Journal: Molecular Imaging

Article Title: PET Study of Sphingosine-1-phosphate Receptor 1 Expression in Response to S. aureus Infection

doi: 10.1155/2021/9982020

Figure Lengend Snippet: MicroPET imaging of S1PR1 activity in S aureus -infected mice. (a) Radiosynthesis of S1PR1-specific radiotracer, [ 18 F]TZ4877; (b) representative sagittal microPET images of [ 18 F]TZ4877 in mice. Comparing with sham mice, the tracer uptake was significantly higher in the infected mice, and the increased uptake of the tracer showed S aureus dose dependent; (c) the tracer uptake in the brain was quantified; time-activity curves showed that the tracer uptake in infected mice was significantly higher than mice without infections; (d) the average tracer uptake in the brain from 30 to 50 min of the PET scan showed a dose-dependent manner. Data represent the mean ± SEM, n = 3 for each group.

Article Snippet: After washing in PBS, all sections were then incubated with rabbit anti-S1PR1 (Alomone, Israel) antibody overnight at 4°C and then washed and incubated with ImmPRESS HRP Horse anti-rabbit polymer for 1 hour at room temperature and developed with ImmPACT DAB (Vector Laboratories, Burlingame, CA).

Techniques: Imaging, Activity Assay, Infection

Journal: Molecular Imaging

Article Title: PET Study of Sphingosine-1-phosphate Receptor 1 Expression in Response to S. aureus Infection

doi: 10.1155/2021/9982020

Figure Lengend Snippet: Biodistribution (%ID/g, mean ± SEM) of S1PR1-specific [ 18 F]TZ4877 in Balb/c mice ( n = 4).

Article Snippet: After washing in PBS, all sections were then incubated with rabbit anti-S1PR1 (Alomone, Israel) antibody overnight at 4°C and then washed and incubated with ImmPRESS HRP Horse anti-rabbit polymer for 1 hour at room temperature and developed with ImmPACT DAB (Vector Laboratories, Burlingame, CA).

Techniques: Mouse Assay

Journal: Molecular Imaging

Article Title: PET Study of Sphingosine-1-phosphate Receptor 1 Expression in Response to S. aureus Infection

doi: 10.1155/2021/9982020

Figure Lengend Snippet: Biodistribution of S1PR1-specific [ 18 F]TZ4877 in sham, infected, and infected with treatments mice ( n = 4).

Article Snippet: After washing in PBS, all sections were then incubated with rabbit anti-S1PR1 (Alomone, Israel) antibody overnight at 4°C and then washed and incubated with ImmPRESS HRP Horse anti-rabbit polymer for 1 hour at room temperature and developed with ImmPACT DAB (Vector Laboratories, Burlingame, CA).

Techniques: Infection, Mouse Assay

Journal: Molecular Imaging

Article Title: PET Study of Sphingosine-1-phosphate Receptor 1 Expression in Response to S. aureus Infection

doi: 10.1155/2021/9982020

Figure Lengend Snippet: MicroPET imaging of S1PR1 activity in S aureus -infected mice. (a) Representative sagittal microPET images of [ 18 F]TZ4877 in the hind limb of mice. The tracer uptake was relatively low in the hind limb muscle with a SUV of ~1.5 in sham mice. Comparing with sham mice, the tracer uptake was significantly higher in the hind limb of infected mice; (b) time-activity curves showed that the tracer uptake in infected mice was significantly higher than sham mice; (c) the average tracer uptake in the hind limb muscle from 30 to 50 min of the PET scan showed a ~39% increase of SUV in infected mice with a P value of 0.0082. Data represent the mean ± SEM, n = 3 for each group.

Article Snippet: After washing in PBS, all sections were then incubated with rabbit anti-S1PR1 (Alomone, Israel) antibody overnight at 4°C and then washed and incubated with ImmPRESS HRP Horse anti-rabbit polymer for 1 hour at room temperature and developed with ImmPACT DAB (Vector Laboratories, Burlingame, CA).

Techniques: Imaging, Activity Assay, Infection

Journal: Molecular Imaging

Article Title: PET Study of Sphingosine-1-phosphate Receptor 1 Expression in Response to S. aureus Infection

doi: 10.1155/2021/9982020

Figure Lengend Snippet: PET measurements of S1PR1-specific [ 18 F]TZ4877 in S aureus -infected and sham mice.

Article Snippet: After washing in PBS, all sections were then incubated with rabbit anti-S1PR1 (Alomone, Israel) antibody overnight at 4°C and then washed and incubated with ImmPRESS HRP Horse anti-rabbit polymer for 1 hour at room temperature and developed with ImmPACT DAB (Vector Laboratories, Burlingame, CA).

Techniques: Infection

Journal: Molecular Imaging

Article Title: PET Study of Sphingosine-1-phosphate Receptor 1 Expression in Response to S. aureus Infection

doi: 10.1155/2021/9982020

Figure Lengend Snippet: Immunohistochemistry analysis of S1PR1 in hind limb muscle of sham and S aureus -infected mice. S1PR1 was significantly upregulated in the muscle of infected mice (red arrow) comparing with sham mice (green arrow), scale bar = 100 μ m.

Article Snippet: After washing in PBS, all sections were then incubated with rabbit anti-S1PR1 (Alomone, Israel) antibody overnight at 4°C and then washed and incubated with ImmPRESS HRP Horse anti-rabbit polymer for 1 hour at room temperature and developed with ImmPACT DAB (Vector Laboratories, Burlingame, CA).

Techniques: Immunohistochemistry, Infection

Journal: Journal of immunology research

Article Title: Diet-Induced Obesity Promotes Liver Metastasis of Pancreatic Ductal Adenocarcinoma via CX3CL1/CX3CR1 Axis.

doi: 10.1155/2022/5665964

Figure Lengend Snippet: Figure 4: DIO increased expression level of CX3CL1 and CX3CR1 in liver metastasis of PDAC. (a) Relative mRNA levels of CX3CL1 in liver of ND mice and DIO mice after intrasplenic injection with 1 × 106 Panc02 cells (n = 6/group). (b) Representative immunohistochemical images of CX3CL1 staining in liver of ND and DIO. Scale bar, 20 μm. (c) Bar graph showed the relative CX3CL1 staining positive rate. (d) Relative mRNA levels of CX3CL1 in liver of ND mice and DIO mice after intrasplenic injection with 1 × 106

Article Snippet: The cells were incubated with CX3CR1 primary antibody (1 : 100, Proteintech,13885-1-AP) for 30min in the dark at 4°C.

Techniques: Expressing, Injection, Immunohistochemical staining, Staining

Journal: Oncotarget

Article Title: The novel sphingosine-1-phosphate receptors antagonist AD2900 affects lymphocyte activation and inhibits T-cell entry into the lymph nodes

doi: 10.18632/oncotarget.18626

Figure Lengend Snippet: AD2900 shows antagonistic activities against S1P1, 2, 3, 4, and 5

Article Snippet: To analyze S1P1 surface expression on AD2900-treated PBMCs, the following were used: anti-S1P1 (Alomone labs, Israel), anti-EDG-1 (Abcam, UK), Dylight405-conjugated AffiniPure Goat Anti-Rabbit lgG (H + L), and APC-conjugated AffiniPure F(ab')2 Goat Anti-Rabbit lgG(H + L) (Jackson ImmonoResearch, USA).

Techniques:

Journal: Oncotarget

Article Title: The novel sphingosine-1-phosphate receptors antagonist AD2900 affects lymphocyte activation and inhibits T-cell entry into the lymph nodes

doi: 10.18632/oncotarget.18626

Figure Lengend Snippet: (A, B) The percentages of S1P1-positive PBMCs after the treatment with different concentrations of AD2900, FTY720, or SEW2871 and at different time points were examined by FACS analysis. S1P1 expression was tested in PBMCs after a 30-min treatment with AD2900 at different concentrations (A) or after a 30-min or 60-min treatment with 100 nM AD2900, FTY720, or SEW2871 (B) . (C) The percentage of CCR7-positive PBMCs was tested by FACS analysis after a 30-min treatment with 100 nM AD2900, FTY720, or SEW2871. All the significances are compared to untreated PBMCs. Results summarize the results of at least four independent experiments. Results of Student’s t -test: *(P < 0.05, two-tailed test), ** (P < 0.01, two-tailed test), *** (P < 0.001, two-tailed test), **** (P < 0.0001, two-tailed test).

Article Snippet: To analyze S1P1 surface expression on AD2900-treated PBMCs, the following were used: anti-S1P1 (Alomone labs, Israel), anti-EDG-1 (Abcam, UK), Dylight405-conjugated AffiniPure Goat Anti-Rabbit lgG (H + L), and APC-conjugated AffiniPure F(ab')2 Goat Anti-Rabbit lgG(H + L) (Jackson ImmonoResearch, USA).

Techniques: Expressing, Two Tailed Test

Journal: Oncotarget

Article Title: The novel sphingosine-1-phosphate receptors antagonist AD2900 affects lymphocyte activation and inhibits T-cell entry into the lymph nodes

doi: 10.18632/oncotarget.18626

Figure Lengend Snippet: C57BL/6 mice were orally administered with 1.8, 2.7, and 3.6 mg/l AD2900 or 1.8 mg/l FTY720 for 2 days, as shown in Figure . Leukocytes from blood, spleen, and pLNs were collected and stained with CD3e and S1P1 or CCR7 fluorescent antibodies and then analyzed by FACS analysis. The percentages of S1P1+ CD3e+ T cells from blood (A) , spleen (B) , and pLNs (C) are shown. The percentages of CCR7+ CD3e+ T cells from blood (D) , spleen (E) , and pLNs (F) are shown. All the significances are compared to untreated healthy mice. Results summarize at least three independent experiments. Results of Student’s t -test:*(P < 0.05, two-tailed test), ** (P < 0.01, two-tailed test), *** (P < 0.001, two-tailed test), **** (P < 0.0001, two-tailed test).

Article Snippet: To analyze S1P1 surface expression on AD2900-treated PBMCs, the following were used: anti-S1P1 (Alomone labs, Israel), anti-EDG-1 (Abcam, UK), Dylight405-conjugated AffiniPure Goat Anti-Rabbit lgG (H + L), and APC-conjugated AffiniPure F(ab')2 Goat Anti-Rabbit lgG(H + L) (Jackson ImmonoResearch, USA).

Techniques: Staining, Two Tailed Test

Journal: Oncotarget

Article Title: The novel sphingosine-1-phosphate receptors antagonist AD2900 affects lymphocyte activation and inhibits T-cell entry into the lymph nodes

doi: 10.18632/oncotarget.18626

Figure Lengend Snippet: As an antagonist to S1P receptors 1–5, AD2900 can compete with S1P to bind S1P receptors leading to reduced S1P signaling and enhanced expression of S1P1 on T cells in S1P-rich environments such as the blood and the spleen. This altered expression, together with decreased CCR7 expression, inhibits T-cell entry into the lymph nodes (LNs) from the blood, causing accumulation of T cells in the blood. However, the entry of T cells to the spleen is not affected because it is not S1P dependent. Since Tcm-like cells express CCR7, these cells are attracted to the spleen and accumulate in it; yet, S1P1 elevated expression may have an effect on the S1P-dependent ingression of these cells from the MZ to the white pulp. Tef/em-like cells, which are CCR7 negative, are the primary T-cell subpopulation in the blood after AD2900 treatment. The significant decrease in naive T-cell counts in the circulation and peripheral lymphoid tissues tested may be explained by the inhibition of S1P signaling in the thymus leading to attenuated T-cell egression from the thymus to the circulation. Arrow key: thick = response; dashed = inhibition.

Article Snippet: To analyze S1P1 surface expression on AD2900-treated PBMCs, the following were used: anti-S1P1 (Alomone labs, Israel), anti-EDG-1 (Abcam, UK), Dylight405-conjugated AffiniPure Goat Anti-Rabbit lgG (H + L), and APC-conjugated AffiniPure F(ab')2 Goat Anti-Rabbit lgG(H + L) (Jackson ImmonoResearch, USA).

Techniques: Expressing, Inhibition

Journal: Gut Microbes

Article Title: Butyrate reduces adherent-invasive E. coli -evoked disruption of epithelial mitochondrial morphology and barrier function: involvement of free fatty acid receptor 3

doi: 10.1080/19490976.2023.2281011

Figure Lengend Snippet: An FFAR3 agonist improves mitochondrial network connectivity and barrier function in E. coli -LF82-infected T84 epithelial cells. Monolayers of the human colon-derived T84 epithelial cell line were treated with E. coli -LF82 (10 8 cfu, 4 h) ± a co-treatment with the FFAR3 agonist AR420626 (25 µM) and representative images collected in a random fashion by first identifying epithelia nuclei (blue, n) and then swapping the confocal laser channel to assess the mitochondrial network as defined by TOMM-20 immunostains. Twenty cells per monolayer were characterized by semi-quantitative assessment (a). (b) Mitochondrial membrane potential was assessed by TMRE fluorescence in a flow cytometer. A 10 min treatment with the metabolic toxin, FCCP (10 µM, n = 5 epithelial monolayers from separate experiments (indicated by different symbols)). Filter-grown T84 cell monolayers (starting transepithelial resistance (TER) range = 957–2155 Ohms.cm ) were cultured with E. coli -LF82 (10 8 cfu) ± AR420626 and TER and transcytosis of the bacteria assessed 24 h later. (c) TER is presented as the change over 24 h with each monolayer being its’ own control ( i.e ., pre-treatment value). (d) Bacterial transcytosis was assessed via serial dilution of culture-well basolateral medium on agar plates, with the data being converted to % transcytosis based on bacterial counts in the apical compartment and then E. coli -LF82 was normalized to 100 for comparison with E. coli +AR420626 in the same experiment (data are mean ± SEM; each data point is an individual experiment ( n = 6) in which measurements from 3 or 4 monolayers were averaged and are shown as a different symbol; * and #, p <.05 compared to control uninfected cells (con) and E. coli -LF82 only infected cells, respectively).

Article Snippet: Following the experimental treatment, protein was extracted from human organoids and epithelial cell lines and immunoblotting performed as previously described using mouse anti-human FFAR3 (1:1,000; #66811–1-1 g, Proteintech, Rosemount, IL) and GAPDH (1:1,000; ab8245, Abcam) antibodies, and a goat anti-mouse HRP-conjugated secondary antibody (1:5,000; sc-2031; Santa Cruz Biotech., Pasa Robles, CA).

Techniques: Infection, Derivative Assay, Membrane, Fluorescence, Flow Cytometry, Cell Culture, Bacteria, Control, Serial Dilution, Comparison

Journal: Gut Microbes

Article Title: Butyrate reduces adherent-invasive E. coli -evoked disruption of epithelial mitochondrial morphology and barrier function: involvement of free fatty acid receptor 3

doi: 10.1080/19490976.2023.2281011

Figure Lengend Snippet: An FFAR3 agonist improves mitochondrial network connectivity in E. coli -LF82-infected human organoids. Monolayers of colonic organoids derived from two healthy controls were treated with E. coli -LF82 (MOI = 100, 4 h) ± a co-treatment with the FFAR3 agonist AR420626 (AR: 25 µM). Representative confocal images of MitoTracker TM (red) and Hoescht (blue) co-stained organoids show the fused mitochondrial network of control organoid cells treated with the FFAR3 agonist alone (a), the puncta-like fragmented mitochondrial network of E. coli -LF82 infected organoid cells (b), and the intermediate fragments of the E. coli- LF82 infected organoids co-treated with the FFAR3 agonist (c) (n, nucleus; *, filamentous mitochondria; arrowhead, fragmented mitochondria). Twenty cells were assessed from four monolayers for semi-quantitative analysis (d). Data are mean ± SEM, * and #, p < .05 compared to control uninfected drug-treated cells (AR) and E. coli -LF82 only infected cells, respectively (two-way ANOVA followed by Tukey’s multiple comparison test) (frag., fragmented mitochondria).

Article Snippet: Following the experimental treatment, protein was extracted from human organoids and epithelial cell lines and immunoblotting performed as previously described using mouse anti-human FFAR3 (1:1,000; #66811–1-1 g, Proteintech, Rosemount, IL) and GAPDH (1:1,000; ab8245, Abcam) antibodies, and a goat anti-mouse HRP-conjugated secondary antibody (1:5,000; sc-2031; Santa Cruz Biotech., Pasa Robles, CA).

Techniques: Infection, Derivative Assay, Staining, Control, Comparison