Journal: Yonsei Medical Journal

Article Title: Catalpol Inhibits Tregs-to-Th17 Cell Transdifferentiation by Up-Regulating Let-7g-5p to Reduce STAT3 Protein Levels

doi: 10.3349/ymj.2022.63.1.56

Figure Lengend Snippet: The effects of CAT on CD4 + IL-17 + Th17 cells and the expression of let-7g-5p in CIA mice. (A) The percentage of CD4 + IL-17 + Th17 cells in PBMCs of CIA mice. (B) The percentage of CD4 + IL-17 + Th17 cells in splenocytes from CIA mice. (C) Proportion of CD4 + IL-17 + Th17 cells in PBMCs from CIA mice. (D) Proportion of CD4 + IL-17 + Th17 cells in spleens from CIA mice. (E) IL-17 levels in plasma from CIA mice were measured by ELISA. (F) The mRNA levels of RORγt in splenic CD4 + T cells were detected by qPCR. (G) The mRNA levels of let-7g-5p in splenic CD4 + T cells were detected by qPCR. Values are presented as a mean±SEM (n=6). * p <0.5, ** p <0.01, *** p <0.001 compared with the model group. CAT, catalpol; CIA, collagen-induced arthritis; ELISA, enzyme-linked immunosorbent assay; qPCR, quantitative polymerase chain reaction; PBMC, peripheral blood mononuclear cells; IL, interleukin.

Article Snippet: Cells were then washed twice with PBS and activated with leukocyte activation cocktail (BD Pharmingen) for 4 h. After staining of Th17 cells with anti-CD4-FITC (BD Pharmingen) and anti-IL-17A-PE (BD Pharmingen) for 30 min, intracellular cytokines were analyzed by flow cytometry (Beckman FC-500, Beckman Coulter, Inc., Brea, CA, USA).

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

Journal: Yonsei Medical Journal

Article Title: Catalpol Inhibits Tregs-to-Th17 Cell Transdifferentiation by Up-Regulating Let-7g-5p to Reduce STAT3 Protein Levels

doi: 10.3349/ymj.2022.63.1.56

Figure Lengend Snippet: CAT suppresses the differentiation of Th17 cells in vitro. (A) Representative flow cytometric pictures indicate the percentage of CD4 + IL-17 + Th17 cells differentiated from naïve CD4 + T cells. (B) The proportion of CD4 + IL-17 + Th17 cells in traditional differentiation of Th17 cells. (C) IL-17 levels in culture supernatants during traditional differentiation of Th17 cells were measured by ELISA. (D) The mRNA levels of RORγ t during traditional differentiation of Th17 cells were detected by qPCR. (E) Representative flow cytometric pictures indicate the percentage of CD4 + IL-17 + Th17 cells differentiated from Tregs. (F) The proportion of CD4 + IL-17 + Th17 cells in Tregs-to-Th17 cell transdifferentiation. (G) IL-17 levels in culture supernatants during Tregsto-Th17 cell transdifferentiation were measured by ELISA. (H) mRNA levels of RORγ t in Tregs-to-Th17 cell transdifferentiation were detected by qPCR. Values are presented as a mean±SEM (n=3). † p <0.5, †† p <0.01, ††† p <0.001 compared with the control group. CAT, catalpol; IL, interleukin; ELISA, enzyme-linked immunosorbent assay; qPCR, quantitative polymerase chain reaction.

Article Snippet: Cells were then washed twice with PBS and activated with leukocyte activation cocktail (BD Pharmingen) for 4 h. After staining of Th17 cells with anti-CD4-FITC (BD Pharmingen) and anti-IL-17A-PE (BD Pharmingen) for 30 min, intracellular cytokines were analyzed by flow cytometry (Beckman FC-500, Beckman Coulter, Inc., Brea, CA, USA).

Techniques: In Vitro, Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction

Journal: Yonsei Medical Journal

Article Title: Catalpol Inhibits Tregs-to-Th17 Cell Transdifferentiation by Up-Regulating Let-7g-5p to Reduce STAT3 Protein Levels

doi: 10.3349/ymj.2022.63.1.56

Figure Lengend Snippet: Expression of let-7g-5p in traditional differentiation of Th17 cells and Tregs-to-Th17 cell transdifferentiation in vitro. (A) mRNA levels of let-7g-5p in traditional differentiation of Th17 cells were detected by qPCR. (B) mRNA levels of let-7g-5p in Tregs-to-Th17 cell transdifferentiation were detected by qPCR. Values are presented as a mean±SEM (n=3). ††† p <0.001 compared with the control group. CAT, catalpol; qPCR, quantitative polymerase chain reaction.

Article Snippet: Cells were then washed twice with PBS and activated with leukocyte activation cocktail (BD Pharmingen) for 4 h. After staining of Th17 cells with anti-CD4-FITC (BD Pharmingen) and anti-IL-17A-PE (BD Pharmingen) for 30 min, intracellular cytokines were analyzed by flow cytometry (Beckman FC-500, Beckman Coulter, Inc., Brea, CA, USA).

Techniques: Expressing, In Vitro, Real-time Polymerase Chain Reaction

Journal: Yonsei Medical Journal

Article Title: Catalpol Inhibits Tregs-to-Th17 Cell Transdifferentiation by Up-Regulating Let-7g-5p to Reduce STAT3 Protein Levels

doi: 10.3349/ymj.2022.63.1.56

Figure Lengend Snippet: Suppression of let-7g-5p on Tregs-to-Th17 cells trans-differentiation in vitro. (A) Representative dot plots of CD4 + IL-17 + Th17 cells differentiated from Tregs in vitro. (B) Proportion of CD4 + IL-17 + Th17 cells. (C) Levels of IL-17 in culture supernatants were measured by ELISA. (D) mRNA levels of IL-17 were detected by qPCR. (E) mRNA levels of RORγ t were detected by qPCR. † p <0.5, †† p <0.01, ††† p <0.001 compared with the control group. IL, interleukin; ELISA, enzyme-linked immunosorbent assay; qPCR, quantitative polymerase chain reaction.

Article Snippet: Cells were then washed twice with PBS and activated with leukocyte activation cocktail (BD Pharmingen) for 4 h. After staining of Th17 cells with anti-CD4-FITC (BD Pharmingen) and anti-IL-17A-PE (BD Pharmingen) for 30 min, intracellular cytokines were analyzed by flow cytometry (Beckman FC-500, Beckman Coulter, Inc., Brea, CA, USA).

Techniques: In Vitro, Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction

Journal: Yonsei Medical Journal

Article Title: Catalpol Inhibits Tregs-to-Th17 Cell Transdifferentiation by Up-Regulating Let-7g-5p to Reduce STAT3 Protein Levels

doi: 10.3349/ymj.2022.63.1.56

Figure Lengend Snippet: CAT suppresses Tregs-to-Th17 cell transdifferentiation by up-regulating let-7g-5p in vitro. (A) Representative dot plots of CD4 + IL-17 + Th17 cells in vitro. (B) Proportion of CD4 + IL-17 + Th17 cells after let-7g-5p mimics transfection. (C) Proportion of CD4 + IL-17 + Th17 cells after let-7g-5p inhibitor transfection. (D) IL-17 levels in culture supernatants after let-7g-5p mimics transfection were measured by ELISA. (E) mRNA levels of RORγ t after let-7g-5p mimics transfection were detected by qPCR. (F) mRNA levels of IL-17 after let-7g-5p mimics transfection were detected by qPCR. (G) IL-17 levels in culture supernatants after let-7g-5p inhibitor transfection were measured by ELISA. (H) mRNA levels of RORγ t after let-7g-5p inhibitor transfection were detected by qPCR. (I) mRNA levels of IL-17 after let-7g-5p inhibitor transfection were detected by qPCR. Values are presented as a mean±SEM (n=3). * p <0.5, ** p <0.01, *** p <0.001 compared with the model group. CAT, catalpol; IL, interleukin; ELISA, enzyme-linked immunosorbent assay; qPCR, quantitative polymerase chain reaction.

Article Snippet: Cells were then washed twice with PBS and activated with leukocyte activation cocktail (BD Pharmingen) for 4 h. After staining of Th17 cells with anti-CD4-FITC (BD Pharmingen) and anti-IL-17A-PE (BD Pharmingen) for 30 min, intracellular cytokines were analyzed by flow cytometry (Beckman FC-500, Beckman Coulter, Inc., Brea, CA, USA).

Techniques: In Vitro, Transfection, Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction

Journal: Yonsei Medical Journal

Article Title: Catalpol Inhibits Tregs-to-Th17 Cell Transdifferentiation by Up-Regulating Let-7g-5p to Reduce STAT3 Protein Levels

doi: 10.3349/ymj.2022.63.1.56

Figure Lengend Snippet: CAT suppresses Tregs-to-Th17 cell transdifferentiation in relation to STAT3. (A) mRNA levels of STAT3 after let-7g-5p mimics transfection were detected by qPCR. (B) mRNA levels of STAT3 after let-7g-5p inhibitor transfection were detected by qPCR. (C) Protein levels of STAT3 after let-7g-5p mimics transfection were analyzed and quantified. (D) Protein levels of STAT3 after let-7g-5p mimics transfection were analyzed and quantified. (E) Expression of STAT3 was detected by Western blot. Values are presented as a mean±SEM (n=3). * p <0.05, *** p <0.001 compared with the model group. CAT, catalpol; qPCR, quantitative polymerase chain reaction; IL, interleukin.

Article Snippet: Cells were then washed twice with PBS and activated with leukocyte activation cocktail (BD Pharmingen) for 4 h. After staining of Th17 cells with anti-CD4-FITC (BD Pharmingen) and anti-IL-17A-PE (BD Pharmingen) for 30 min, intracellular cytokines were analyzed by flow cytometry (Beckman FC-500, Beckman Coulter, Inc., Brea, CA, USA).

Techniques: Transfection, Expressing, Western Blot, Real-time Polymerase Chain Reaction

Journal: World Journal of Gastroenterology : WJG

Article Title: Caffeic acid phenethyl ester modifies the Th1/Th2 balance in ileal mucosa after γ-irradiation in the rat by modulating the cytokine pattern

doi: 10.3748/wjg.v12.i31.4996

Figure Lengend Snippet: Th1/Th2-like pattern of gene expression in the ileal mucosal layer after γ-irradiation in rats. (■): CAPE-untreated, (□): CAPE-treated and CD4+ immunostaining in control (C), 6 h and 7 d (D7) after irradiation (D (x 20) (mean ± SEM, n = 6); aP < 0.05, bP < 0.01 vs control; cP < 0.05, dP < 0.001 vs untreated.

Article Snippet: Sections were incubated in protein blocking serum (Dako, USA) before treatment for 90 min at room temperature with the monoclonal anti-CD4 antibody (ab6413; 1/100 dilution, Abcam).

Techniques: Expressing, Irradiation, Immunostaining

Journal:

Article Title: Enhanced Infectivity of an R5-Tropic Simian/Human Immunodeficiency Virus Carrying Human Immunodeficiency Virus Type 1 Subtype C Envelope after Serial Passages in Pig-Tailed Macaques ( Macaca nemestrina )

doi:

Figure Lengend Snippet: The replication kinetics of SHIVCHN19 in human PBMC (A) and in CD4+ T lymphocytes derived from a pig-tailed macaque (B). Human PBMC or macaque CD4+ T lymphocytes (2 × 106) were infected with a virus input of 100 TCID50. The replication of SHIV and SIV was monitored by using the p27 assay (Cellular Products). The y axis represents the level of p27 production in the culture supernatants.

Article Snippet: Then, 10 μl of leu-3a-PE anti-CD4 (Becton Dickinson), 10 μl of leu-2a-PerCP anti-CD8 (Becton Dickinson), and 10 μl of fluorescein isothiocyanate anti-CD3ɛ (Pharmingen, San Diego, Calif.) antibodies were added, and the mixture was incubated at room temperature in the dark for 20 min. Then, 450 μl of 1× fluorescence-activated cell sorter (FACS) lysing solution (Becton Dickinson) was added, and another incubation at room temperature for 15 min was carried out to ensure the total lysis of red blood cells.

Techniques: Derivative Assay, Infection

Journal:

Article Title: Enhanced Infectivity of an R5-Tropic Simian/Human Immunodeficiency Virus Carrying Human Immunodeficiency Virus Type 1 Subtype C Envelope after Serial Passages in Pig-Tailed Macaques ( Macaca nemestrina )

doi:

Figure Lengend Snippet: Comparison of percentage of CD4+ cells among total CD3+ T lymphocytes from different compartments. Four animals were tested, including two P4 (T909 and T910) and two naïve (AE15 and AV25) pig-tailed macaques. T lymphocytes derived from three compartments, including blood, colonic lymph node, and jejunal lamina propria, were assayed. The y axis represents the percentage of CD4+ T lymphocytes. The number above each vertical bar indicates the average percentage of two separate stainings of the same specimen. Specimens from the two P4 animals were collected 2 weeks p.i.

Article Snippet: Then, 10 μl of leu-3a-PE anti-CD4 (Becton Dickinson), 10 μl of leu-2a-PerCP anti-CD8 (Becton Dickinson), and 10 μl of fluorescein isothiocyanate anti-CD3ɛ (Pharmingen, San Diego, Calif.) antibodies were added, and the mixture was incubated at room temperature in the dark for 20 min. Then, 450 μl of 1× fluorescence-activated cell sorter (FACS) lysing solution (Becton Dickinson) was added, and another incubation at room temperature for 15 min was carried out to ensure the total lysis of red blood cells.

Techniques: Derivative Assay

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: The Microbiome Activates CD4 T-cell–mediated Immunity to Compensate for Increased Intestinal Permeability

doi: 10.1016/j.jcmgh.2017.06.001

Figure Lengend Snippet: Increased intestinal permeability limits early enteric pathogen invasion. ( A ) Fluorescence micrographs of CD3 + CD4 + LP T cells in WT and CA-MLCK mice. CD3, red ; CD4, green ; nuclei, blue . White arrowheads indicate CD4 + T cells. Scale bar, 20 μm. ( B ) Morphometric analysis of LP CD4 + T cells. n = 4–5 mice. ** P < .01. ( C ) WT and CA-MLCK mice were orally administered T gondii cysts and killed after 1 hour (SAC). Alternatively, mice were exposed to S typhimurium through the addition of 10 8 CFUs directly to the mucosal surface for 30 minutes. ( D ) Morphometric analysis of parasite translocation 1 hour after infection. n = 5–6 mice from 2 independent experiments. * P < .05. ( E ) Fluorescence micrographs of S typhimurium ( red, arrows )–infected WT and CA-MLCK mice crossed to Rag1 - or γδ T-cell ( Tcrd )–deficient backgrounds. Nuclei, blue ; f-actin, green . White arrowheads indicate translocation of S typhimurium , whereas yellow arrowheads are bacteria not counted. Scale bar, 20 μm. ( F ) Morphometric analysis of S typhimurium invasion. n = 5–7 mice from at least 2 independent experiments. Approximately 200 villi were counted for each condition. * P < .05, ** P < .01. ( G ) Morphometric analysis of γδ T cells in WT and CA-MLCK mice. n = 4 mice. Mean ± SEM are shown.

Article Snippet: Frozen sections were immunostained as previously described by using primary antibodies, rabbit anti-myeloperoxidase, rabbit anti-CD3 (Abcam, Cambridge, MA), rabbit anti-LDH1 antiserum, rat anti-CD4, rat anti-IgA (BD Pharmingen, San Jose, CA), followed by AlexaFluor-conjugated phalloidin or secondary antibodies and Hoechst 33342 dye (Invitrogen, Carlsbad, CA).

Techniques: Permeability, Fluorescence, Translocation Assay, Infection

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: The Microbiome Activates CD4 T-cell–mediated Immunity to Compensate for Increased Intestinal Permeability

doi: 10.1016/j.jcmgh.2017.06.001

Figure Lengend Snippet: CD4 + T cells are required to confer protection against bacterial invasion in CA-MLCK mice. ( A ) After administration of anti-CD4 (GK1.5) or control IgG intraperitoneally, WT and CA-MLCK mice were exposed to S typhimurium through direct application of bacteria to small intestinal mucosal surface for 30 minutes and then killed (SAC). ( B ) Percentage of CD3 + CD4 + T splenocytes 48 hours after anti-CD4 administration. # P < .001. ( C ) Morphometric analysis of S typhimurium invasion after administration of anti-CD4 or control IgG. n = 6–9 mice from 2 independent experiments. * P < .05, # P < .001. ( D ) Rag1 KO mice and those also expressing the CA-MLCK Tg received CD4 + T-cell adoptive transfer. After 2 weeks of engraftment, mice were exposed to S typhimurium for 30 minutes. ( E ) Morphometric analysis of CD4 + LP T cells in Rag1 KO and Rag1 KO CA-MLCK mice after CD4 + T-cell adoptive transfer. n = 7–8 mice from 2 independent experiments. * P < .05. ( F ) Analysis of S typhimurium translocation in Rag1 KO and Rag1 KO; CA-MLCK mice receiving CD4 + T-cell adoptive transfer. n = 7–11 mice from 2 independent experiments. * P < .05.

Article Snippet: Frozen sections were immunostained as previously described by using primary antibodies, rabbit anti-myeloperoxidase, rabbit anti-CD3 (Abcam, Cambridge, MA), rabbit anti-LDH1 antiserum, rat anti-CD4, rat anti-IgA (BD Pharmingen, San Jose, CA), followed by AlexaFluor-conjugated phalloidin or secondary antibodies and Hoechst 33342 dye (Invitrogen, Carlsbad, CA).

Techniques: Expressing, Adoptive Transfer Assay, Translocation Assay

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: The Microbiome Activates CD4 T-cell–mediated Immunity to Compensate for Increased Intestinal Permeability

doi: 10.1016/j.jcmgh.2017.06.001

Figure Lengend Snippet: IgA + plasma cell number is increased, yet not required for CA-MLCK–mediated protection against bacterial translocation. ( A ) Morphometric analysis of IgA + plasma cells in the LP. n = 4–6 mice. Mean ± SEM, * P = .05. ( B ) Enzyme-linked immunosorbent assay for fecal IgA. n = 7–10 mice. Mean ± SEM. ( C ) Shannon Index was calculated from CDR3 sequence pools of given samples. The mean is shown. ( D ) Mutation frequencies in CDR1/2 and FR sequences calculated from the average number of SHM within the Vh region. Frequencies were calculated through division of mutation numbers by the number of nucleotides within the given FR or CDR regions. Mean ± standard deviation. ( E ) Morphometric analysis of S typhimurium invasion in WT and CA-MLCK mice on an IghA KO background. n = 5–7 mice from 2 independent experiments. Mean is shown, * P = .05. ( F ) Morphometric analysis of LP IgA + plasma cells 48 hours after administration of anti-CD4 or control IgG. Mean ± SEM, ** P < .01. IghA, immunoglobulin A; SHM, somatic hypermutations.

Article Snippet: Frozen sections were immunostained as previously described by using primary antibodies, rabbit anti-myeloperoxidase, rabbit anti-CD3 (Abcam, Cambridge, MA), rabbit anti-LDH1 antiserum, rat anti-CD4, rat anti-IgA (BD Pharmingen, San Jose, CA), followed by AlexaFluor-conjugated phalloidin or secondary antibodies and Hoechst 33342 dye (Invitrogen, Carlsbad, CA).

Techniques: Translocation Assay, Enzyme-linked Immunosorbent Assay, Sequencing, Mutagenesis, Standard Deviation

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: The Microbiome Activates CD4 T-cell–mediated Immunity to Compensate for Increased Intestinal Permeability

doi: 10.1016/j.jcmgh.2017.06.001

Figure Lengend Snippet: IL17A is required to limit bacterial translocation through promotion of neutrophil recruitment. ( A ) Morphometric analysis of S typhimurium invasion in WT and CA-MLCK mice on an IL17A-deficient background. n = 6–7 mice from 2 independent experiments. * P < .05, ** P < .01. ( B ) Morphometric analysis of CD4 + LP T cells in WT and CA-MLCK mice crossed to IL17A-deficient mice. ** P < .01. ( C ) Morphometric analysis of neutrophils within the small intestinal LP in uninfected and infected (30 minutes) WT, CA-MLCK, IL17A KO or IL17A KO; CA-MLCK mice. n = 3–5 mice. # P < .001.

Article Snippet: Frozen sections were immunostained as previously described by using primary antibodies, rabbit anti-myeloperoxidase, rabbit anti-CD3 (Abcam, Cambridge, MA), rabbit anti-LDH1 antiserum, rat anti-CD4, rat anti-IgA (BD Pharmingen, San Jose, CA), followed by AlexaFluor-conjugated phalloidin or secondary antibodies and Hoechst 33342 dye (Invitrogen, Carlsbad, CA).

Techniques: Translocation Assay, Infection

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: The Microbiome Activates CD4 T-cell–mediated Immunity to Compensate for Increased Intestinal Permeability

doi: 10.1016/j.jcmgh.2017.06.001

Figure Lengend Snippet: Commensal bacteria are essential for promoting mucosal immune responses to early Salmonella invasion in response to increased intestinal permeability. ( A ) Fluorescence micrographs of CD3 + CD4 + LP T cells in WT and CA-MLCK mice raised under either SPF or GF conditions. Scale bar, 20 μm. Morphometric analysis of CD4 + LP T cells in GF WT and CA-MLCK mice are shown. n = 6–8 mice from 2 independent experiments. ( B ) Fluorescence micrographs of IgA + plasma cells in the LP in WT and CA-MLCK mice raised under either SPF or GF conditions. Scale bar, 20 μm. Morphometric analysis of IgA + plasma cells in GF WT and CA-MLCK mice is shown. n = 5–8 mice from 2 independent experiments. ( C ) Morphometric analysis of S typhimurium invasion (30 minutes) in WT and CA-MLCK mice maintained under GF conditions or conventionalized with Taconic (TAC) or Jackson (JAX) microbiota. n = 5–8 mice from 2 independent experiments. ** P < .01.

Article Snippet: Frozen sections were immunostained as previously described by using primary antibodies, rabbit anti-myeloperoxidase, rabbit anti-CD3 (Abcam, Cambridge, MA), rabbit anti-LDH1 antiserum, rat anti-CD4, rat anti-IgA (BD Pharmingen, San Jose, CA), followed by AlexaFluor-conjugated phalloidin or secondary antibodies and Hoechst 33342 dye (Invitrogen, Carlsbad, CA).

Techniques: Permeability, Fluorescence