Journal: EBioMedicine

Article Title: Therapeutic ultrasound attenuates DSS-induced colitis through the cholinergic anti-inflammatory pathway

doi: 10.1016/j.ebiom.2019.06.033

Figure Lengend Snippet: Immune cell population changes in colon, spleen and MLN in 2% DSS colitis mice under TUS. (A-E) Colon IHC analysis revealed no differences amongst all groups regarding B220 + B cells, increased levels of CD4 + , CD8 + and F4/80 + cells in comparison to controls, while CD8 + levels were decreased when comparing TUS treated animals to 2% DSS group and CD25 + T cells were increased in 2% DSS only group. (F-G) Spleen FACS analysis demonstrated no changes for CD4 + , CD25 + and F4/80 + cells. Increase percentage was seen for CD8 + T cells and decrease in B220 + B cells when comparing 2% DSS to control. In addition, TUS treatment normalized CD8 + T cells and B220 + B cells when compared to 2% DSS. (K—O) MLN IHC analysis demonstrated no difference amongst all groups for CD8 + , CD25 + and F4/80 + cells. TUS treatment increased CD4 + and B220 + levels compared to control. * p < .05 compared to control. #p < .05 compared to 2% DSS. One-way ANOVA followed by Tukey post-hoc test. N = 4/group for IHC analysis and N = 6/group for FACS analysis. Results are presented as mean ± SD.

Article Snippet: After blocking, samples were incubated overnight at 4 °C with the following primary antibodies: CD4 (Rabbit, 0.623 mg/mL, Abcam, cat. ab183685), CD8 (Rabbit, 1 mg/mL, Abcam, ab203035), CD25 (Goat, 0.2 mg/mL, Invitrogen, cat. PA5–46922), F4/80 (Rabbit, 0.23 mg/mL, Novus Biologicals, cat. NBP2–12506), B220 (Rat, 0.5 mg/mL, Invitrogen, cat. 14–0452-81), GFAP (Rabbit, 1 mg/mL, Abcam, cat. ab211271) and α7nAChR (Goat, 0.5 mg/mL, Abcam, cat. ab110851).

Techniques:

Journal: EBioMedicine

Article Title: Therapeutic ultrasound attenuates DSS-induced colitis through the cholinergic anti-inflammatory pathway

doi: 10.1016/j.ebiom.2019.06.033

Figure Lengend Snippet: IHC staining for Immune cell profiling of the colon. Representative images of IHC analysis performed at days 0 (control) and 14 (2% DSS and 2% DSS + TUS) in the colon of C57BL6 WT mice for CD4 + , CD8 + , CD25 + , F4/80 + and B220 + cells. Images were taken with a 20× objective.

Article Snippet: After blocking, samples were incubated overnight at 4 °C with the following primary antibodies: CD4 (Rabbit, 0.623 mg/mL, Abcam, cat. ab183685), CD8 (Rabbit, 1 mg/mL, Abcam, ab203035), CD25 (Goat, 0.2 mg/mL, Invitrogen, cat. PA5–46922), F4/80 (Rabbit, 0.23 mg/mL, Novus Biologicals, cat. NBP2–12506), B220 (Rat, 0.5 mg/mL, Invitrogen, cat. 14–0452-81), GFAP (Rabbit, 1 mg/mL, Abcam, cat. ab211271) and α7nAChR (Goat, 0.5 mg/mL, Abcam, cat. ab110851).

Techniques: Immunohistochemistry

Journal: EBioMedicine

Article Title: Therapeutic ultrasound attenuates DSS-induced colitis through the cholinergic anti-inflammatory pathway

doi: 10.1016/j.ebiom.2019.06.033

Figure Lengend Snippet: IHC staining for Immune cell profiling of the MLN. Representative images of IHC analysis performed at days 0 (control) and 14 (2% DSS and 2% DSS + TUS) in the MLN of C57BL6 WT mice for CD4 + , CD8 + , CD25 + , F4/80 + and B220 + cells. Images were taken with a 20× objective.

Article Snippet: After blocking, samples were incubated overnight at 4 °C with the following primary antibodies: CD4 (Rabbit, 0.623 mg/mL, Abcam, cat. ab183685), CD8 (Rabbit, 1 mg/mL, Abcam, ab203035), CD25 (Goat, 0.2 mg/mL, Invitrogen, cat. PA5–46922), F4/80 (Rabbit, 0.23 mg/mL, Novus Biologicals, cat. NBP2–12506), B220 (Rat, 0.5 mg/mL, Invitrogen, cat. 14–0452-81), GFAP (Rabbit, 1 mg/mL, Abcam, cat. ab211271) and α7nAChR (Goat, 0.5 mg/mL, Abcam, cat. ab110851).

Techniques: Immunohistochemistry

Journal: EBioMedicine

Article Title: Therapeutic ultrasound attenuates DSS-induced colitis through the cholinergic anti-inflammatory pathway

doi: 10.1016/j.ebiom.2019.06.033

Figure Lengend Snippet: Colon and MLN immune cell changes in splenectomised mice. There was no difference between the groups when analysing the colons for (A) CD4 + , (B) CD8 + , (D) F4/80 + and (E) B220 + cells. TUS induced an increase in (C) colonic CD25 + T cells while decreasing the percentage of (H) CD25 + T cells in MLN. Furthermore, (F) CD4 + T cell levels were decreased in the MLN, while no difference was seen regarding (G) CD8 + , (I) F4/80 + and (J) B220 + cells. *p < .05 compared to 2% DSS. Student's t-test. N = 4/group. Results are presented as mean ± SD.

Article Snippet: After blocking, samples were incubated overnight at 4 °C with the following primary antibodies: CD4 (Rabbit, 0.623 mg/mL, Abcam, cat. ab183685), CD8 (Rabbit, 1 mg/mL, Abcam, ab203035), CD25 (Goat, 0.2 mg/mL, Invitrogen, cat. PA5–46922), F4/80 (Rabbit, 0.23 mg/mL, Novus Biologicals, cat. NBP2–12506), B220 (Rat, 0.5 mg/mL, Invitrogen, cat. 14–0452-81), GFAP (Rabbit, 1 mg/mL, Abcam, cat. ab211271) and α7nAChR (Goat, 0.5 mg/mL, Abcam, cat. ab110851).

Techniques:

Journal: EBioMedicine

Article Title: Therapeutic ultrasound attenuates DSS-induced colitis through the cholinergic anti-inflammatory pathway

doi: 10.1016/j.ebiom.2019.06.033

Figure Lengend Snippet: IHC staining for Immune cell profiling of the colon of splenectomised mice. Representative images of IHC analysis performed at day 14 in the colon of splenectomised mice for CD4 + , CD8 + , CD25 + , F4/80 + and B220 + cells. Images were taken with a 20× objective.

Article Snippet: After blocking, samples were incubated overnight at 4 °C with the following primary antibodies: CD4 (Rabbit, 0.623 mg/mL, Abcam, cat. ab183685), CD8 (Rabbit, 1 mg/mL, Abcam, ab203035), CD25 (Goat, 0.2 mg/mL, Invitrogen, cat. PA5–46922), F4/80 (Rabbit, 0.23 mg/mL, Novus Biologicals, cat. NBP2–12506), B220 (Rat, 0.5 mg/mL, Invitrogen, cat. 14–0452-81), GFAP (Rabbit, 1 mg/mL, Abcam, cat. ab211271) and α7nAChR (Goat, 0.5 mg/mL, Abcam, cat. ab110851).

Techniques: Immunohistochemistry

Journal: EBioMedicine

Article Title: Therapeutic ultrasound attenuates DSS-induced colitis through the cholinergic anti-inflammatory pathway

doi: 10.1016/j.ebiom.2019.06.033

Figure Lengend Snippet: IHC staining for Immune cell profiling of the MLN of splenectomised mice. Representative images of IHC analysis performed at day 14 in the MLN of splenectomised mice for CD4 + , CD8 + , CD25 + , F4/80 + and B220 + cells. Images were taken with a 20× objective.

Article Snippet: After blocking, samples were incubated overnight at 4 °C with the following primary antibodies: CD4 (Rabbit, 0.623 mg/mL, Abcam, cat. ab183685), CD8 (Rabbit, 1 mg/mL, Abcam, ab203035), CD25 (Goat, 0.2 mg/mL, Invitrogen, cat. PA5–46922), F4/80 (Rabbit, 0.23 mg/mL, Novus Biologicals, cat. NBP2–12506), B220 (Rat, 0.5 mg/mL, Invitrogen, cat. 14–0452-81), GFAP (Rabbit, 1 mg/mL, Abcam, cat. ab211271) and α7nAChR (Goat, 0.5 mg/mL, Abcam, cat. ab110851).

Techniques: Immunohistochemistry

Journal: EBioMedicine

Article Title: Therapeutic ultrasound attenuates DSS-induced colitis through the cholinergic anti-inflammatory pathway

doi: 10.1016/j.ebiom.2019.06.033

Figure Lengend Snippet: Colon, spleen and MLN immune cell changes in α7nAChR KO mice. There was no difference amongst all groups when analysing the colons for (A-E) CD4 + , CD8 + , CD25 + , F4/80 + and B220 + cells. Splenic levels of (F) CD4 + were decreased (compared to 2% DSS) and (H) CD25 + T cells levels were not different from controls with TUS treatment. (G) CD8 + T cells were increased in both 2% DSS and 2% DSS + TUS KO groups. MLN levels of (M) CD25 + T cells were increased in both 2% DSS and 2% DSS + TUS KO groups, while decreased (O) B220 + levels were seen in the 2% DSS KO group only. There was no difference regarding splenic (I) F4/80 + and (J) B220 + cells, and no difference in MLN (K) CD4 + , (L) CD8 + and (N) F4/80 + cells. *p < .05 compared to 2% DSS WT. # p < .05 compared to 2% DSS KO. One-way ANOVA followed by Tukey post-hoc test. N = 4/group for IHC analysis. Results are presented as mean ± SD.

Article Snippet: After blocking, samples were incubated overnight at 4 °C with the following primary antibodies: CD4 (Rabbit, 0.623 mg/mL, Abcam, cat. ab183685), CD8 (Rabbit, 1 mg/mL, Abcam, ab203035), CD25 (Goat, 0.2 mg/mL, Invitrogen, cat. PA5–46922), F4/80 (Rabbit, 0.23 mg/mL, Novus Biologicals, cat. NBP2–12506), B220 (Rat, 0.5 mg/mL, Invitrogen, cat. 14–0452-81), GFAP (Rabbit, 1 mg/mL, Abcam, cat. ab211271) and α7nAChR (Goat, 0.5 mg/mL, Abcam, cat. ab110851).

Techniques:

Journal: EBioMedicine

Article Title: Therapeutic ultrasound attenuates DSS-induced colitis through the cholinergic anti-inflammatory pathway

doi: 10.1016/j.ebiom.2019.06.033

Figure Lengend Snippet: IHC staining for Immune cell profiling of the colon of WT or α7nAChR KO mice. Representative images of IHC analysis performed at day 14 in the colon of WT or α7nAChR KO mice for CD4 + , CD8 + , CD25 + , F4/80 + and B220 + cells. Images were taken with a 20× objective.

Article Snippet: After blocking, samples were incubated overnight at 4 °C with the following primary antibodies: CD4 (Rabbit, 0.623 mg/mL, Abcam, cat. ab183685), CD8 (Rabbit, 1 mg/mL, Abcam, ab203035), CD25 (Goat, 0.2 mg/mL, Invitrogen, cat. PA5–46922), F4/80 (Rabbit, 0.23 mg/mL, Novus Biologicals, cat. NBP2–12506), B220 (Rat, 0.5 mg/mL, Invitrogen, cat. 14–0452-81), GFAP (Rabbit, 1 mg/mL, Abcam, cat. ab211271) and α7nAChR (Goat, 0.5 mg/mL, Abcam, cat. ab110851).

Techniques: Immunohistochemistry

Journal: EBioMedicine

Article Title: Therapeutic ultrasound attenuates DSS-induced colitis through the cholinergic anti-inflammatory pathway

doi: 10.1016/j.ebiom.2019.06.033

Figure Lengend Snippet: IHC staining for Immune cell profiling of the MLN of WT or α7nAChR KO mice. Representative images of IHC analysis performed at day 14 in the MLN of WT or α7nAChR KO mice for CD4 + , CD8 + , CD25 + , F4/80 + and B220 + cells. Images were taken with a 20× objective.

Article Snippet: After blocking, samples were incubated overnight at 4 °C with the following primary antibodies: CD4 (Rabbit, 0.623 mg/mL, Abcam, cat. ab183685), CD8 (Rabbit, 1 mg/mL, Abcam, ab203035), CD25 (Goat, 0.2 mg/mL, Invitrogen, cat. PA5–46922), F4/80 (Rabbit, 0.23 mg/mL, Novus Biologicals, cat. NBP2–12506), B220 (Rat, 0.5 mg/mL, Invitrogen, cat. 14–0452-81), GFAP (Rabbit, 1 mg/mL, Abcam, cat. ab211271) and α7nAChR (Goat, 0.5 mg/mL, Abcam, cat. ab110851).

Techniques: Immunohistochemistry

Journal: EBioMedicine

Article Title: Therapeutic ultrasound attenuates DSS-induced colitis through the cholinergic anti-inflammatory pathway

doi: 10.1016/j.ebiom.2019.06.033

Figure Lengend Snippet: IHC staining for Immune cell profiling of the spleen of WT or α7nAChR KO mice. Representative images of IHC analysis performed at day 14 in the spleen of WT or α7nAChR KO mice for CD4 + , CD8 + , CD25 + , F4/80 + and B220 + cells. Images were taken with a 20× objective.

Article Snippet: After blocking, samples were incubated overnight at 4 °C with the following primary antibodies: CD4 (Rabbit, 0.623 mg/mL, Abcam, cat. ab183685), CD8 (Rabbit, 1 mg/mL, Abcam, ab203035), CD25 (Goat, 0.2 mg/mL, Invitrogen, cat. PA5–46922), F4/80 (Rabbit, 0.23 mg/mL, Novus Biologicals, cat. NBP2–12506), B220 (Rat, 0.5 mg/mL, Invitrogen, cat. 14–0452-81), GFAP (Rabbit, 1 mg/mL, Abcam, cat. ab211271) and α7nAChR (Goat, 0.5 mg/mL, Abcam, cat. ab110851).

Techniques: Immunohistochemistry

Journal: Cancer Immunology, Immunotherapy

Article Title: TGF-β orchestrates the phenotype and function of monocytic myeloid-derived suppressor cells in colorectal cancer

doi: 10.1007/s00262-021-03081-5

Figure Lengend Snippet: CD14+HLA-DR- cells are the major immunosuppressive cells in CRC. a Allogeneic T cell proliferation under anti-CD3/CD28 antibody stimulation is suppressed with the addition of CD14+HLA-DR- cells from the blood (n=16) and the tissue (n=13) of CRC patients, as measured by 3HTymidine uptake. The CD14+HLA-DR+ population is not suppressive. The 1:1 ratio of T cells:CD14+HLA-DR- cells is shown. Each colour represents a CRC patient. b Significantly higher level of IL-10 was detected by ELISA in the supernatant of CD14+ (blood samples n=8, tumour tissue n=8)and CD14+HLA-DR- cells (blood samples n=5, tumour tissue n=3) compared healthy control. c IL-10 receptor blocking antibody (10μg/ml) inhibits the suppressive activity of CD14+HLA-DR- cells of CRC patients restoring T cell proliferation (n=10). d Colorectal tumour conditioned media (TCMs, n=15)drive the release of IL10 from healthy monocytes after 48h incubation. e A representative histogram of HLA-DR expression on monocytes after 48h incubation with TCM. f Down regulation of HLA-DR on monocytes cultured with 20 TCMs

Article Snippet: Where indicated anti-IL-10 Receptor Antibody (10 μg/ml) ((Thermo Fisher Scientific Cat# 16-7108-85, RRID:AB_469229) (Isotype control, Rat IgG Ƙ Isotype, (Thermo Fisher Scientific Cat# 16-4301-85, RRID:AB_470154) were added to cultures to inhibit IL-10 binding.

Techniques: Enzyme-linked Immunosorbent Assay, Blocking Assay, Activity Assay, Incubation, Expressing, Cell Culture

Journal: Cancer Immunology, Immunotherapy

Article Title: TGF-β orchestrates the phenotype and function of monocytic myeloid-derived suppressor cells in colorectal cancer

doi: 10.1007/s00262-021-03081-5

Figure Lengend Snippet: Colorectal tumour conditioned media drives IL10 secretion. a Significantly higher level of IL-10 was detected by ELISA in the supernatant of CD14+HLA-DR- cells polarized by TCM (blood samples n=4, tumour tissue n=3) compared healthy control. b IL-10 receptor blocking antibody (10μg/ml) inhibits the suppressive activity of TCMs polarized CD14+HLA-DR- cells restoring T cell proliferation (n=4). Higher concentration of TGF-β was measured by ELISA in TCM (n=19) c and in the plasma d of colorectal patients (n=39 and healthy plasma n=10). e A representative histogram of HLA-DR expression on monocytes cultured with cytokines for 48h. f Down regulation of HLA-DR on monocytes cultured with cytokines for 48h was observed by Flow analysis (n=23 TGFβ, n=26 IL-6, n=11 IL-10, n=6 G-CSF, n=5 GM-CSF and n=4 VEGF)

Article Snippet: Where indicated anti-IL-10 Receptor Antibody (10 μg/ml) ((Thermo Fisher Scientific Cat# 16-7108-85, RRID:AB_469229) (Isotype control, Rat IgG Ƙ Isotype, (Thermo Fisher Scientific Cat# 16-4301-85, RRID:AB_470154) were added to cultures to inhibit IL-10 binding.

Techniques: Enzyme-linked Immunosorbent Assay, Blocking Assay, Activity Assay, Concentration Assay, Expressing, Cell Culture

Journal: Cancer Immunology, Immunotherapy

Article Title: TGF-β orchestrates the phenotype and function of monocytic myeloid-derived suppressor cells in colorectal cancer

doi: 10.1007/s00262-021-03081-5

Figure Lengend Snippet: TGF-β derived M-MDSC suppress T cell proliferation. a TGF-β polarized CD14+HLA-DRcells suppress T-cell proliferation. Less suppressive activity has shown from TGF-β polarized CD14+HLA-DR+ cells. The 1:0.5 ratio of T cells: myeloid cells shown (n=13). b High expression of p-SMAD and p-STAT3 was detected by Western blot in the CD14+HLA-DR- cells polarized by TGF-β.Representative of 4 experiments. c and d TGF-β receptor inhibitor inhibits the ability of TGF-β and TCMs to down regulate HLA-DR on polarized monocytes (n=4). e TGF- β receptor inhibitor inhibits the release of IL-10 from TGF-β and TCM polarized monocytes. Representative of 2 experiments. f TGF- β receptor inhibitor inhibits the suppressive activity of CD14+HLA-DR- cells polarized with TGF-β and CRC cell TCM (n=3)

Article Snippet: Where indicated anti-IL-10 Receptor Antibody (10 μg/ml) ((Thermo Fisher Scientific Cat# 16-7108-85, RRID:AB_469229) (Isotype control, Rat IgG Ƙ Isotype, (Thermo Fisher Scientific Cat# 16-4301-85, RRID:AB_470154) were added to cultures to inhibit IL-10 binding.

Techniques: Derivative Assay, Activity Assay, Expressing, Western Blot

Journal: PLoS ONE

Article Title: CNTF Mediates Neurotrophic Factor Secretion and Fluid Absorption in Human Retinal Pigment Epithelium

doi: 10.1371/journal.pone.0023148

Figure Lengend Snippet: The cytokines are depicted schematically as grey circles. Signal transducing-receptor subunits are light blue (LIFRβ and OsMRβ) or dark blue (gp130). α-receptor subunits are shown in light grey.

Article Snippet: Membranes were incubated in StartingBlock™ T20 (TBS) (Pierce Biotechnology) and probed with antibodies against CNTFRα (ab58560, Abcam, Cambridge, MA), LIFRβ (AF-249-NA, R & D Systems, Minneapolis, MN), gp130 (3732, Cell Signaling Technology, Danvers, MA), or OsMRβ (MAB4389, R & D Systems).

Techniques:

Journal: PLoS ONE

Article Title: CNTF Mediates Neurotrophic Factor Secretion and Fluid Absorption in Human Retinal Pigment Epithelium

doi: 10.1371/journal.pone.0023148

Figure Lengend Snippet: 15 µg of enriched membrane proteins were electrophoresed and labeled with corresponding antibodies. Antibody specific bands (see arrows) for CNTFRα, LIFRβ, gp130 and OsMRβ are detected at approximately 41, 190, 130, and 200 kDa, respectively. Panel A (CNTFRα) - Lane 1: Magic Mark™ XP Western protein Standard; Lane 2: Brain (Human) membrane lysate-adult normal tissue; Lane 3: hfRPE membrane extract. Panel B (LIFRβ) - Lane 1: Magic Mark™ XP Western protein Standard; Lane 2: hfRPE membrane extract. Panel C (gp130) - Lane 1: HiMark™ Molecular Weight (HMW) Standard; Lane 2: hfRPE membrane extract; Lane 3: Magic Mark™ XP Western protein Standard; Lane 4: Hela whole cell lysate; Lane 5: NIH3T3 whole cell lysate. Panel D (OsMRβ) - Lane 1: HiMark™ Molecular Weight (HMW) Standard; Lane 2: hfRPE membrane extract; Lane 3: Magic Mark™ XP Western protein Standard.

Article Snippet: Membranes were incubated in StartingBlock™ T20 (TBS) (Pierce Biotechnology) and probed with antibodies against CNTFRα (ab58560, Abcam, Cambridge, MA), LIFRβ (AF-249-NA, R & D Systems, Minneapolis, MN), gp130 (3732, Cell Signaling Technology, Danvers, MA), or OsMRβ (MAB4389, R & D Systems).

Techniques: Labeling, Western Blot, Molecular Weight

Journal: PLoS ONE

Article Title: CNTF Mediates Neurotrophic Factor Secretion and Fluid Absorption in Human Retinal Pigment Epithelium

doi: 10.1371/journal.pone.0023148

Figure Lengend Snippet: Central part of each panel is an enface view of cell culture monolayer, shown as a single optical section obtained from a Z-stack. Nuclei were stained with DAPI (blue) and ZO-1 tight junction marker stained green. Images of the cross section through the Z-plane are shown at the top of each panel. CNTFRα (A, red), LIFRβ (B, red), gp130 (C, red) and OsMRβ (D, red) were detected mainly on the apical membrane of confluent monolayer of hfRPE.

Article Snippet: Membranes were incubated in StartingBlock™ T20 (TBS) (Pierce Biotechnology) and probed with antibodies against CNTFRα (ab58560, Abcam, Cambridge, MA), LIFRβ (AF-249-NA, R & D Systems, Minneapolis, MN), gp130 (3732, Cell Signaling Technology, Danvers, MA), or OsMRβ (MAB4389, R & D Systems).

Techniques: Cell Culture, Staining, Marker