Journal: International Journal of Molecular Sciences

Article Title: TNF-α Affects Signature Cytokines of Th1 and Th17 T Cell Subsets through Differential Actions on TNFR1 and TNFR2

doi: 10.3390/ijms23169306

Figure Lengend Snippet: TNF-α does not impact the levels of TNFR1 and TNFR2 on Th1 and Th17 cells. Purified CD4 + T cell subsets were stimulated with 1 µg/mL of TNF-α for 24 h and stained, for flow cytometry, with anti-human TNFR1 or TNFR2 mAbs. ( A ) Representative histograms showing the expression levels of TNFR1 and TNFR2 on Th1 and Th17 cells at baseline conditions and upon TNF-α stimulus. Solid black line histograms, isotype control; black histograms, Th1 cells; grey histograms, Th17 cells; dashed line histograms, TNF-α-treated Th1 cells; long dashed line histograms, TNF-α-treated Th17 cells. The levels of TNFR1 ( B ) and TNFR2 ( C ) were measured on purified CD4 + T cell subpopulations obtained from two to four healthy controls. Bars represent the mean values ± SD. Statistical analyses were carried out with Kruskal–Wallis followed by Dunn’s multiple comparison tests. ** p < 0.01.

Article Snippet: Cells were then stained with a mouse anti-human TNFR1-APC mAb (clone 16803) (R&D Systems, Minneapolis, MN, USA) and a rat anti-human TNFR2-PE mAb (clone hTNFR-M1) (BD Biosciences) for 30 min at 4 °C in the dark.

Techniques: Purification, Staining, Flow Cytometry, Expressing, Control, Comparison

Journal: International Journal of Molecular Sciences

Article Title: TNF-α Affects Signature Cytokines of Th1 and Th17 T Cell Subsets through Differential Actions on TNFR1 and TNFR2

doi: 10.3390/ijms23169306

Figure Lengend Snippet: Effect of TNFR1 or TNFR2 blockade on the production of IFN-γ and IL-17 by Th1 and Th17 cells. Purified CD4 + T lymphocyte subpopulations were incubated with neutralizing antibodies to TNFR1 or TNFR2 for 1 h prior to a 4-day stimulus with TNF-α (1 µg/mL). Intracellular staining of IFN-γ and IL-17 was assessed by flow cytometry. ( A ) Representative dot plots of Th1 and Th17 cells treated with anti-TNFR1 or anti-TNFR2 in the presence or absence of TNF-α. An isotype control was used to discard non-specific effects of the neutralizing antibodies. The fold increase in the percentages of IFN-γ ( B , D ) or IL-17 ( C , E ) producers was measured on Th1 and Th17 cells obtained from two to six healthy donors. Bars represent the mean values ± SD. Data were normalized against cells that did not receive treatment with TNF-α or TNF-α plus TNFRs blocking mAbs. For statistical analysis, Kruskal–Wallis and Dunn’s multiple comparison tests were performed. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Cells were then stained with a mouse anti-human TNFR1-APC mAb (clone 16803) (R&D Systems, Minneapolis, MN, USA) and a rat anti-human TNFR2-PE mAb (clone hTNFR-M1) (BD Biosciences) for 30 min at 4 °C in the dark.

Techniques: Purification, Incubation, Staining, Flow Cytometry, Control, Blocking Assay, Comparison

Journal: International Journal of Molecular Sciences

Article Title: TNF-α Affects Signature Cytokines of Th1 and Th17 T Cell Subsets through Differential Actions on TNFR1 and TNFR2

doi: 10.3390/ijms23169306

Figure Lengend Snippet: Expression of TNFR1 and TNFR2 on Th1 and Th17 cells present in the peripheral blood of rheumatoid arthritis (RA) patients treated with adalimumab. Cell staining for flow cytometry analysis was performed on PBMC samples from healthy controls ( n = 9) and RA patients ( n = 10) before (PRE) and after (POST) treatment with adalimumab. The levels of TNFR1 ( A ) and TNFR2 ( C ) are expressed in MFI values. The frequency of TNFR1 ( B ) and TNFR2 ( D )-expressing lymphocytes are also shown. Each symbol represents data for one individual. Mean values ± SD are indicated. Significance was assessed with non-parametric Kruskal–Wallis test followed by Dunn’s multiple comparison test (for MFI data) or parametric one-way ANOVA plus Tukey’s post-test (for lymphocyte frequencies data). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Cells were then stained with a mouse anti-human TNFR1-APC mAb (clone 16803) (R&D Systems, Minneapolis, MN, USA) and a rat anti-human TNFR2-PE mAb (clone hTNFR-M1) (BD Biosciences) for 30 min at 4 °C in the dark.

Techniques: Expressing, Staining, Flow Cytometry, Comparison

Journal: Molecular Metabolism

Article Title: Indoleamine 2,3-dioxygenase mediates the therapeutic effects of adipose-derived stromal/stem cells in experimental periodontitis by modulating macrophages through the kynurenine-AhR-NRF2 pathway

doi: 10.1016/j.molmet.2022.101617

Figure Lengend Snippet: ASC therapeutic effects on experimental periodontitis in rats. (A) The schematic diagram showed the induction of the experimental rat model of periodontitis for 21 days and ASC injection after the removal of ligatures. (B) Micro-CT imaging and bone parameters (CEJ-ABC distance, BV/TV, Tb. Th, and Tb. Sp) between healthy and experimental rats of periodontitis. Scale bar, 1 mm. (C) Micro-CT imaging displayed the alveolar bone of PBS-injected and ASC-injected rats on day 7, day 14, and day 21 Scale bar, 1 mm. (D) The CEJ-ABC distance and bone parameters (BV/TV, Tb. Th, and Tb. Sp) between the PBS-injected and ASC-injected rats. (E) Representative immunofluorescence staining of M1 phenotype macrophages (iNOS + ; Green) and M2 macrophages (CD206 + ; Red) in sagittal sections of maxillary molars in PBS-injected and ASC-injected rats. Scale bar, 20 μm. (F) The calculated ratio of iNOS + /CD206 + macrophages in PBS-injected and ASC-injected groups. CEJ-ABC, cementoenamel junction and alveolar bone crest. All data were expressed as mean ± SD; ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001.

Article Snippet: For tissues, the sections were incubated with a mouse anti-CD206 monoclonal antibody (1:100, 60143-1-lg, Proteintech), a rabbit anti-NRF2 monoclonal antibody (1:100, A0674, Abclonal), a rabbit anti-inducible nitric oxide synthase (iNOS) polyclonal antibody (1:100, 18985-1-AP, Proteintech) overnight at 4 °C.

Techniques: Injection, Micro-CT, Imaging, Immunofluorescence, Staining

Journal: Molecular Metabolism

Article Title: Indoleamine 2,3-dioxygenase mediates the therapeutic effects of adipose-derived stromal/stem cells in experimental periodontitis by modulating macrophages through the kynurenine-AhR-NRF2 pathway

doi: 10.1016/j.molmet.2022.101617

Figure Lengend Snippet: ASCs modulated macrophage polarization through NRF2. (A) Representative immunohistochemical staining of NRF2 in the PBS-injected and ASC-injected rats on day 7, day 14, and day 21. Scale bar, 50 μm. (B) Representative immunofluorescence staining of NRF2 (Green) and M2 macrophages (CD206 + ; Red) in sagittal sections of maxillary molars in PBS-injected and ASC-injected rats. Scale bar, 20 μm. (C) Schematic diagram of knocking down NRF2 in macrophages and macrophages cocultured with ASCs and LPS stimulation. (D, E) RT-qPCR and western blotting were used to measure the NRF2 siRNA or siNC in macrophages. (F, G) The mRNA expression and protein levels of M1 markers were increased, while those of M2 markers were decreased after silencing NRF2 in the cocultured groups. R, root; PDL, periodontal ligament; AB, alveolar bone; siRNA, small interfering RNA; NC, negative control. All data were expressed as mean ± SD; ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001.

Article Snippet: For tissues, the sections were incubated with a mouse anti-CD206 monoclonal antibody (1:100, 60143-1-lg, Proteintech), a rabbit anti-NRF2 monoclonal antibody (1:100, A0674, Abclonal), a rabbit anti-inducible nitric oxide synthase (iNOS) polyclonal antibody (1:100, 18985-1-AP, Proteintech) overnight at 4 °C.

Techniques: Immunohistochemical staining, Staining, Injection, Immunofluorescence, Quantitative RT-PCR, Western Blot, Expressing, Small Interfering RNA, Negative Control

Journal: Molecular Metabolism

Article Title: Indoleamine 2,3-dioxygenase mediates the therapeutic effects of adipose-derived stromal/stem cells in experimental periodontitis by modulating macrophages through the kynurenine-AhR-NRF2 pathway

doi: 10.1016/j.molmet.2022.101617

Figure Lengend Snippet: Inhibition of IDO activity reduced the therapeutic potential of ASCs in experimental periodontitis and downregulated NRF2 expression. (A) Three-dimensional reconstruction of maxillary alveolar bone in ASC-injected and 1-MT pretreated ASC-injected rats on day 7, day 14, and day 21. Scale bar, 1 mm. (B) Analysis of bone parameters. (C) Immunohistochemical staining of IL-1β, OCN, and NRF2 in the ASC-injected and 1-MT pretreated ASC-injected rats. Scale bar, 50 μm. (D) Representative immunofluorescence staining of M1 phenotype macrophages (iNOS + ; Green) and M2 macrophages (CD206 + ; Red) in sagittal sections of maxillary molars in ASC-injected and 1-MT pretreated ASC-injected rats. Scale bar, 20 μm. (E) The mean IOD of IL-1β, OCN, and NRF2 and the calculated ratio of iNOS + /CD206 + . IOD, integrated optical density.

Article Snippet: For tissues, the sections were incubated with a mouse anti-CD206 monoclonal antibody (1:100, 60143-1-lg, Proteintech), a rabbit anti-NRF2 monoclonal antibody (1:100, A0674, Abclonal), a rabbit anti-inducible nitric oxide synthase (iNOS) polyclonal antibody (1:100, 18985-1-AP, Proteintech) overnight at 4 °C.

Techniques: Inhibition, Activity Assay, Expressing, Injection, Immunohistochemical staining, Staining, Immunofluorescence

Journal: bioRxiv

Article Title: Clonal evaluation of early onset prostate cancer by expression profiling of ERG, SPINK1, ETV1 , and ETV4 on whole mount radical prostatectomy tissue

doi: 10.1101/667832

Figure Lengend Snippet: Dual IHC for ERG/SPINK1 on a whole mount prostatectomy tissue. ERG, strong nuclear positivity is seen in the dominant tumor and adjacent HGPIN and atrophic prostatic glands. Insets show the hematoxylin and eosin whole mount image marked with tumor foci (dotted circle, top left) and the high magnification view of ERG positive neoplastic glands with adjacent HGPIN. Abbreviations: HGPIN, high-grade prostatic intraepithelial neoplasia.

Article Snippet: Anti-ERG (EPR3864) rabbit monoclonal primary antibody diluted 1:50 (Abcam, Cambridge, MA, ab92513) and a mouse monoclonal against SPINK1 diluted 1:100 (Novus Biologicals, Centennial, CO, H00006690-M01) were added to each slide, which were then coverslipped with parafilm, placed in a humidifying chamber, and incubated overnight at 4°C.

Techniques:

Journal: bioRxiv

Article Title: Clonal evaluation of early onset prostate cancer by expression profiling of ERG, SPINK1, ETV1 , and ETV4 on whole mount radical prostatectomy tissue

doi: 10.1101/667832

Figure Lengend Snippet: Dual IHC for ERG/SPINK1 on a whole mount prostatectomy tissue. SPINK1, strong cytoplasmic and membranous positivity is seen in a secondary tumor nodule while the index/dominant tumor nodule is negative for both ERG and SPINK1expression. Insets show the hematoxylin and eosin whole mount image (top left), high magnification view for SPINK1 positive tumor nodule (top right) and strong cytoplasmic staining of SPINK1 (bottom left) in tumor foci. ERG positivity is observed as brown nuclear staining in endothelial cells in blood vessels (internal control).

Article Snippet: Anti-ERG (EPR3864) rabbit monoclonal primary antibody diluted 1:50 (Abcam, Cambridge, MA, ab92513) and a mouse monoclonal against SPINK1 diluted 1:100 (Novus Biologicals, Centennial, CO, H00006690-M01) were added to each slide, which were then coverslipped with parafilm, placed in a humidifying chamber, and incubated overnight at 4°C.

Techniques: Staining

Journal: bioRxiv

Article Title: Clonal evaluation of early onset prostate cancer by expression profiling of ERG, SPINK1, ETV1 , and ETV4 on whole mount radical prostatectomy tissue

doi: 10.1101/667832

Figure Lengend Snippet: Dual IHC for ERG/SPINK1 on a whole mount prostatectomy tissue. Multiple independent tumor foci showing mutually exclusive expression of ERG (stained in brown) and SPINK1 (stained in blue). Insets showing the high magnification view for ERG (bottom right) and SPINK1 (top left) expression. There are multiple secondary tumor foci separated by benign glands/stroma showing ERG which indicates inter tumor heterogeneity with independent clonal origin of the tumors.

Article Snippet: Anti-ERG (EPR3864) rabbit monoclonal primary antibody diluted 1:50 (Abcam, Cambridge, MA, ab92513) and a mouse monoclonal against SPINK1 diluted 1:100 (Novus Biologicals, Centennial, CO, H00006690-M01) were added to each slide, which were then coverslipped with parafilm, placed in a humidifying chamber, and incubated overnight at 4°C.

Techniques: Expressing, Staining

Journal: bioRxiv

Article Title: Clonal evaluation of early onset prostate cancer by expression profiling of ERG, SPINK1, ETV1 , and ETV4 on whole mount radical prostatectomy tissue

doi: 10.1101/667832

Figure Lengend Snippet: Sunburst chart showing the summary of distribution of four biomarkers (ERG, SPINK1, ETV1 , and ETV4 ) mutually exclusive expression on whole mount radical prostatectomy tissue in the young men with prostate cancer, highlighting ERG positive and ERG negative tumors with SPINK1, ETV1 and ETV4 expression status.

Article Snippet: Anti-ERG (EPR3864) rabbit monoclonal primary antibody diluted 1:50 (Abcam, Cambridge, MA, ab92513) and a mouse monoclonal against SPINK1 diluted 1:100 (Novus Biologicals, Centennial, CO, H00006690-M01) were added to each slide, which were then coverslipped with parafilm, placed in a humidifying chamber, and incubated overnight at 4°C.

Techniques: Expressing

Journal: PLoS ONE

Article Title: Prevention of Herpes Simplex Virus Induced Stromal Keratitis by a Glycoprotein B-Specific Monoclonal Antibody

doi: 10.1371/journal.pone.0116800

Figure Lengend Snippet: Confluent Vero cells were infected with 100 TCID 50 of HSV-1 KOS and treated with either pooled human polyclonal HSV-neutralizing sera (1:40 in medium) or mAb 2c (500 nM). Cells were stained 48 h after infection for viral transmission with an HSV-1/2-glycoprotein D specific murine antibody and an Alexa488-conjugated secondary anti-mouse or anti-human antibody. Bound human antibodies or mAb 2c were detected with a Cy3-conjugated secondary antibody. Uninfected cells served as negative controls and showed no background staining (not shown). Magnification: 100x. Scale bar: 100 μm.

Article Snippet: HSV-1 infected cells were stained with a mouse anti-HSV-1/2-gD-antibody (Acris Antibodies, San Diego, CA, USA) and an Alexa Fluor 488 goat anti-mouse IgG specific secondary antibody (Invitrogen).

Techniques: Infection, Staining, Transmission Assay