mann-Whitney U-Test: **P ≤ 0.005. ***P ≤ 0.0001.

    Article Snippet: FTY-720 toxicity on cultured chromaffin cells We used cell monolayer cultures incubated with supplemented DMEM (see above) and trypan blue solution (1:5: Sigma Aldrich, T6146) cultured for 24 h and evaluated at: t0 , 5 min, 10 min, 15 min, 1 h, 2 h, 4 h, 8 h, 12 h and 24 h. We prepared control cultures and others that were treated with FTY-270 (20 μM; Sigma Aldrich, SML0700, Lot#056M4724V) for 5, 10 and 15 min.

    Techniques: Cell Culture, MANN-WHITNEY

    Altered mitochondrial membrane potential and reduced cell viability promoted by FTY-720. ( A ) Time-lapse confocal fluorescence microscopy images from a representative control and FTY-720 treated cultured chromaffin cell (t0, 5, 10 and 15 min) labeled with MitoTracker Red CMXRos to identify mitochondria. ( B,C ) Mean ± SEM mitochondrial membrane potential in terms of fluorescence intensity (white bars) and density (black bars) for control condition ( B ) (N control = 5 cells; N T0 = 797, N 5min = 781, N 10min = 764, N 15min = 760 mitochondria) and FTY720 treatment ( C ) (N FTY720 = 5 cells; N TO = 804, N 5min = 621, N 10min = 424, N 15min = 350 mitochondria). ( D ) Time-lapse optical microscopy images from representative chromaffin cell cultures under control conditions and after FTY-720 treatment for 15 min evaluated at different times: 15 min, 4 h, 12 h, and 24 h. ( E ) Mean ± SEM of the viability of control (white bars) and FTY-720 treated (black bars) cells evaluated at distinct times (N t0 = 467 ( C ), 488 (FTY); N 5min = 503 ( C ), 453 (FTY); N 10min = 497 ( C ), 468 (FTY); N 15min = 510 ( C ), 505 (FTY); N 1h = 477 ( C ), 464 (FTY); N 2h = 396 ( C ), 478 (FTY); N 4h = 470 ( C ), 482 (FTY); N 8h = 567 ( C ), 538 (FTY); N 12h = 477 ( C ), 445 (FTY); N 24h = 455 ( C ), 476 (FTY) cells). Statistical significance was assessed with a Mann-Whitney Test: n.s. non-significant, (P ≥ 0.05), *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. Scale bars represent 1 μm ( A ) and 15 μm ( B ).

    Journal: Scientific Reports

    Article Title: Multiple sclerosis drug FTY-720 toxicity is mediated by the heterotypic fusion of organelles in neuroendocrine cells

    doi: 10.1038/s41598-019-55106-w

    Figure Lengend Snippet: Altered mitochondrial membrane potential and reduced cell viability promoted by FTY-720. ( A ) Time-lapse confocal fluorescence microscopy images from a representative control and FTY-720 treated cultured chromaffin cell (t0, 5, 10 and 15 min) labeled with MitoTracker Red CMXRos to identify mitochondria. ( B,C ) Mean ± SEM mitochondrial membrane potential in terms of fluorescence intensity (white bars) and density (black bars) for control condition ( B ) (N control = 5 cells; N T0 = 797, N 5min = 781, N 10min = 764, N 15min = 760 mitochondria) and FTY720 treatment ( C ) (N FTY720 = 5 cells; N TO = 804, N 5min = 621, N 10min = 424, N 15min = 350 mitochondria). ( D ) Time-lapse optical microscopy images from representative chromaffin cell cultures under control conditions and after FTY-720 treatment for 15 min evaluated at different times: 15 min, 4 h, 12 h, and 24 h. ( E ) Mean ± SEM of the viability of control (white bars) and FTY-720 treated (black bars) cells evaluated at distinct times (N t0 = 467 ( C ), 488 (FTY); N 5min = 503 ( C ), 453 (FTY); N 10min = 497 ( C ), 468 (FTY); N 15min = 510 ( C ), 505 (FTY); N 1h = 477 ( C ), 464 (FTY); N 2h = 396 ( C ), 478 (FTY); N 4h = 470 ( C ), 482 (FTY); N 8h = 567 ( C ), 538 (FTY); N 12h = 477 ( C ), 445 (FTY); N 24h = 455 ( C ), 476 (FTY) cells). Statistical significance was assessed with a Mann-Whitney Test: n.s. non-significant, (P ≥ 0.05), *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. Scale bars represent 1 μm ( A ) and 15 μm ( B ).

    Article Snippet: FTY-720 toxicity on cultured chromaffin cells We used cell monolayer cultures incubated with supplemented DMEM (see above) and trypan blue solution (1:5: Sigma Aldrich, T6146) cultured for 24 h and evaluated at: t0 , 5 min, 10 min, 15 min, 1 h, 2 h, 4 h, 8 h, 12 h and 24 h. We prepared control cultures and others that were treated with FTY-270 (20 μM; Sigma Aldrich, SML0700, Lot#056M4724V) for 5, 10 and 15 min.

    Techniques: Fluorescence, Microscopy, Cell Culture, Labeling, MANN-WHITNEY

    Quantification of lung edema and injury in LPS-challenged mice (A) Lung edema was determined as the lung wet/dry weight ratio. Results are shown for samples taken at 48 h and 7 days after LPS challenge. (B) Lung injury scores show an obvious decrease in the severity of lung injury in the LPS+hUC-MSCs and LPS+FTY720 groups, and especially, in the LPS+hUC-MSCs+FTY720 group. Data are represented by the mean ± SD, n = 10 at each time point for each group. * P

    Journal: Oncotarget

    Article Title: Combination therapy of human umbilical cord mesenchymal stem cells and FTY720 attenuates acute lung injury induced by lipopolysaccharide in a murine model

    doi: 10.18632/oncotarget.20491

    Figure Lengend Snippet: Quantification of lung edema and injury in LPS-challenged mice (A) Lung edema was determined as the lung wet/dry weight ratio. Results are shown for samples taken at 48 h and 7 days after LPS challenge. (B) Lung injury scores show an obvious decrease in the severity of lung injury in the LPS+hUC-MSCs and LPS+FTY720 groups, and especially, in the LPS+hUC-MSCs+FTY720 group. Data are represented by the mean ± SD, n = 10 at each time point for each group. * P

    Article Snippet: A single intraperitoneal injection of FTY720 reduces murine lung injury obviously and low concentrations of FTY720 (0.1 mg/kg) significantly reduce lung permeability [ , ]; therefore, mice in the LPS+FTY720 and LPS+hUC-MSCs+FTY720 groups were given an intraperitoneal injection of FTY720 (0.1 mg/kg, 200 μl; Sigma, St. Louis, MO) at 24h after LPS administration.

    Techniques: Mouse Assay

    Mouse survival rates (n = 15 for each group) at 48 h after LPS challenge using Kaplan–Meier analysis followed by a log-rank test The survival of mice in the LPS+hUC-MSCs+FTY720 group was higher than that in other experimental groups, but there were not statistically significant in the differences ( P = 0.07, LPS+hUC-MSCs+FTY720 vs. LPS+PBS; P = 0.29 LPS+hUC-MSCs+FTY720 vs. LPS+hUC-MSCs; P = 0.31 LPS+ hUC-MSCs+FTY720 vs. LPS+FTY720).

    Journal: Oncotarget

    Article Title: Combination therapy of human umbilical cord mesenchymal stem cells and FTY720 attenuates acute lung injury induced by lipopolysaccharide in a murine model

    doi: 10.18632/oncotarget.20491

    Figure Lengend Snippet: Mouse survival rates (n = 15 for each group) at 48 h after LPS challenge using Kaplan–Meier analysis followed by a log-rank test The survival of mice in the LPS+hUC-MSCs+FTY720 group was higher than that in other experimental groups, but there were not statistically significant in the differences ( P = 0.07, LPS+hUC-MSCs+FTY720 vs. LPS+PBS; P = 0.29 LPS+hUC-MSCs+FTY720 vs. LPS+hUC-MSCs; P = 0.31 LPS+ hUC-MSCs+FTY720 vs. LPS+FTY720).

    Article Snippet: A single intraperitoneal injection of FTY720 reduces murine lung injury obviously and low concentrations of FTY720 (0.1 mg/kg) significantly reduce lung permeability [ , ]; therefore, mice in the LPS+FTY720 and LPS+hUC-MSCs+FTY720 groups were given an intraperitoneal injection of FTY720 (0.1 mg/kg, 200 μl; Sigma, St. Louis, MO) at 24h after LPS administration.

    Techniques: Mouse Assay

    CT scans of lung tissues at 48 h after LPS challenge (A) Control, (B) LPS+PBS, (C) LPS+hUC-MSCs, (D) LPS+FTY720, (E) LPS+ hUC-MSCs+ FTY720.

    Journal: Oncotarget

    Article Title: Combination therapy of human umbilical cord mesenchymal stem cells and FTY720 attenuates acute lung injury induced by lipopolysaccharide in a murine model

    doi: 10.18632/oncotarget.20491

    Figure Lengend Snippet: CT scans of lung tissues at 48 h after LPS challenge (A) Control, (B) LPS+PBS, (C) LPS+hUC-MSCs, (D) LPS+FTY720, (E) LPS+ hUC-MSCs+ FTY720.

    Article Snippet: A single intraperitoneal injection of FTY720 reduces murine lung injury obviously and low concentrations of FTY720 (0.1 mg/kg) significantly reduce lung permeability [ , ]; therefore, mice in the LPS+FTY720 and LPS+hUC-MSCs+FTY720 groups were given an intraperitoneal injection of FTY720 (0.1 mg/kg, 200 μl; Sigma, St. Louis, MO) at 24h after LPS administration.

    Techniques:

    Effect of S1P receptor agonist pretreatment with FTY720 and SEW2871 on contractile responses at 1 day following recovery from Sham or CPB. Left Panels : responses to PE in mesenteric arteries. Right panels : responses to SE in coronary arteries. Abbreviations: CPB, cardiopulmonary bypass; PE, phenylephrine; SE, serotonin. Data are mean±SEM. a - indicates P

    Journal: PLoS ONE

    Article Title: S1P1 Receptor Modulation Preserves Vascular Function in Mesenteric and Coronary Arteries after CPB in the Rat Independent of Depletion of Lymphocytes

    doi: 10.1371/journal.pone.0097196

    Figure Lengend Snippet: Effect of S1P receptor agonist pretreatment with FTY720 and SEW2871 on contractile responses at 1 day following recovery from Sham or CPB. Left Panels : responses to PE in mesenteric arteries. Right panels : responses to SE in coronary arteries. Abbreviations: CPB, cardiopulmonary bypass; PE, phenylephrine; SE, serotonin. Data are mean±SEM. a - indicates P

    Article Snippet: FTY720 and SEW2871 were purchased from Tocris Bioscience (United Kingdom) and were dissolved in 125 µl DMSO and diluted with 375 µl saline (0.9% NaCl).

    Techniques:

    Effect of S1P receptor agonist pretreatment with FTY720 and SEW2871 on relaxant reactivity at 1 day following recovery from Sham or CPB. ACh-induced relaxation was studied in mesenteric arteries (left panels A and B) and coronary arteries (right panels C and D). Abbreviations: CPB, cardiopulmonary bypass, ACh, acetylcholine. Data are mean±SEM. a- indicates P = 0.026 SEW2871 vs Vehicle, Repeated Measurements ANOVA, Bonferroni test; b- indicates P = 0.018 FTY720 vs Vehicle Repeated Measurements ANOVA, Bonferroni test; c- indicates P = 0.03 SEW2871 vs Vehicle, t-test; d- indicates P = 0.03 FTY720 vs Vehicle, t-test.

    Journal: PLoS ONE

    Article Title: S1P1 Receptor Modulation Preserves Vascular Function in Mesenteric and Coronary Arteries after CPB in the Rat Independent of Depletion of Lymphocytes

    doi: 10.1371/journal.pone.0097196

    Figure Lengend Snippet: Effect of S1P receptor agonist pretreatment with FTY720 and SEW2871 on relaxant reactivity at 1 day following recovery from Sham or CPB. ACh-induced relaxation was studied in mesenteric arteries (left panels A and B) and coronary arteries (right panels C and D). Abbreviations: CPB, cardiopulmonary bypass, ACh, acetylcholine. Data are mean±SEM. a- indicates P = 0.026 SEW2871 vs Vehicle, Repeated Measurements ANOVA, Bonferroni test; b- indicates P = 0.018 FTY720 vs Vehicle Repeated Measurements ANOVA, Bonferroni test; c- indicates P = 0.03 SEW2871 vs Vehicle, t-test; d- indicates P = 0.03 FTY720 vs Vehicle, t-test.

    Article Snippet: FTY720 and SEW2871 were purchased from Tocris Bioscience (United Kingdom) and were dissolved in 125 µl DMSO and diluted with 375 µl saline (0.9% NaCl).

    Techniques:

    Effect of fingolimod, fingolimod-phosphate and the serums of patients under fingolimod therapy on CD56 expression in vitro . ( a ) Kinetic change of CD56 + T cells co-cultured with fingolimod (F) or fingolimod-phosphate (F-p) for 5 days in healthy subjects (HS) and multiple sclerosis patients without disease-modifying drug (MS without DMD). ( b ) CD56 expression on T cells stimulated by PHA in the presence or absence of fingolimod or fingolimod phosphate at a concentration of 0.1 and 1 μM. Representative graphs of CD56 expression on T cells cultured in the presence of PHA, PHA with 0.1 μM fingolimod and PHA with 0.1 μM fingolimod-phosphate (left panel) ( c ) CD56 expression on T cells of MS without DMD co-cultured with serum of the same patient after fingolimod therapy. Each column represents mean ± SEM. p-values: *p

    Journal: Scientific Reports

    Article Title: Altered T cell phenotypes associated with clinical relapse of multiple sclerosis patients receiving fingolimod therapy

    doi: 10.1038/srep35314

    Figure Lengend Snippet: Effect of fingolimod, fingolimod-phosphate and the serums of patients under fingolimod therapy on CD56 expression in vitro . ( a ) Kinetic change of CD56 + T cells co-cultured with fingolimod (F) or fingolimod-phosphate (F-p) for 5 days in healthy subjects (HS) and multiple sclerosis patients without disease-modifying drug (MS without DMD). ( b ) CD56 expression on T cells stimulated by PHA in the presence or absence of fingolimod or fingolimod phosphate at a concentration of 0.1 and 1 μM. Representative graphs of CD56 expression on T cells cultured in the presence of PHA, PHA with 0.1 μM fingolimod and PHA with 0.1 μM fingolimod-phosphate (left panel) ( c ) CD56 expression on T cells of MS without DMD co-cultured with serum of the same patient after fingolimod therapy. Each column represents mean ± SEM. p-values: *p

    Article Snippet: In vitro analysis of effect of fingolimod regarding CD56 expression on T cells PBMC from healthy subjects and MS patients without DMD were cultured for 5 days at 1 × 106 cells/ml in the presence of fingolimod (0.01, 0.1, 1, 5, 10 μM; Cayman Chemical, Ann Arbor, MI,USA) or fingolimod-phosphate (0.01 μM, 0.1 μM, 1 μM, 5 μM, 10 μM; Cayman Chemical) in AIM-V medium.

    Techniques: Expressing, In Vitro, Cell Culture, Mass Spectrometry, Concentration Assay

    The frequency of Foxp3+ regulatory T cells in multiple sclerosis patients and healthy subjects. The frequency of regulatory T cells (Treg cells: CD3 + CD4 + CD25 + Foxp3 + cells) in CD3 + T cells (upper) and CD4 + T cells (bottom). HS: healthy subjects, nF-MS: multiple sclerosis patients without fingolimod therapy, F-MS: multiple sclerosis patients with fingolimod therapy, rem: remission, rel: relapse, rel-free: relapse-free. Bars represent median values. p-values: *p

    Journal: Scientific Reports

    Article Title: Altered T cell phenotypes associated with clinical relapse of multiple sclerosis patients receiving fingolimod therapy

    doi: 10.1038/srep35314

    Figure Lengend Snippet: The frequency of Foxp3+ regulatory T cells in multiple sclerosis patients and healthy subjects. The frequency of regulatory T cells (Treg cells: CD3 + CD4 + CD25 + Foxp3 + cells) in CD3 + T cells (upper) and CD4 + T cells (bottom). HS: healthy subjects, nF-MS: multiple sclerosis patients without fingolimod therapy, F-MS: multiple sclerosis patients with fingolimod therapy, rem: remission, rel: relapse, rel-free: relapse-free. Bars represent median values. p-values: *p

    Article Snippet: In vitro analysis of effect of fingolimod regarding CD56 expression on T cells PBMC from healthy subjects and MS patients without DMD were cultured for 5 days at 1 × 106 cells/ml in the presence of fingolimod (0.01, 0.1, 1, 5, 10 μM; Cayman Chemical, Ann Arbor, MI,USA) or fingolimod-phosphate (0.01 μM, 0.1 μM, 1 μM, 5 μM, 10 μM; Cayman Chemical) in AIM-V medium.

    Techniques: Mass Spectrometry

    Association of CD56 and CCR7 expression on T cells in healthy subjects (HS) and multiple sclerosis (MS) patients. ( a ) The frequency of CCR7 + T cells within CD3 + T cells in F-MS patients both in remission and at relapse. ( b ) The frequency of central memory T cells (CCR7 + CD45RA − T cells) within CD3 + , CD4 + or CD8 + T cells in F-MS patients both in remission and at relapse. ( c ) The frequency of CCR7 − T cells within CD56 + T cells in HS and each patient group. ( d ) The frequency of CD56 + cells within CCR7 − and CCR7 + T cells. ( e ) The frequency of CD56 + cells within naïve (CCR7 + CD45RA + ), central memory (CCR7 + CD45RA − ) (TCM), effector memory (CCR7 − CD45RA − ) (TEM) and CD45RA + TEM (TEMRA) subsets in CD4 + and CD8 + T cells. nF-MS: multiple sclerosis patients without fingolimod therapy, F-MS: multiple sclerosis patients with fingolimod therapy, rem: remission, rel: relapse, rel-free: relapse-free. Bars represent median values. p-values: *p

    Journal: Scientific Reports

    Article Title: Altered T cell phenotypes associated with clinical relapse of multiple sclerosis patients receiving fingolimod therapy

    doi: 10.1038/srep35314

    Figure Lengend Snippet: Association of CD56 and CCR7 expression on T cells in healthy subjects (HS) and multiple sclerosis (MS) patients. ( a ) The frequency of CCR7 + T cells within CD3 + T cells in F-MS patients both in remission and at relapse. ( b ) The frequency of central memory T cells (CCR7 + CD45RA − T cells) within CD3 + , CD4 + or CD8 + T cells in F-MS patients both in remission and at relapse. ( c ) The frequency of CCR7 − T cells within CD56 + T cells in HS and each patient group. ( d ) The frequency of CD56 + cells within CCR7 − and CCR7 + T cells. ( e ) The frequency of CD56 + cells within naïve (CCR7 + CD45RA + ), central memory (CCR7 + CD45RA − ) (TCM), effector memory (CCR7 − CD45RA − ) (TEM) and CD45RA + TEM (TEMRA) subsets in CD4 + and CD8 + T cells. nF-MS: multiple sclerosis patients without fingolimod therapy, F-MS: multiple sclerosis patients with fingolimod therapy, rem: remission, rel: relapse, rel-free: relapse-free. Bars represent median values. p-values: *p

    Article Snippet: In vitro analysis of effect of fingolimod regarding CD56 expression on T cells PBMC from healthy subjects and MS patients without DMD were cultured for 5 days at 1 × 106 cells/ml in the presence of fingolimod (0.01, 0.1, 1, 5, 10 μM; Cayman Chemical, Ann Arbor, MI,USA) or fingolimod-phosphate (0.01 μM, 0.1 μM, 1 μM, 5 μM, 10 μM; Cayman Chemical) in AIM-V medium.

    Techniques: Expressing, Mass Spectrometry, Transmission Electron Microscopy

    The frequency of proinflammatory cytokine-producing cells in response to PMA/ionomycin stimulation. ( a ) Representative data of FACS dot plots analyzing IFN-γ and IL-17A production gated on CD3 + T cells. Depicted as follows from left to right, a healthy subject (HS), a multiple sclerosis (MS) patient not on fingolimod in remission (nF-MS rem) and one at relapse (nF-MS rel), and a relapse-free patient with fingolimod therapy (rel-free F-MS) and one at relapse (F-MS rel). ( b ) The frequency of IL-17A-producing cells in CD3 + T cells (left) and that of IFN-γ-producing cells in CD3 + T cells (right). ( c ) The frequency of IFN-γ producing cells was compared between CD56 + and CD56 − T cells. ( d ) The frequency of IFN-γ-producing cells in CD4 + CD56 + T cells and CD4 + CD56 − T cells, CD8 + CD56 + T cells and CD8 + CD56 − T cells. Bars represent median values. Each column represents mean ± SEM. P-values: *p

    Journal: Scientific Reports

    Article Title: Altered T cell phenotypes associated with clinical relapse of multiple sclerosis patients receiving fingolimod therapy

    doi: 10.1038/srep35314

    Figure Lengend Snippet: The frequency of proinflammatory cytokine-producing cells in response to PMA/ionomycin stimulation. ( a ) Representative data of FACS dot plots analyzing IFN-γ and IL-17A production gated on CD3 + T cells. Depicted as follows from left to right, a healthy subject (HS), a multiple sclerosis (MS) patient not on fingolimod in remission (nF-MS rem) and one at relapse (nF-MS rel), and a relapse-free patient with fingolimod therapy (rel-free F-MS) and one at relapse (F-MS rel). ( b ) The frequency of IL-17A-producing cells in CD3 + T cells (left) and that of IFN-γ-producing cells in CD3 + T cells (right). ( c ) The frequency of IFN-γ producing cells was compared between CD56 + and CD56 − T cells. ( d ) The frequency of IFN-γ-producing cells in CD4 + CD56 + T cells and CD4 + CD56 − T cells, CD8 + CD56 + T cells and CD8 + CD56 − T cells. Bars represent median values. Each column represents mean ± SEM. P-values: *p

    Article Snippet: In vitro analysis of effect of fingolimod regarding CD56 expression on T cells PBMC from healthy subjects and MS patients without DMD were cultured for 5 days at 1 × 106 cells/ml in the presence of fingolimod (0.01, 0.1, 1, 5, 10 μM; Cayman Chemical, Ann Arbor, MI,USA) or fingolimod-phosphate (0.01 μM, 0.1 μM, 1 μM, 5 μM, 10 μM; Cayman Chemical) in AIM-V medium.

    Techniques: FACS, Mass Spectrometry

    Expression of cytotoxic molecules in each T cell subset of healthy subjects (HS) and multiple sclerosis (MS) patients. ( a ) Representative analysis of CD56 and granzyme B expression, gated on CD3 + T cells from left to right: a healthy subject, an MS patient not on fingolimod in remission (nF-MS rem) and one at relapse (nF-MS rel), a relapse-free MS patient with fingolimod therapy (rel-free F-MS) and one at relapse (F-MS rel). ( b ) The expression of granzyme B (GrzB), perforin, and Fas-Ligand (FasL) in or on CD3 + T cells are shown. ( c ) The frequency of cells expressing each cytotoxic molecule was compared between CD56 + and CD56 − T cells. ( d ) The frequency of cells expressing each cytotoxic molecule in CD4 + CD56 + T cells, CD4 + CD56 − T cells, CD8 + CD56 + T cells, and CD8 + CD56 − T cells. Bars represent median values. Each column represents mean ± SEM. p-values: *p

    Journal: Scientific Reports

    Article Title: Altered T cell phenotypes associated with clinical relapse of multiple sclerosis patients receiving fingolimod therapy

    doi: 10.1038/srep35314

    Figure Lengend Snippet: Expression of cytotoxic molecules in each T cell subset of healthy subjects (HS) and multiple sclerosis (MS) patients. ( a ) Representative analysis of CD56 and granzyme B expression, gated on CD3 + T cells from left to right: a healthy subject, an MS patient not on fingolimod in remission (nF-MS rem) and one at relapse (nF-MS rel), a relapse-free MS patient with fingolimod therapy (rel-free F-MS) and one at relapse (F-MS rel). ( b ) The expression of granzyme B (GrzB), perforin, and Fas-Ligand (FasL) in or on CD3 + T cells are shown. ( c ) The frequency of cells expressing each cytotoxic molecule was compared between CD56 + and CD56 − T cells. ( d ) The frequency of cells expressing each cytotoxic molecule in CD4 + CD56 + T cells, CD4 + CD56 − T cells, CD8 + CD56 + T cells, and CD8 + CD56 − T cells. Bars represent median values. Each column represents mean ± SEM. p-values: *p

    Article Snippet: In vitro analysis of effect of fingolimod regarding CD56 expression on T cells PBMC from healthy subjects and MS patients without DMD were cultured for 5 days at 1 × 106 cells/ml in the presence of fingolimod (0.01, 0.1, 1, 5, 10 μM; Cayman Chemical, Ann Arbor, MI,USA) or fingolimod-phosphate (0.01 μM, 0.1 μM, 1 μM, 5 μM, 10 μM; Cayman Chemical) in AIM-V medium.

    Techniques: Expressing, Mass Spectrometry

    Myelin basic protein (MBP)-reactive proinflammatory cytokine production by CD56 + T cells from multiple sclerosis (MS) patients on fingolimod in remission. CD56 + and CD56 − T cells were co-cultured with antigen-presenting cells (APC) in the presence or absence of MBP peptide mixture, and intracellular cytokines were analyzed by flow cytometry. ( a ) Representative FACS dot plots of IFN-γ-producing cells in CD56 + and CD56 − T cells when the cells were co-cultured with APC in the presence or absence of MBP. ( b ) The delta frequency of cells that produced IFN-γ (upper row) and IL-17A (bottom row) within CD56 + and CD56 − T cells. The delta frequency was calculated by subtracting the frequency of cytokine-producing cells in the absence of MBP from that in the presence of MBP. Diamonds are data from relapse-experienced MS patients with fingolimod therapy in remission, and circles represent relapse-free MS patients while on fingolimod treatment. Bars represent median values. p-values: **p

    Journal: Scientific Reports

    Article Title: Altered T cell phenotypes associated with clinical relapse of multiple sclerosis patients receiving fingolimod therapy

    doi: 10.1038/srep35314

    Figure Lengend Snippet: Myelin basic protein (MBP)-reactive proinflammatory cytokine production by CD56 + T cells from multiple sclerosis (MS) patients on fingolimod in remission. CD56 + and CD56 − T cells were co-cultured with antigen-presenting cells (APC) in the presence or absence of MBP peptide mixture, and intracellular cytokines were analyzed by flow cytometry. ( a ) Representative FACS dot plots of IFN-γ-producing cells in CD56 + and CD56 − T cells when the cells were co-cultured with APC in the presence or absence of MBP. ( b ) The delta frequency of cells that produced IFN-γ (upper row) and IL-17A (bottom row) within CD56 + and CD56 − T cells. The delta frequency was calculated by subtracting the frequency of cytokine-producing cells in the absence of MBP from that in the presence of MBP. Diamonds are data from relapse-experienced MS patients with fingolimod therapy in remission, and circles represent relapse-free MS patients while on fingolimod treatment. Bars represent median values. p-values: **p

    Article Snippet: In vitro analysis of effect of fingolimod regarding CD56 expression on T cells PBMC from healthy subjects and MS patients without DMD were cultured for 5 days at 1 × 106 cells/ml in the presence of fingolimod (0.01, 0.1, 1, 5, 10 μM; Cayman Chemical, Ann Arbor, MI,USA) or fingolimod-phosphate (0.01 μM, 0.1 μM, 1 μM, 5 μM, 10 μM; Cayman Chemical) in AIM-V medium.

    Techniques: Mass Spectrometry, Cell Culture, Flow Cytometry, Cytometry, FACS, Produced

    Frequency of CD56 + T cells in healthy subjects (HS) and patients with multiple sclerosis (MS). ( a ) CD56 expression on CD3-gated T cells. Representative FACS dot plots of HS, MS patients without disease-modifying drug (MS without DMD), MS patients with IFN-β therapy (MS with IFN-β), and those with fingolimod therapy (MS with fingolimod) are shown. ( b ) The frequency of CD56 + cells in CD3 + T cells in the indicated groups. ( c ) The frequency of CD56 + cells in CD4 + T cells in the indicated groups (left), and that of CD56 + cells in CD8 + T cells in the indicated groups (right). ( d ) The frequency of CD56 + T cells before and after fingolimod treatment in MS patients. The frequency of CD56 + T cells within total T cells, and CD4 + and CD8 + T cell subsets is shown. nF-MS: multiple sclerosis patients without fingolimod therapy, F-MS: multiple sclerosis patients with fingolimod therapy, rem: remission, rel: relapse, rel-free: relapse-free. Bars represent median values. p-values: *p

    Journal: Scientific Reports

    Article Title: Altered T cell phenotypes associated with clinical relapse of multiple sclerosis patients receiving fingolimod therapy

    doi: 10.1038/srep35314

    Figure Lengend Snippet: Frequency of CD56 + T cells in healthy subjects (HS) and patients with multiple sclerosis (MS). ( a ) CD56 expression on CD3-gated T cells. Representative FACS dot plots of HS, MS patients without disease-modifying drug (MS without DMD), MS patients with IFN-β therapy (MS with IFN-β), and those with fingolimod therapy (MS with fingolimod) are shown. ( b ) The frequency of CD56 + cells in CD3 + T cells in the indicated groups. ( c ) The frequency of CD56 + cells in CD4 + T cells in the indicated groups (left), and that of CD56 + cells in CD8 + T cells in the indicated groups (right). ( d ) The frequency of CD56 + T cells before and after fingolimod treatment in MS patients. The frequency of CD56 + T cells within total T cells, and CD4 + and CD8 + T cell subsets is shown. nF-MS: multiple sclerosis patients without fingolimod therapy, F-MS: multiple sclerosis patients with fingolimod therapy, rem: remission, rel: relapse, rel-free: relapse-free. Bars represent median values. p-values: *p

    Article Snippet: In vitro analysis of effect of fingolimod regarding CD56 expression on T cells PBMC from healthy subjects and MS patients without DMD were cultured for 5 days at 1 × 106 cells/ml in the presence of fingolimod (0.01, 0.1, 1, 5, 10 μM; Cayman Chemical, Ann Arbor, MI,USA) or fingolimod-phosphate (0.01 μM, 0.1 μM, 1 μM, 5 μM, 10 μM; Cayman Chemical) in AIM-V medium.

    Techniques: Mass Spectrometry, Expressing, FACS

    Longitudinal analysis of CD56 expression, granzyme B (GrzB) expression, and frequency of IFN-γ-producing cells in relapse-experienced multiple sclerosis (MS) patients on fingolimod. ( a ) Temporal changes in the frequency of CD56-expressing cells within the CD3 + T cell fraction. ( b ) The frequency of GrzB-expressing cells and IFN-γ-producing cells in CD56 + and CD56 − T cell subsets decreased 3 months after relapse. ( c ) The frequency of CD56 expression in relapse-experienced MS patients with fingolimod therapy in remission remained higher than in patients who did not have any relapse while on fingolimod treatment. Each relapse-experienced patient is indicated by different-shaped dots. The frequencies at relapse are presented as red dots, and those in remission are presented as black dots. Bars represent median values. p-values: **p

    Journal: Scientific Reports

    Article Title: Altered T cell phenotypes associated with clinical relapse of multiple sclerosis patients receiving fingolimod therapy

    doi: 10.1038/srep35314

    Figure Lengend Snippet: Longitudinal analysis of CD56 expression, granzyme B (GrzB) expression, and frequency of IFN-γ-producing cells in relapse-experienced multiple sclerosis (MS) patients on fingolimod. ( a ) Temporal changes in the frequency of CD56-expressing cells within the CD3 + T cell fraction. ( b ) The frequency of GrzB-expressing cells and IFN-γ-producing cells in CD56 + and CD56 − T cell subsets decreased 3 months after relapse. ( c ) The frequency of CD56 expression in relapse-experienced MS patients with fingolimod therapy in remission remained higher than in patients who did not have any relapse while on fingolimod treatment. Each relapse-experienced patient is indicated by different-shaped dots. The frequencies at relapse are presented as red dots, and those in remission are presented as black dots. Bars represent median values. p-values: **p

    Article Snippet: In vitro analysis of effect of fingolimod regarding CD56 expression on T cells PBMC from healthy subjects and MS patients without DMD were cultured for 5 days at 1 × 106 cells/ml in the presence of fingolimod (0.01, 0.1, 1, 5, 10 μM; Cayman Chemical, Ann Arbor, MI,USA) or fingolimod-phosphate (0.01 μM, 0.1 μM, 1 μM, 5 μM, 10 μM; Cayman Chemical) in AIM-V medium.

    Techniques: Expressing, Mass Spectrometry

    Effect of FTY720 on inflammatory cytokines, including monocyte chemoattractant protein-1 (MCP-1), macrophage-related TNF-α and IL-6, arginase-1 and IL-10, in the three groups. (A) Western blot analysis of MCP-1 and M1 and M2 macrophage-related cytokines; β-actin was used as a control. (B) Quantitative analysis of protein expression of MCP-1 and macrophage-related cytokines. (C) Expression of MCP-1 and macrophage-related cytokines mRNA; β-actin was used as a control. Values are presented as the mean±SEM. n =6. b P

    Journal: Acta Pharmacologica Sinica

    Article Title: FTY720 inhibits tubulointerstitial inflammation in albumin overload-induced nephropathy of rats via the Sphk1 pathway

    doi: 10.1038/aps.2014.100

    Figure Lengend Snippet: Effect of FTY720 on inflammatory cytokines, including monocyte chemoattractant protein-1 (MCP-1), macrophage-related TNF-α and IL-6, arginase-1 and IL-10, in the three groups. (A) Western blot analysis of MCP-1 and M1 and M2 macrophage-related cytokines; β-actin was used as a control. (B) Quantitative analysis of protein expression of MCP-1 and macrophage-related cytokines. (C) Expression of MCP-1 and macrophage-related cytokines mRNA; β-actin was used as a control. Values are presented as the mean±SEM. n =6. b P

    Article Snippet: Then, they were divided into three groups: 1) Saline control, daily intraperitoneal injection of saline solution (0.09%, equivalent volume of BSA); 2) AO (albumin overload), intraperitoneal injection of BSA (bovine serum albumin, Roche, Switzerland) 1 week after surgery (5 g·kg−1 ·d−1 ) for 9 weeks; and 3) AO+FTY720, FTY720 (Fingolimod, Selleck Chemicals, Houston, USA) administration (0.5 mg·kg−1 ·d−1 ) along with BSA.

    Techniques: Western Blot, Expressing

    Effect of FTY720 on urinary protein and NAG. Time course of proteinuria (A) and N -acetyl-β- D -glycosaminidase (NAG) activity (B) in saline controls, AO rats, and FTY720-treated rats. Values are presented as the mean±SEM. n =6. b P

    Journal: Acta Pharmacologica Sinica

    Article Title: FTY720 inhibits tubulointerstitial inflammation in albumin overload-induced nephropathy of rats via the Sphk1 pathway

    doi: 10.1038/aps.2014.100

    Figure Lengend Snippet: Effect of FTY720 on urinary protein and NAG. Time course of proteinuria (A) and N -acetyl-β- D -glycosaminidase (NAG) activity (B) in saline controls, AO rats, and FTY720-treated rats. Values are presented as the mean±SEM. n =6. b P

    Article Snippet: Then, they were divided into three groups: 1) Saline control, daily intraperitoneal injection of saline solution (0.09%, equivalent volume of BSA); 2) AO (albumin overload), intraperitoneal injection of BSA (bovine serum albumin, Roche, Switzerland) 1 week after surgery (5 g·kg−1 ·d−1 ) for 9 weeks; and 3) AO+FTY720, FTY720 (Fingolimod, Selleck Chemicals, Houston, USA) administration (0.5 mg·kg−1 ·d−1 ) along with BSA.

    Techniques: Activity Assay

    Effect of FTY720 on histological changes in AO rats. (A) PAS stains of tissue as indicated (×200). (B) Tubulointerstitial injury score for the three groups. Values are presented as the mean±SEM. n =6. b P

    Journal: Acta Pharmacologica Sinica

    Article Title: FTY720 inhibits tubulointerstitial inflammation in albumin overload-induced nephropathy of rats via the Sphk1 pathway

    doi: 10.1038/aps.2014.100

    Figure Lengend Snippet: Effect of FTY720 on histological changes in AO rats. (A) PAS stains of tissue as indicated (×200). (B) Tubulointerstitial injury score for the three groups. Values are presented as the mean±SEM. n =6. b P

    Article Snippet: Then, they were divided into three groups: 1) Saline control, daily intraperitoneal injection of saline solution (0.09%, equivalent volume of BSA); 2) AO (albumin overload), intraperitoneal injection of BSA (bovine serum albumin, Roche, Switzerland) 1 week after surgery (5 g·kg−1 ·d−1 ) for 9 weeks; and 3) AO+FTY720, FTY720 (Fingolimod, Selleck Chemicals, Houston, USA) administration (0.5 mg·kg−1 ·d−1 ) along with BSA.

    Techniques:

    Effect of FTY720 on peripheral blood cells. (A) Lymphocytes in the blood. (B) Monocytes in the blood. Values are the means±SEM. n =6. b P

    Journal: Acta Pharmacologica Sinica

    Article Title: FTY720 inhibits tubulointerstitial inflammation in albumin overload-induced nephropathy of rats via the Sphk1 pathway

    doi: 10.1038/aps.2014.100

    Figure Lengend Snippet: Effect of FTY720 on peripheral blood cells. (A) Lymphocytes in the blood. (B) Monocytes in the blood. Values are the means±SEM. n =6. b P

    Article Snippet: Then, they were divided into three groups: 1) Saline control, daily intraperitoneal injection of saline solution (0.09%, equivalent volume of BSA); 2) AO (albumin overload), intraperitoneal injection of BSA (bovine serum albumin, Roche, Switzerland) 1 week after surgery (5 g·kg−1 ·d−1 ) for 9 weeks; and 3) AO+FTY720, FTY720 (Fingolimod, Selleck Chemicals, Houston, USA) administration (0.5 mg·kg−1 ·d−1 ) along with BSA.

    Techniques:

    Effect of FTY720 on Sphk1/S1P/S1pr signal-related protein expression in the three groups. (A) Immunohistochemical staining of Sphk1, S1pr1, and S1pr3 in the saline, AO, and AO+FTY720 groups (original magnification is ×200). (B) Semiquantitative immunohistochemical analysis of the Sphk1/S1P/S1pr signal-related protein expression in the three groups (I–III). Values are presented as the mean±SEM. n =6. b P

    Journal: Acta Pharmacologica Sinica

    Article Title: FTY720 inhibits tubulointerstitial inflammation in albumin overload-induced nephropathy of rats via the Sphk1 pathway

    doi: 10.1038/aps.2014.100

    Figure Lengend Snippet: Effect of FTY720 on Sphk1/S1P/S1pr signal-related protein expression in the three groups. (A) Immunohistochemical staining of Sphk1, S1pr1, and S1pr3 in the saline, AO, and AO+FTY720 groups (original magnification is ×200). (B) Semiquantitative immunohistochemical analysis of the Sphk1/S1P/S1pr signal-related protein expression in the three groups (I–III). Values are presented as the mean±SEM. n =6. b P

    Article Snippet: Then, they were divided into three groups: 1) Saline control, daily intraperitoneal injection of saline solution (0.09%, equivalent volume of BSA); 2) AO (albumin overload), intraperitoneal injection of BSA (bovine serum albumin, Roche, Switzerland) 1 week after surgery (5 g·kg−1 ·d−1 ) for 9 weeks; and 3) AO+FTY720, FTY720 (Fingolimod, Selleck Chemicals, Houston, USA) administration (0.5 mg·kg−1 ·d−1 ) along with BSA.

    Techniques: Expressing, Immunohistochemistry, Staining

    Fingolimod impairs induction of activation markers on human monocytes Peripheral blood mononuclear cells from healthy donors were briefly exposed to increasing concentrations of fingolimod (0.1 μM, 1 μM, and 10 μM) or vehicle, and left in culture for 18 hours, eventually in the presence of 100 ng/mL lipopolysaccharide (LPS). Monocyte activation was monitored by staining for 2 surface markers, CD25 (A, B) and CD150 (C, D). Gates on monocytes in forward vs side scatterplot and then on viable (7-AAD negative) cells were applied. Thresholds were set on relative isotype controls. (A, C) Representative CD25 and CD150 stainings in unstimulated (left panels), LPS-stimulated (middle panels), or LPS-stimulated cultures exposed to 1 μM fingolimod (right panels). (B, D) Frequency of CD25 and CD150 expressing monocytes in unstimulated and LPS-stimulated cultures in the absence or presence of increasing concentrations of fingolimod. Data were obtained from 5 independent experiments. (E) Graphs represent fingolimod-mediated inhibition of activation markers induced at 2 LPS doses. Data were obtained from 2 to 5 experiments. Bars represent SEM. * p

    Journal: Neurology® Neuroimmunology & Neuroinflammation

    Article Title: Myeloid cells as target of fingolimod action in multiple sclerosis

    doi: 10.1212/NXI.0000000000000157

    Figure Lengend Snippet: Fingolimod impairs induction of activation markers on human monocytes Peripheral blood mononuclear cells from healthy donors were briefly exposed to increasing concentrations of fingolimod (0.1 μM, 1 μM, and 10 μM) or vehicle, and left in culture for 18 hours, eventually in the presence of 100 ng/mL lipopolysaccharide (LPS). Monocyte activation was monitored by staining for 2 surface markers, CD25 (A, B) and CD150 (C, D). Gates on monocytes in forward vs side scatterplot and then on viable (7-AAD negative) cells were applied. Thresholds were set on relative isotype controls. (A, C) Representative CD25 and CD150 stainings in unstimulated (left panels), LPS-stimulated (middle panels), or LPS-stimulated cultures exposed to 1 μM fingolimod (right panels). (B, D) Frequency of CD25 and CD150 expressing monocytes in unstimulated and LPS-stimulated cultures in the absence or presence of increasing concentrations of fingolimod. Data were obtained from 5 independent experiments. (E) Graphs represent fingolimod-mediated inhibition of activation markers induced at 2 LPS doses. Data were obtained from 2 to 5 experiments. Bars represent SEM. * p

    Article Snippet: EAE was induced in 7-week-old female C57BL/6N mice (Harlan Laboratories, Udine, Italy) and animals were assessed for clinical signs of EAE as previously described., Fingolimod (FTY720, 3 mg/kg body weight, same batch used in ; Selleckchem, DBA Italia, Milan, Italy) was given by oral gavage once daily starting 3 days after the appearance of the first clinical sign of disease.

    Techniques: Activation Assay, Staining, Expressing, Inhibition

    Treatment with fingolimod raises the activation threshold of monocytes in MS Peripheral blood mononuclear cells from 8 healthy donors, 7 patients with untreated multiple sclerosis (MS), and 11 patients with fingolimod-treated MS were briefly stimulated ex vivo with lipopolysaccharide (LPS). Monocytes were gated by forward vs side scatterplot and CD14-positive staining. Gate on viable (7-AAD negative) cells was then applied. Monocyte activation was monitored by staining for the 2 surface markers CD25 and CD150. Thresholds were set on relative isotype controls. (A) Frequency of CD25-positive (upper graph) and CD150-positive (lower graph) monocytes cultured ex vivo with 100 ng/mL LPS; each dot represents a single participant; black bars represent mean values for each group. (B) CD25 (upper) and CD150 (lower) stainings in cultures stimulated with 600 pg/mL LPS from a representative healthy control (left), a patient with untreated MS (middle), and a patient with fingolimod-treated MS (right). (C) Frequency of CD25 and CD150 positive monocytes at increasing LPS concentrations. Dose respons e curves are shown for each participant. Right panels show the means for each group. * p

    Journal: Neurology® Neuroimmunology & Neuroinflammation

    Article Title: Myeloid cells as target of fingolimod action in multiple sclerosis

    doi: 10.1212/NXI.0000000000000157

    Figure Lengend Snippet: Treatment with fingolimod raises the activation threshold of monocytes in MS Peripheral blood mononuclear cells from 8 healthy donors, 7 patients with untreated multiple sclerosis (MS), and 11 patients with fingolimod-treated MS were briefly stimulated ex vivo with lipopolysaccharide (LPS). Monocytes were gated by forward vs side scatterplot and CD14-positive staining. Gate on viable (7-AAD negative) cells was then applied. Monocyte activation was monitored by staining for the 2 surface markers CD25 and CD150. Thresholds were set on relative isotype controls. (A) Frequency of CD25-positive (upper graph) and CD150-positive (lower graph) monocytes cultured ex vivo with 100 ng/mL LPS; each dot represents a single participant; black bars represent mean values for each group. (B) CD25 (upper) and CD150 (lower) stainings in cultures stimulated with 600 pg/mL LPS from a representative healthy control (left), a patient with untreated MS (middle), and a patient with fingolimod-treated MS (right). (C) Frequency of CD25 and CD150 positive monocytes at increasing LPS concentrations. Dose respons e curves are shown for each participant. Right panels show the means for each group. * p

    Article Snippet: EAE was induced in 7-week-old female C57BL/6N mice (Harlan Laboratories, Udine, Italy) and animals were assessed for clinical signs of EAE as previously described., Fingolimod (FTY720, 3 mg/kg body weight, same batch used in ; Selleckchem, DBA Italia, Milan, Italy) was given by oral gavage once daily starting 3 days after the appearance of the first clinical sign of disease.

    Techniques: Activation Assay, Mass Spectrometry, Ex Vivo, Staining, Cell Culture

    Fingolimod does not alter human monocyte viability Peripheral blood mononuclear cells from healthy donors were briefly exposed to increasing concentrations of fingolimod (0.1 μM, 1 μM, and 10 μM) or vehicle, and left in culture for 18 hours, eventually in the presence of 100 ng/mL lipopolysaccharide (LPS). Dead cells were stained with the vital dye 7-AAD and measured at flow cytometer. Monocytes were gated in forward vs side scatter plot. Graph depicts the frequency of cell death in unstimulated or LPS-stimulated cultures after exposure to fingolimod. Data were obtained from 3 independent experiments. Bars represent SEM.

    Journal: Neurology® Neuroimmunology & Neuroinflammation

    Article Title: Myeloid cells as target of fingolimod action in multiple sclerosis

    doi: 10.1212/NXI.0000000000000157

    Figure Lengend Snippet: Fingolimod does not alter human monocyte viability Peripheral blood mononuclear cells from healthy donors were briefly exposed to increasing concentrations of fingolimod (0.1 μM, 1 μM, and 10 μM) or vehicle, and left in culture for 18 hours, eventually in the presence of 100 ng/mL lipopolysaccharide (LPS). Dead cells were stained with the vital dye 7-AAD and measured at flow cytometer. Monocytes were gated in forward vs side scatter plot. Graph depicts the frequency of cell death in unstimulated or LPS-stimulated cultures after exposure to fingolimod. Data were obtained from 3 independent experiments. Bars represent SEM.

    Article Snippet: EAE was induced in 7-week-old female C57BL/6N mice (Harlan Laboratories, Udine, Italy) and animals were assessed for clinical signs of EAE as previously described., Fingolimod (FTY720, 3 mg/kg body weight, same batch used in ; Selleckchem, DBA Italia, Milan, Italy) was given by oral gavage once daily starting 3 days after the appearance of the first clinical sign of disease.

    Techniques: Staining, Flow Cytometry, Cytometry

    Fingolimod inhibits TNF-α secretion by human monocytes Peripheral blood mononuclear cells from healthy donors were briefly exposed to increasing concentrations of fingolimod (0.1 μM, 1 μM, and 10 μM) or vehicle, and left in culture for 18 hours, eventually in the presence of 600 pg/mL or 100 ng/mL lipopolysaccharide (LPS). Supernatants were collected and tumor necrosis factor–α (TNF-α) levels were detected by ELISA assay. Data of a representative experiment are shown. Bars represent SEM. * p

    Journal: Neurology® Neuroimmunology & Neuroinflammation

    Article Title: Myeloid cells as target of fingolimod action in multiple sclerosis

    doi: 10.1212/NXI.0000000000000157

    Figure Lengend Snippet: Fingolimod inhibits TNF-α secretion by human monocytes Peripheral blood mononuclear cells from healthy donors were briefly exposed to increasing concentrations of fingolimod (0.1 μM, 1 μM, and 10 μM) or vehicle, and left in culture for 18 hours, eventually in the presence of 600 pg/mL or 100 ng/mL lipopolysaccharide (LPS). Supernatants were collected and tumor necrosis factor–α (TNF-α) levels were detected by ELISA assay. Data of a representative experiment are shown. Bars represent SEM. * p

    Article Snippet: EAE was induced in 7-week-old female C57BL/6N mice (Harlan Laboratories, Udine, Italy) and animals were assessed for clinical signs of EAE as previously described., Fingolimod (FTY720, 3 mg/kg body weight, same batch used in ; Selleckchem, DBA Italia, Milan, Italy) was given by oral gavage once daily starting 3 days after the appearance of the first clinical sign of disease.

    Techniques: Enzyme-linked Immunosorbent Assay

    Therapeutic administration of fingolimod during EAE reduces myeloid cell activation in the spleen and CNS (A) Clinical expression of MOG 35-55 peptide-induced experimental autoimmune encephalomyelitis (EAE) in fingolimod-treated (n = 13, black dots) or vehicle-treated (n = 12, white dots) mice. Drug or vehicle were administered daily starting 3 days after disease onset. Left graph shows mean clinical scores from day of immunization, right graph depicts mean clinical score according to treatment duration. (B) Representative stainings for tumor necrosis factor–α (TNF-α) production by CD45+ CD11b+ spleen myeloid cells from vehicle- and fingolimod-treated EAE mice. (C) Frequency of TNF-α–producing myeloid cells in vehicle-treated (n = 5) and fingolimod-treated EAE (n = 4) mice. (D) Representative stainings for TNF-α production by CNS myeloid cells from vehicle-treated and fingolimod-treated EAE mice. Left panels show gating strategy. R′: CD45 hi CD11b+ blood-borne CNS infiltrating myeloid cells. Gate R″: CD45 low CD11b+Ly6C − CNS resident microglia. Middle and right panels depict representative stainings for TNF-α production by CNS infiltrating (upper) or resident (lower) myeloid cells from vehicle-treated and fingolimod-treated EAE mice. (E) Frequency of TNF-α–producing CNS myeloid cells in vehicle-treated (n = 5) and fingolimod-treated EAE mice (n = 5). Representative animals of the mean EAE clinical score (A) were analyzed in C and E. Bars represent SEM. * p

    Journal: Neurology® Neuroimmunology & Neuroinflammation

    Article Title: Myeloid cells as target of fingolimod action in multiple sclerosis

    doi: 10.1212/NXI.0000000000000157

    Figure Lengend Snippet: Therapeutic administration of fingolimod during EAE reduces myeloid cell activation in the spleen and CNS (A) Clinical expression of MOG 35-55 peptide-induced experimental autoimmune encephalomyelitis (EAE) in fingolimod-treated (n = 13, black dots) or vehicle-treated (n = 12, white dots) mice. Drug or vehicle were administered daily starting 3 days after disease onset. Left graph shows mean clinical scores from day of immunization, right graph depicts mean clinical score according to treatment duration. (B) Representative stainings for tumor necrosis factor–α (TNF-α) production by CD45+ CD11b+ spleen myeloid cells from vehicle- and fingolimod-treated EAE mice. (C) Frequency of TNF-α–producing myeloid cells in vehicle-treated (n = 5) and fingolimod-treated EAE (n = 4) mice. (D) Representative stainings for TNF-α production by CNS myeloid cells from vehicle-treated and fingolimod-treated EAE mice. Left panels show gating strategy. R′: CD45 hi CD11b+ blood-borne CNS infiltrating myeloid cells. Gate R″: CD45 low CD11b+Ly6C − CNS resident microglia. Middle and right panels depict representative stainings for TNF-α production by CNS infiltrating (upper) or resident (lower) myeloid cells from vehicle-treated and fingolimod-treated EAE mice. (E) Frequency of TNF-α–producing CNS myeloid cells in vehicle-treated (n = 5) and fingolimod-treated EAE mice (n = 5). Representative animals of the mean EAE clinical score (A) were analyzed in C and E. Bars represent SEM. * p

    Article Snippet: EAE was induced in 7-week-old female C57BL/6N mice (Harlan Laboratories, Udine, Italy) and animals were assessed for clinical signs of EAE as previously described., Fingolimod (FTY720, 3 mg/kg body weight, same batch used in ; Selleckchem, DBA Italia, Milan, Italy) was given by oral gavage once daily starting 3 days after the appearance of the first clinical sign of disease.

    Techniques: Activation Assay, Expressing, Mouse Assay

    Comparison of NEDA status in the core study and the first extension year by treatment group (A) IFN-switch group (B) Continuous-fingolimod group. Data presented here are for the pooled fingolimod 0.5 and 1.25 mg groups. N, total number of patients in the group; n, number of patients achieving NEDA; IFN, interferon; NEDA, no evidence of disease activity.

    Journal: Journal of Neurology, Neurosurgery, and Psychiatry

    Article Title: Long-term (up to 4.5 years) treatment with fingolimod in multiple sclerosis: results from the extension of the randomised TRANSFORMS study

    doi: 10.1136/jnnp-2015-310597

    Figure Lengend Snippet: Comparison of NEDA status in the core study and the first extension year by treatment group (A) IFN-switch group (B) Continuous-fingolimod group. Data presented here are for the pooled fingolimod 0.5 and 1.25 mg groups. N, total number of patients in the group; n, number of patients achieving NEDA; IFN, interferon; NEDA, no evidence of disease activity.

    Article Snippet: However, in a pooled analysis comprising 3553 patients treated with fingolimod in the core and extension studies, the incidence of basal cell carcinoma with fingolimod 0.5 mg was 1.8%, and the total incidence was 1.5% (Novartis, data on file); our results show comparable frequencies of basal cell carcinoma (2% for the continuous-fingolimod 0.5 mg group) and a low incidence of other cancer types ( < 0.6%).

    Techniques: Activity Assay

    Dynamics of H60-specific CD8 + T cells under different conditions. ( a ) J15 CD8 + T cells were harvested from the spleen and peritoneum of adoptive hosts at the indicated times after exposure to different priming conditions, ( b ) quantified and ( c ) analysed by flow cytometry to compare CFSE-dilution profiles. Numbers and CFSE profiles of J15 CD8 + T cells from FTY-720-treated mice were obtained on day 5 post priming and were incorporated in plots designated as day 5+F in ( b , c ). ( d ) Contraction degrees (peak numbers/contraction numbers) of splenic J15 CD8 + T cells obtained from adoptive hosts are plotted. Data shown were obtained from adoptive hosts transferred in high numbers ( b–d ). ( e ) Dynamics of J15-LucTg CD8 + T cells in B6-albino-adoptive hosts (transferred with 1 × 10 5 cells) were monitored periodically after priming under different conditions through secondary expansion. Those in B6-albino-adoptive hosts without priming are also shown as control (naive). Photon flux values as the primary responses progressed were plotted after designating the whole body as the ROI. Fold changes in peak values in 1 o peak relative to the values of 1 o day 0 (before priming) are shown. The mean relative ratios of 2 o peak: 2 o day 0 (before boosting) photon flux values are presented in the table. Data shown represent at least three ( n =2 per group; b–d ) or two ( n =3 per group; e ) independent experiments. A.T., Adoptive Transfer. Data are presented as means±s.e.m. Not Significant (NS) P > 0.05, * P

    Journal: Nature Communications

    Article Title: Memory programming in CD8+ T-cell differentiation is intrinsic and is not determined by CD4 help

    doi: 10.1038/ncomms8994

    Figure Lengend Snippet: Dynamics of H60-specific CD8 + T cells under different conditions. ( a ) J15 CD8 + T cells were harvested from the spleen and peritoneum of adoptive hosts at the indicated times after exposure to different priming conditions, ( b ) quantified and ( c ) analysed by flow cytometry to compare CFSE-dilution profiles. Numbers and CFSE profiles of J15 CD8 + T cells from FTY-720-treated mice were obtained on day 5 post priming and were incorporated in plots designated as day 5+F in ( b , c ). ( d ) Contraction degrees (peak numbers/contraction numbers) of splenic J15 CD8 + T cells obtained from adoptive hosts are plotted. Data shown were obtained from adoptive hosts transferred in high numbers ( b–d ). ( e ) Dynamics of J15-LucTg CD8 + T cells in B6-albino-adoptive hosts (transferred with 1 × 10 5 cells) were monitored periodically after priming under different conditions through secondary expansion. Those in B6-albino-adoptive hosts without priming are also shown as control (naive). Photon flux values as the primary responses progressed were plotted after designating the whole body as the ROI. Fold changes in peak values in 1 o peak relative to the values of 1 o day 0 (before priming) are shown. The mean relative ratios of 2 o peak: 2 o day 0 (before boosting) photon flux values are presented in the table. Data shown represent at least three ( n =2 per group; b–d ) or two ( n =3 per group; e ) independent experiments. A.T., Adoptive Transfer. Data are presented as means±s.e.m. Not Significant (NS) P > 0.05, * P

    Article Snippet: FTY-720 (0.3 mg kg−1 ; Biovision, Milpitas, CA, USA) was injected i.p. daily from day 2 post priming until the mice were euthanized.

    Techniques: Flow Cytometry, Cytometry, Mouse Assay, Adoptive Transfer Assay

    Effector differentiation between helper-deficient cells and helped cells. ( a – c ) Phenotypic profiles of CD45.1 + J15 CD8 + T cells in the spleens of adoptive hosts transferred in high numbers ( a ), treated with FTY-720 ( b ) or transferred in low numbers ( c ). ( d ) Phenotypic profiles of polyclonal H60-tetramer-binding CD8 + T cells. ( e–g ) Representative flow cytometry data and plots of the proportion and numbers of short-lived effector cell (SLEC) and memory precursor effector cell (MPEC) populations ( e , f ) in J15 CD8 + T cells from adoptive hosts transferred with high ( e ) or low ( f ) numbers of cells, or ( g ) in polyclonal H60-tetramer-binding CD8 + T cells. Data represent three ( a , b , e ) or two ( c , d , f , g ) independent experiments ( n =2 per group per experiment). Data are presented as means±s.e.m. Not Significant (NS) P > 0.05, * P

    Journal: Nature Communications

    Article Title: Memory programming in CD8+ T-cell differentiation is intrinsic and is not determined by CD4 help

    doi: 10.1038/ncomms8994

    Figure Lengend Snippet: Effector differentiation between helper-deficient cells and helped cells. ( a – c ) Phenotypic profiles of CD45.1 + J15 CD8 + T cells in the spleens of adoptive hosts transferred in high numbers ( a ), treated with FTY-720 ( b ) or transferred in low numbers ( c ). ( d ) Phenotypic profiles of polyclonal H60-tetramer-binding CD8 + T cells. ( e–g ) Representative flow cytometry data and plots of the proportion and numbers of short-lived effector cell (SLEC) and memory precursor effector cell (MPEC) populations ( e , f ) in J15 CD8 + T cells from adoptive hosts transferred with high ( e ) or low ( f ) numbers of cells, or ( g ) in polyclonal H60-tetramer-binding CD8 + T cells. Data represent three ( a , b , e ) or two ( c , d , f , g ) independent experiments ( n =2 per group per experiment). Data are presented as means±s.e.m. Not Significant (NS) P > 0.05, * P

    Article Snippet: FTY-720 (0.3 mg kg−1 ; Biovision, Milpitas, CA, USA) was injected i.p. daily from day 2 post priming until the mice were euthanized.

    Techniques: Binding Assay, Flow Cytometry, Cytometry

    A diagram shows differentially expressed genes (in a category of ECM organization/inflammation response) by Affymetrix gene array. Gene expression profiling in sham hearts was normalized to yellow, which is defined as unchanged. Orange and red represent increased expression, and grey and blue represent decreased expression. Fold change of expression is shown for TAC/FTY-720 versus TAC only (n=2 for each group). All genes identified as significantly altered (P

    Journal: Circulation. Heart failure

    Article Title: A Novel Immunomodulator, FTY-720 Reverses Existing Cardiac Hypertrophy and Fibrosis from Pressure Overload by Targeting NFAT Signaling and Periostin

    doi: 10.1161/CIRCHEARTFAILURE.112.000123

    Figure Lengend Snippet: A diagram shows differentially expressed genes (in a category of ECM organization/inflammation response) by Affymetrix gene array. Gene expression profiling in sham hearts was normalized to yellow, which is defined as unchanged. Orange and red represent increased expression, and grey and blue represent decreased expression. Fold change of expression is shown for TAC/FTY-720 versus TAC only (n=2 for each group). All genes identified as significantly altered (P

    Article Snippet: Brinkmann V. FTY-720: Mechanism of action and potential benefit in organ transplantation.

    Techniques: Expressing

    FTY-720 negatively regulates NFAT activity through Pak1 activation in cardiomyocytes. (A) Representative fluorescent images show the cytoplasmic-nuclear localization of endogenous NFATc4 in NRCMs infected with Ad-shPak1 or Ad-shC2 followed by treatment with PE ± FTY-720, which demonstrate FTY-720 inhibited PE-induced NFAT nuclear retention, whereas such inhibitory effects were eliminated in NRCMs with Pak1 knockdown. Red staining is for NFAT, blue for visualizing the nuclei, arrows point to cytoplasmic NFAT; arrowheads show nuclear localization of NFAT (scale bar: 20μm). (B) These results were quantified from approximately 500 infected cardiomyocytes per group and presented as the percentage of cells with nuclear-NFAT. (C) Immunostaining of NFAT4c was also applied to NRCMs infected by Ad-CnA with or without FTY-720. Ad-LacZ was used as a control virus. Quantitative data is presented as the percentage of cells expressing nuclear-NFAT. N=5 per group, data is presented as mean ± SEM.

    Journal: Circulation. Heart failure

    Article Title: A Novel Immunomodulator, FTY-720 Reverses Existing Cardiac Hypertrophy and Fibrosis from Pressure Overload by Targeting NFAT Signaling and Periostin

    doi: 10.1161/CIRCHEARTFAILURE.112.000123

    Figure Lengend Snippet: FTY-720 negatively regulates NFAT activity through Pak1 activation in cardiomyocytes. (A) Representative fluorescent images show the cytoplasmic-nuclear localization of endogenous NFATc4 in NRCMs infected with Ad-shPak1 or Ad-shC2 followed by treatment with PE ± FTY-720, which demonstrate FTY-720 inhibited PE-induced NFAT nuclear retention, whereas such inhibitory effects were eliminated in NRCMs with Pak1 knockdown. Red staining is for NFAT, blue for visualizing the nuclei, arrows point to cytoplasmic NFAT; arrowheads show nuclear localization of NFAT (scale bar: 20μm). (B) These results were quantified from approximately 500 infected cardiomyocytes per group and presented as the percentage of cells with nuclear-NFAT. (C) Immunostaining of NFAT4c was also applied to NRCMs infected by Ad-CnA with or without FTY-720. Ad-LacZ was used as a control virus. Quantitative data is presented as the percentage of cells expressing nuclear-NFAT. N=5 per group, data is presented as mean ± SEM.

    Article Snippet: Brinkmann V. FTY-720: Mechanism of action and potential benefit in organ transplantation.

    Techniques: Activity Assay, Activation Assay, Infection, Staining, Immunostaining, Expressing

    FTY-720 antagonizes hypertrophy through a G i -dependent mechanism. (A) Protein extracts from NRCMs treated with FTY-720 ± SPHK inhibitor 2 (SPHK inh) or ± various doses of S1P were examined by immunoblotting for total and phosphorylated (Thr 423) Pak1. (B) Immunoblotting demonstrates effect of FTY-720 alone, or in combination with PTX on Pak1 phosphorylation at Thr 423 in NRCMs and HFCMs. The ratios of P/T Pak1 are presented by the bar graphs. (C) Representative fluorescent images show triple staining of ANP in HFCMs receiving PE ± FTY-720, or combination with PTX (red staining for ANP, pointed by arrows; green for α-actinin; blue for DAPI to highlight the nuclei, scale bar: 20μm). Quantification of ANP-expressing cells is presented by the bar graph. (D–E) PTX blocks the anti-hypertrophic effect of FTY-720, demonstrating by increased HW/TL ratio and larger cardiomyocyte surface area in comparison to the TAC/FTY-720 heart. (F) M-mode echocardiographic tracings from the mice receiving various treatments. (G–H) By echocardiography left ventricular PW and EF% confirm that PTX blocks the beneficial effect of FTY-720 in antagonizing cardiac hypertrophy. N=6–8 per group, data is presented as mean ± SEM.

    Journal: Circulation. Heart failure

    Article Title: A Novel Immunomodulator, FTY-720 Reverses Existing Cardiac Hypertrophy and Fibrosis from Pressure Overload by Targeting NFAT Signaling and Periostin

    doi: 10.1161/CIRCHEARTFAILURE.112.000123

    Figure Lengend Snippet: FTY-720 antagonizes hypertrophy through a G i -dependent mechanism. (A) Protein extracts from NRCMs treated with FTY-720 ± SPHK inhibitor 2 (SPHK inh) or ± various doses of S1P were examined by immunoblotting for total and phosphorylated (Thr 423) Pak1. (B) Immunoblotting demonstrates effect of FTY-720 alone, or in combination with PTX on Pak1 phosphorylation at Thr 423 in NRCMs and HFCMs. The ratios of P/T Pak1 are presented by the bar graphs. (C) Representative fluorescent images show triple staining of ANP in HFCMs receiving PE ± FTY-720, or combination with PTX (red staining for ANP, pointed by arrows; green for α-actinin; blue for DAPI to highlight the nuclei, scale bar: 20μm). Quantification of ANP-expressing cells is presented by the bar graph. (D–E) PTX blocks the anti-hypertrophic effect of FTY-720, demonstrating by increased HW/TL ratio and larger cardiomyocyte surface area in comparison to the TAC/FTY-720 heart. (F) M-mode echocardiographic tracings from the mice receiving various treatments. (G–H) By echocardiography left ventricular PW and EF% confirm that PTX blocks the beneficial effect of FTY-720 in antagonizing cardiac hypertrophy. N=6–8 per group, data is presented as mean ± SEM.

    Article Snippet: Brinkmann V. FTY-720: Mechanism of action and potential benefit in organ transplantation.

    Techniques: Staining, Aqueous Normal-phase Chromatography, Expressing, Mouse Assay

    FTY-720 reduces fibrotic response and improves extracellular compartment milieu. (A) Immunoblotting shows FTY-720 profoundly diminished TAC-induced periostin expression. GAPDH expression is the protein loading control. The ratio of periostin/GAPDH is represented by the bar graph. (B) qPCR analysis validates decreased gene expression of Col1α2, Col3α1 and Col5α2 in the heart receiving FTY-720 treatment. The data are normalized to the GAPDH content. (C) Immunoblot analyses show that the protein level of periostin in NRCFs and HACFs was increased by Ang II and decreased by additional FTY-720 treatment. The ratios of periostin/GAPDH are represented by the bar graphs. (D) Expression of activated TGF-β1 and Smad 2 phosphorylation were substantial increases upon TAC stress, whilst both decreased in response to FTY-720 treatment. The ratios of TGF-β1/GAPDH and phosphorylated/total Smad 2 are represented by the bar graphs. (E) Increased luciferase activity reflecting TGF-β activation was detected upon Ang II stimulation, whereas FTY-720 caused a reduction in luciferase activity. (F) Protein lysates were prepared for analysis of siRNA-mediated knockdown of periostin (upper panel). Periostin knockdown resulted in blunted TGF-β activation (upper panel) and down-regulated gene expression of Col1α2 and Col5α2 (lower panel). N=5–8 per group, data is presented as mean ± SEM.

    Journal: Circulation. Heart failure

    Article Title: A Novel Immunomodulator, FTY-720 Reverses Existing Cardiac Hypertrophy and Fibrosis from Pressure Overload by Targeting NFAT Signaling and Periostin

    doi: 10.1161/CIRCHEARTFAILURE.112.000123

    Figure Lengend Snippet: FTY-720 reduces fibrotic response and improves extracellular compartment milieu. (A) Immunoblotting shows FTY-720 profoundly diminished TAC-induced periostin expression. GAPDH expression is the protein loading control. The ratio of periostin/GAPDH is represented by the bar graph. (B) qPCR analysis validates decreased gene expression of Col1α2, Col3α1 and Col5α2 in the heart receiving FTY-720 treatment. The data are normalized to the GAPDH content. (C) Immunoblot analyses show that the protein level of periostin in NRCFs and HACFs was increased by Ang II and decreased by additional FTY-720 treatment. The ratios of periostin/GAPDH are represented by the bar graphs. (D) Expression of activated TGF-β1 and Smad 2 phosphorylation were substantial increases upon TAC stress, whilst both decreased in response to FTY-720 treatment. The ratios of TGF-β1/GAPDH and phosphorylated/total Smad 2 are represented by the bar graphs. (E) Increased luciferase activity reflecting TGF-β activation was detected upon Ang II stimulation, whereas FTY-720 caused a reduction in luciferase activity. (F) Protein lysates were prepared for analysis of siRNA-mediated knockdown of periostin (upper panel). Periostin knockdown resulted in blunted TGF-β activation (upper panel) and down-regulated gene expression of Col1α2 and Col5α2 (lower panel). N=5–8 per group, data is presented as mean ± SEM.

    Article Snippet: Brinkmann V. FTY-720: Mechanism of action and potential benefit in organ transplantation.

    Techniques: Expressing, Real-time Polymerase Chain Reaction, Luciferase, Activity Assay, Activation Assay

    FTY-720 reverses cardiac hypertrophy and interstitial fibrosis caused by pressure overload. (A) HW/TL ratios and (B) Measurements of mean cross-sectional area of cardiomyocytes (scale bar: 20μm) demonstrate that TAC increased whereas FTY-720 reduced cardiac hypertrophy. (C) qPCR analysis of ANP and RCAN1.4 . The data is normalized to the GAPDH. (D) Masson’s trichrome staining (scale bar: 50μm) shows that FTY-720 remarkably reduced TAC-induced fibrosis. Quantification of the relative area of fibrosis is expressed as percentage fibrosis. (E) Quantification of immunostaining of heart sections with anti-Mac-3 and anti-neutrophil antibodies indicates increased populations of macrophages and neutrophils in TAC-stressed ventricular myocardium, whereas FTY-720 treatment ameliorated this TAC-provoked leucocyte infiltration. N=8–12 per group, data is presented as mean ± SEM.

    Journal: Circulation. Heart failure

    Article Title: A Novel Immunomodulator, FTY-720 Reverses Existing Cardiac Hypertrophy and Fibrosis from Pressure Overload by Targeting NFAT Signaling and Periostin

    doi: 10.1161/CIRCHEARTFAILURE.112.000123

    Figure Lengend Snippet: FTY-720 reverses cardiac hypertrophy and interstitial fibrosis caused by pressure overload. (A) HW/TL ratios and (B) Measurements of mean cross-sectional area of cardiomyocytes (scale bar: 20μm) demonstrate that TAC increased whereas FTY-720 reduced cardiac hypertrophy. (C) qPCR analysis of ANP and RCAN1.4 . The data is normalized to the GAPDH. (D) Masson’s trichrome staining (scale bar: 50μm) shows that FTY-720 remarkably reduced TAC-induced fibrosis. Quantification of the relative area of fibrosis is expressed as percentage fibrosis. (E) Quantification of immunostaining of heart sections with anti-Mac-3 and anti-neutrophil antibodies indicates increased populations of macrophages and neutrophils in TAC-stressed ventricular myocardium, whereas FTY-720 treatment ameliorated this TAC-provoked leucocyte infiltration. N=8–12 per group, data is presented as mean ± SEM.

    Article Snippet: Brinkmann V. FTY-720: Mechanism of action and potential benefit in organ transplantation.

    Techniques: Real-time Polymerase Chain Reaction, Aqueous Normal-phase Chromatography, Staining, Immunostaining

    Electrophysiological characteristics of fingolimod treatment in CD86 −/− NOD mice. ( A ) The compound motor action potential amplitude (CMAP) of the sciatic nerve declined similarly in both fingolimod and vehicle treated CD86 −/− NOD mice. ( B ) Motor fibers showed electrophysiological signs of demyelination indicated by the decrease of motor nerve conduction velocity (MCV), which was more pronounced in the fingolimod treated mice compared to vehicle treatment. ( C ) F-wave latency increased in both groups with disease progression. Again, fingolimod treated mice were more affected than mice in the vehicle group. Similar to motor fibers, the ( D ) sensory nerve action potential amplitude (SNAP) and ( E ) sensory nerve conduction velocity (SCV) declined in both treatment groups. Statistical analysis: ( A ) Kruskall-Wallis test with Dunn’s method, ( B – E ) 2-way ANOVA with Sidak post hoc, group sizes: n = 10 (vehicle), n = 9–10 (fingolimod). *p ≤ 0.05, ns: not significant.

    Journal: Scientific Reports

    Article Title: Fingolimod therapy is not effective in a mouse model of spontaneous autoimmune peripheral polyneuropathy

    doi: 10.1038/s41598-018-23949-4

    Figure Lengend Snippet: Electrophysiological characteristics of fingolimod treatment in CD86 −/− NOD mice. ( A ) The compound motor action potential amplitude (CMAP) of the sciatic nerve declined similarly in both fingolimod and vehicle treated CD86 −/− NOD mice. ( B ) Motor fibers showed electrophysiological signs of demyelination indicated by the decrease of motor nerve conduction velocity (MCV), which was more pronounced in the fingolimod treated mice compared to vehicle treatment. ( C ) F-wave latency increased in both groups with disease progression. Again, fingolimod treated mice were more affected than mice in the vehicle group. Similar to motor fibers, the ( D ) sensory nerve action potential amplitude (SNAP) and ( E ) sensory nerve conduction velocity (SCV) declined in both treatment groups. Statistical analysis: ( A ) Kruskall-Wallis test with Dunn’s method, ( B – E ) 2-way ANOVA with Sidak post hoc, group sizes: n = 10 (vehicle), n = 9–10 (fingolimod). *p ≤ 0.05, ns: not significant.

    Article Snippet: Treatment protocols, drug application and placement of osmotic pump Fingolimod (S5002; Selleckchem, Munich, Germany) was dissolved in 0.9% saline solution (NaCl) to a final concentration of 9.5 mg/ml.

    Techniques: Mouse Assay

    Influence of fingolimod on serum cytokine expression. Serum samples were taken after an eight-week treatment period with fingolimod or vehicle. No differences in cytokine levels were observed for ( A ) Interferon-γ (IFN-γ), ( B ) Tumor necrosis factor-α (TNF-α), ( C ) Interleukin (IL)-1b (IL-1b), ( D ) IL-2. ( E ) Fingolimod treated mice showed elevated levels of IL-6 compared to vehicle treated control animals, while ( F ) IL-10, ( G ) IL-12p70 and ( H ) IL-17 were not different between treatment groups. Statistical analysis: ( A , C , D , E , G , H ) Mann-Whitney-U test, ( B , F ) unpaired t-test, group sizes: n = 5–9 (vehicle), n = 3–8 (fingolimod). *p ≤ 0.05, ns: not significant.

    Journal: Scientific Reports

    Article Title: Fingolimod therapy is not effective in a mouse model of spontaneous autoimmune peripheral polyneuropathy

    doi: 10.1038/s41598-018-23949-4

    Figure Lengend Snippet: Influence of fingolimod on serum cytokine expression. Serum samples were taken after an eight-week treatment period with fingolimod or vehicle. No differences in cytokine levels were observed for ( A ) Interferon-γ (IFN-γ), ( B ) Tumor necrosis factor-α (TNF-α), ( C ) Interleukin (IL)-1b (IL-1b), ( D ) IL-2. ( E ) Fingolimod treated mice showed elevated levels of IL-6 compared to vehicle treated control animals, while ( F ) IL-10, ( G ) IL-12p70 and ( H ) IL-17 were not different between treatment groups. Statistical analysis: ( A , C , D , E , G , H ) Mann-Whitney-U test, ( B , F ) unpaired t-test, group sizes: n = 5–9 (vehicle), n = 3–8 (fingolimod). *p ≤ 0.05, ns: not significant.

    Article Snippet: Treatment protocols, drug application and placement of osmotic pump Fingolimod (S5002; Selleckchem, Munich, Germany) was dissolved in 0.9% saline solution (NaCl) to a final concentration of 9.5 mg/ml.

    Techniques: Expressing, Mouse Assay, MANN-WHITNEY

    Effect of fingolimod treatment on disease progression. ( A ) Fingolimod (1 mg/kg bodyweight/day) and vehicle treated CD86 −/− NOD knockout mice showed no difference in weight progression over the course of the treatment. ( B ) Survival of mice did not differ between fingolimod and vehicle treated animals, although one animal in the fingolimod group died due to complications of the pump implantation. ( C ) Mice in both groups had a median clinical score of 2.0 at the start of treatment. Both fingolimod and vehicle treated mice showed a similar disease progression over the course of the 8-week treatment (indicated by gray shaded rectangle). ( D ) Locomotor function declined similarly between fingolimod and vehicle treated mice as disease and paralysis progressed. Statistical analysis: ( A ) 2-way ANOVA with Sidak post hoc test, ( B ) Kaplan-Meier analysis with Mantel-Cox test, ( D ) Kruskall-Wallis test with Dunn’s method; group sizes: n = 10 (vehicle), n = 9–10 (fingolimod). *p ≤ 0.05, ns: not significant.

    Journal: Scientific Reports

    Article Title: Fingolimod therapy is not effective in a mouse model of spontaneous autoimmune peripheral polyneuropathy

    doi: 10.1038/s41598-018-23949-4

    Figure Lengend Snippet: Effect of fingolimod treatment on disease progression. ( A ) Fingolimod (1 mg/kg bodyweight/day) and vehicle treated CD86 −/− NOD knockout mice showed no difference in weight progression over the course of the treatment. ( B ) Survival of mice did not differ between fingolimod and vehicle treated animals, although one animal in the fingolimod group died due to complications of the pump implantation. ( C ) Mice in both groups had a median clinical score of 2.0 at the start of treatment. Both fingolimod and vehicle treated mice showed a similar disease progression over the course of the 8-week treatment (indicated by gray shaded rectangle). ( D ) Locomotor function declined similarly between fingolimod and vehicle treated mice as disease and paralysis progressed. Statistical analysis: ( A ) 2-way ANOVA with Sidak post hoc test, ( B ) Kaplan-Meier analysis with Mantel-Cox test, ( D ) Kruskall-Wallis test with Dunn’s method; group sizes: n = 10 (vehicle), n = 9–10 (fingolimod). *p ≤ 0.05, ns: not significant.

    Article Snippet: Treatment protocols, drug application and placement of osmotic pump Fingolimod (S5002; Selleckchem, Munich, Germany) was dissolved in 0.9% saline solution (NaCl) to a final concentration of 9.5 mg/ml.

    Techniques: Knock-Out, Mouse Assay

    Effect of FTY720 on inflammatory cytokines, including monocyte chemoattractant protein-1 (MCP-1), macrophage-related TNF-α and IL-6, arginase-1 and IL-10, in the three groups. (A) Western blot analysis of MCP-1 and M1 and M2 macrophage-related cytokines; β-actin was used as a control. (B) Quantitative analysis of protein expression of MCP-1 and macrophage-related cytokines. (C) Expression of MCP-1 and macrophage-related cytokines mRNA; β-actin was used as a control. Values are presented as the mean±SEM. n =6. b P

    Journal: Acta Pharmacologica Sinica

    Article Title: FTY720 inhibits tubulointerstitial inflammation in albumin overload-induced nephropathy of rats via the Sphk1 pathway

    doi: 10.1038/aps.2014.100

    Figure Lengend Snippet: Effect of FTY720 on inflammatory cytokines, including monocyte chemoattractant protein-1 (MCP-1), macrophage-related TNF-α and IL-6, arginase-1 and IL-10, in the three groups. (A) Western blot analysis of MCP-1 and M1 and M2 macrophage-related cytokines; β-actin was used as a control. (B) Quantitative analysis of protein expression of MCP-1 and macrophage-related cytokines. (C) Expression of MCP-1 and macrophage-related cytokines mRNA; β-actin was used as a control. Values are presented as the mean±SEM. n =6. b P

    Article Snippet: Lymphocytes “sense” an S1P gradient (low concentrations in the lymph nodes versus high concentrations in circulation) with their S1P1 receptors, thereby migrating out of the lymphoid organs into the circulation, and this action was found to be blocked by FTY720.

    Techniques: Western Blot, Expressing

    Effect of FTY720 on urinary protein and NAG. Time course of proteinuria (A) and N -acetyl-β- D -glycosaminidase (NAG) activity (B) in saline controls, AO rats, and FTY720-treated rats. Values are presented as the mean±SEM. n =6. b P

    Journal: Acta Pharmacologica Sinica

    Article Title: FTY720 inhibits tubulointerstitial inflammation in albumin overload-induced nephropathy of rats via the Sphk1 pathway

    doi: 10.1038/aps.2014.100

    Figure Lengend Snippet: Effect of FTY720 on urinary protein and NAG. Time course of proteinuria (A) and N -acetyl-β- D -glycosaminidase (NAG) activity (B) in saline controls, AO rats, and FTY720-treated rats. Values are presented as the mean±SEM. n =6. b P

    Article Snippet: Lymphocytes “sense” an S1P gradient (low concentrations in the lymph nodes versus high concentrations in circulation) with their S1P1 receptors, thereby migrating out of the lymphoid organs into the circulation, and this action was found to be blocked by FTY720.

    Techniques: Activity Assay

    Effect of FTY720 on histological changes in AO rats. (A) PAS stains of tissue as indicated (×200). (B) Tubulointerstitial injury score for the three groups. Values are presented as the mean±SEM. n =6. b P

    Journal: Acta Pharmacologica Sinica

    Article Title: FTY720 inhibits tubulointerstitial inflammation in albumin overload-induced nephropathy of rats via the Sphk1 pathway

    doi: 10.1038/aps.2014.100

    Figure Lengend Snippet: Effect of FTY720 on histological changes in AO rats. (A) PAS stains of tissue as indicated (×200). (B) Tubulointerstitial injury score for the three groups. Values are presented as the mean±SEM. n =6. b P

    Article Snippet: Lymphocytes “sense” an S1P gradient (low concentrations in the lymph nodes versus high concentrations in circulation) with their S1P1 receptors, thereby migrating out of the lymphoid organs into the circulation, and this action was found to be blocked by FTY720.

    Techniques:

    Effect of FTY720 on peripheral blood cells. (A) Lymphocytes in the blood. (B) Monocytes in the blood. Values are the means±SEM. n =6. b P

    Journal: Acta Pharmacologica Sinica

    Article Title: FTY720 inhibits tubulointerstitial inflammation in albumin overload-induced nephropathy of rats via the Sphk1 pathway

    doi: 10.1038/aps.2014.100

    Figure Lengend Snippet: Effect of FTY720 on peripheral blood cells. (A) Lymphocytes in the blood. (B) Monocytes in the blood. Values are the means±SEM. n =6. b P

    Article Snippet: Lymphocytes “sense” an S1P gradient (low concentrations in the lymph nodes versus high concentrations in circulation) with their S1P1 receptors, thereby migrating out of the lymphoid organs into the circulation, and this action was found to be blocked by FTY720.

    Techniques:

    Effect of FTY720 on Sphk1/S1P/S1pr signal-related protein expression in the three groups. (A) Immunohistochemical staining of Sphk1, S1pr1, and S1pr3 in the saline, AO, and AO+FTY720 groups (original magnification is ×200). (B) Semiquantitative immunohistochemical analysis of the Sphk1/S1P/S1pr signal-related protein expression in the three groups (I–III). Values are presented as the mean±SEM. n =6. b P

    Journal: Acta Pharmacologica Sinica

    Article Title: FTY720 inhibits tubulointerstitial inflammation in albumin overload-induced nephropathy of rats via the Sphk1 pathway

    doi: 10.1038/aps.2014.100

    Figure Lengend Snippet: Effect of FTY720 on Sphk1/S1P/S1pr signal-related protein expression in the three groups. (A) Immunohistochemical staining of Sphk1, S1pr1, and S1pr3 in the saline, AO, and AO+FTY720 groups (original magnification is ×200). (B) Semiquantitative immunohistochemical analysis of the Sphk1/S1P/S1pr signal-related protein expression in the three groups (I–III). Values are presented as the mean±SEM. n =6. b P

    Article Snippet: Lymphocytes “sense” an S1P gradient (low concentrations in the lymph nodes versus high concentrations in circulation) with their S1P1 receptors, thereby migrating out of the lymphoid organs into the circulation, and this action was found to be blocked by FTY720.

    Techniques: Expressing, Immunohistochemistry, Staining

    Ischemic lesion size in propranolol-treated (a) FTY720-treated (b) and the respective vehicle-treated control mice with tMCAO of 1 h occlusion time after an observation period of 24 h after tMCAO (propranolol: n = 7, vehicle: n = 9; FTY720: n = 8, vehicle: n = 10). The box boundaries mark the 25th and 75th percentile, the line within the box indicates the mean. Whiskers above and below the box mark the minimum and maximum. Statistical significance was assessed with Student’s t test, * p = 0.01. FTY720, fingolimod; tMCAO, transient middle cerebral artery occlusion.

    Journal: Therapeutic Advances in Neurological Disorders

    Article Title: Beta adrenoceptor blockade ameliorates impaired glucose tolerance and alterations of the cerebral ceramide metabolism in an experimental model of ischemic stroke

    doi: 10.1177/1756286418769830

    Figure Lengend Snippet: Ischemic lesion size in propranolol-treated (a) FTY720-treated (b) and the respective vehicle-treated control mice with tMCAO of 1 h occlusion time after an observation period of 24 h after tMCAO (propranolol: n = 7, vehicle: n = 9; FTY720: n = 8, vehicle: n = 10). The box boundaries mark the 25th and 75th percentile, the line within the box indicates the mean. Whiskers above and below the box mark the minimum and maximum. Statistical significance was assessed with Student’s t test, * p = 0.01. FTY720, fingolimod; tMCAO, transient middle cerebral artery occlusion.

    Article Snippet: Pharmacological interventions with propranolol and FTY 720 Propranolol [(±)-propranolol hydrochloride, Sigma-Aldrich], was dissolved in 0.9% sodium chloride solution and administered 10 mg/kg intra peritoneally (i.p.) at 0, 4 and 8 h after tMCAO as previously described.

    Techniques: Mouse Assay