Journal: Frontiers in Immunology

Article Title: Direct antigen presentation is the canonical pathway of cytomegalovirus CD8 T-cell priming regulated by balanced immune evasion ensuring a strong antiviral response

doi: 10.3389/fimmu.2023.1272166

Figure Lengend Snippet: CD8 T-cell response induced by mCMV in presence and absence of the cross-presentation pathway. (A) CD8 T-cell response in C57BL/6 mice (n=5 per group/virus, tested as cohorts). (B) CD8 T-cell response in Unc93b1 3d/3d mice (n=4 per group/virus, tested as cohorts) that are deficient in antigen cross-presentation. CD8 T cells were isolated from the draining regional lymph node (RLN), that is, the popliteal lymph node, on day 7 after intraplantar infection with 1x10 5 PFU each of either mCMV-WT (WT) or mCMV-Δm152 (Δm152), and used as effector cells in an IFNγ-based ELISpot assay. EL4 cells exogenously loaded with synthetic peptides at a saturating concentration of 10 −7 M were used as APCs. The panel of tested antigenic peptides and the corresponding peptide-presenting MHC-I molecules are indicated. Bars represent cohort average most probable numbers (MPN) of responding cells determined by intercept-free linear regression analysis, and error bars represent the corresponding 95% confidence intervals. Test groups are considered significantly different if the 95% confidence intervals do not overlap. Ø, no peptide added.

Article Snippet: P815 (No. TIB-64, haplotype H-2 d ) and EL4 (No. TIB-39, haplotype H-2 b ) cells were obtained from the American Tissue Culture Collection (ATCC) and cultivated in RPMI supplemented with 5% fetal calf serum (FCS) and antibiotics, or in DMEM with 10% FCS and antibiotics, respectively.

Techniques: Virus, Isolation, Infection, Enzyme-linked Immunospot, Concentration Assay

Journal: Frontiers in Cellular Neuroscience

Article Title: Glucagon-Like Peptide-1 Secreting L-Cells Coupled to Sensory Nerves Translate Microbial Signals to the Host Rat Nervous System

doi: 10.3389/fncel.2020.00095

Figure Lengend Snippet: L-cells facilitate indole-evoked activation of vagal afferents. (A) The plot illustrates basolateral secretion of Glucagon-like peptide-1 (GLP-1) from colonic tissue apically exposed to indole (1 mM, 30 min) or saline (control). (B) The schematic (amended from Buckley and O’Malley, ) illustrates the ex vivo distal colonic tissue preparation. Recording is made from vagal afferents attached to the esophagus. The mucosa can be left intact, or removed to expose enteric neurons and associated afferent endings. (C) The rectified and raw (in boxes) traces show vagal nerve activity in response to stimulation of the distal colonic mucosa. Scatter plots illustrate peak frequency. Indole-evoked vagal nerve activity was attenuated by (D) exendin 3(9–39) amide [Ex-3(9–39)]. (E) Exendin-4 (Ex-4) stimulated vagal nerve firing when applied to the exposed neurons. (F) Tetrodotoxin (TTX) inhibited the indole-evoked response when it is applied to a hemisected piece of colonic tissue but not (G) colonic tissue with an intact mucosa. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Reagents [indole (1 mM, Sigma-Aldrich: cat. No. I3408), exendin 3(9–39) amide (10 μM, Tocris; cat. No. 2081), exendin-4 (10 μM, Tocris: cat. No. 1933) or tetrodotoxin (10 nM, Tocris: cat. No. 1078)] were applied to the colonic bath in the superfusate.

Techniques: Activation Assay, Saline, Control, Ex Vivo, Activity Assay

Journal: Frontiers in Cellular Neuroscience

Article Title: Glucagon-Like Peptide-1 Secreting L-Cells Coupled to Sensory Nerves Translate Microbial Signals to the Host Rat Nervous System

doi: 10.3389/fncel.2020.00095

Figure Lengend Snippet: Mucosal L-cells facilitate indole-evoked activation of submucosal neurons. (A) Indole did not affect intracellular calcium ([Ca 2+ ] i ) levels in submucosal neurons. (B) Immunofluorescence images show the GLP-1 receptor (GLP-1R) expression in colonic submucosal neurons following neuronal and mucosal exposure to indole. Arrows indicate increased expression at the neuronal cell membranes. Scalebar: 50 μm. (C) The GLP-1R agonist, exendin 4 (Ex-4) stimulated an increase in [Ca 2+ ] i , a reproducible effect that was (D) attenuated by the GLP-1R antagonist, exendin 3(9–39) amide [Ex-3(9–39)]. (E) The schematic illustrates a cross-section of the hemisected distal colon. By removing half of the mucosa, changes in intracellular calcium can be recorded from submucosal neurons loaded with a calcium indicator dye. By leaving some of the mucosa intact, the role of endocrine hormones secreted by epithelial cells can be investigated. (F) In hemisected colonic tissue, the Ex-4-evoked calcium response was enhanced by mucosal indole. (G) Immunofluorescent images show GLP-1R expression in colonic submucosal neurons following mucosal stimulation with saline, cryoprotectant, broth, L. paracasei secretions, and L. paracasei bacteria. Scalebar: 50 μm. (H) Mucosal application of L. paracasei secretions evoked a calcium response in submucosal neurons which was attenuated by the GLP-1R antagonist, exendin 3(9–39) amide [Ex-3(9–39)]. *** p < 0.001.

Article Snippet: Reagents [indole (1 mM, Sigma-Aldrich: cat. No. I3408), exendin 3(9–39) amide (10 μM, Tocris; cat. No. 2081), exendin-4 (10 μM, Tocris: cat. No. 1933) or tetrodotoxin (10 nM, Tocris: cat. No. 1078)] were applied to the colonic bath in the superfusate.

Techniques: Activation Assay, Immunofluorescence, Expressing, Saline, Bacteria

Journal: Frontiers in Cellular Neuroscience

Article Title: Glucagon-Like Peptide-1 Secreting L-Cells Coupled to Sensory Nerves Translate Microbial Signals to the Host Rat Nervous System

doi: 10.3389/fncel.2020.00095

Figure Lengend Snippet: Lactobacillus paracasei secretions stimulate host neurons. (A) The representative immunofluorescent image of a rat colonic cross-section illustrates that some L-cells expressed GLP-1Rs (arrowhead) but others did not (arrow). (B) Mucosal application of exendin-4 (Ex-4) increased vagal activity. (C) Increased vagal nerve activity evoked by L. paracasei secretions was abolished by Ex-3(9–39). * p < 0.05, ** p < 0.01. Scale bars: 50 μm.

Article Snippet: Reagents [indole (1 mM, Sigma-Aldrich: cat. No. I3408), exendin 3(9–39) amide (10 μM, Tocris; cat. No. 2081), exendin-4 (10 μM, Tocris: cat. No. 1933) or tetrodotoxin (10 nM, Tocris: cat. No. 1078)] were applied to the colonic bath in the superfusate.

Techniques: Activity Assay

Journal: Frontiers in Cellular Neuroscience

Article Title: Glucagon-Like Peptide-1 Secreting L-Cells Coupled to Sensory Nerves Translate Microbial Signals to the Host Rat Nervous System

doi: 10.3389/fncel.2020.00095

Figure Lengend Snippet: Exendin-4 stimulates calcium release from intracellular stores and influx via voltage-gated calcium channels. (A) The calcium response evoked by exendin-4 (Ex-4, 10 μM) was attenuated by thapsigargin (100 nM, n = 35) and (B) removal of extracellular calcium ( n = 17). Specific inhibitors of (C) P/Q-(ω agatoxin IVA 100 nM, n = 9) and (D) N-(ω-conotoxin GVIA, 100 nM, n = 14) type voltage gated calcium channels inhibited Ex-4-evoked calcium responses. (E) The phosphoinositide 3-kinase (PI 3-kinase) inhibitor, wortmannin (100 nM, n = 11), and (F) the STAT3 inhibitor, WP1006 (1 μM, n = 8) attenuated Ex-4-evoked calcium responses. (G) The ERK-MAPK inhibitor, PD98059 (1 μM, n = 15), had no effect. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Reagents [indole (1 mM, Sigma-Aldrich: cat. No. I3408), exendin 3(9–39) amide (10 μM, Tocris; cat. No. 2081), exendin-4 (10 μM, Tocris: cat. No. 1933) or tetrodotoxin (10 nM, Tocris: cat. No. 1078)] were applied to the colonic bath in the superfusate.

Techniques:

Journal: Frontiers in Cellular Neuroscience

Article Title: Glucagon-Like Peptide-1 Secreting L-Cells Coupled to Sensory Nerves Translate Microbial Signals to the Host Rat Nervous System

doi: 10.3389/fncel.2020.00095

Figure Lengend Snippet: Exendin-4 enhances colonic secretory currents. (A) The representative secretory traces from Ussing chamber studies and the associated scatter plots illustrate the secretory current evoked by the GLP-1R agonist, exendin 4 (Ex-4, 10 μM, n = 8). (B) Ex-4 does not modify the cholinergically-mediated secretory current (I SC ) evoked by carbachol (10 μM, n = 8) but enhances currents evoked by the (C) sodium channel agonist, veratridine (10 μM, n = 8) and (D) the sensory nerve stimulant, capsaicin (1 μM, n = 8). (E) Ex-4 also increases transepithelial resistance (TER) throughout an experiment (60–90 min). * p < 0.05, ** p < 0.01.

Article Snippet: Reagents [indole (1 mM, Sigma-Aldrich: cat. No. I3408), exendin 3(9–39) amide (10 μM, Tocris; cat. No. 2081), exendin-4 (10 μM, Tocris: cat. No. 1933) or tetrodotoxin (10 nM, Tocris: cat. No. 1078)] were applied to the colonic bath in the superfusate.

Techniques:

Journal:

Article Title: Antigen-pulsed dendritic cells expressing macrophage-derived chemokine elicit Th2 responses and promote specific humoral immunity

doi:

Figure Lengend Snippet: Chemotaxis of human and murine T lymphocytes to conditioned media of genetically modified DCs. (a) CEM cells. Human T lymphocyte CEM cells placed in the upper chamber of a transwell chamber were assayed for chemotaxis in response to serial dilution of supernatant from DCs transduced with AdMDC, AdNull, or PBS alone (naive control) in the lower chamber. (b) EL4 cells. The study was similar to that in a, but the murine T lymphocyte EL4 cell line was used. (c) Suppression of AdMDC-mediated chemotaxis. CEM and EL4 cells were assayed for chemotaxis in response to 1:2 dilution of supernatant from AdMDC-modified DCs. Where indicated, anti–MDC neutralizing Ab or mouse IgG control Ab was added to the supernatant in the lower chambers at 10 μg/ml at the initiation of assay or cells were placed to the upper chamber in the presence of recombinant (r) MDC or TARC at 100 ng/ml. For all parts, the number of cells migrating to the lower chamber at 37°C for 4 hours was counted by FACS analysis. Migration index was calculated as the number of cells migrating to the conditioned media over the number of cells migrating to control medium. Results represent the mean ± SE (n = 3 per data point).

Article Snippet: To demonstrate the function of the MDC protein expressed by the AdMDC-modified DCs, human T lymphocyte CEM cells or murine T lymphocyte EL4 cells (no. CCL-119 and TIB-39, respectively; American Type Culture Collection, Manassas, Virginia, USA) were suspended in 100 μl of RPMI-1640 media containing 1 mg/ml BSA at 10 7 cells/ml and loaded to the upper wells of 5-μm pore, polycarbonate, transwell chambers in 24-well plates (Corning Costar Inc., Corning, New York, USA).

Techniques: Chemotaxis Assay, Genetically Modified, Serial Dilution, Transduction, Control, Modification, Recombinant, Migration