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: Frontiers in Immunology

Article Title: The monocyte-derived cytokine response in whole blood from preterm newborns against sepsis-related bacteria is similar to term newborns and adults

doi: 10.3389/fimmu.2024.1353039

Figure Lengend Snippet: The production of TNF-α and IL-6 by term neonatal and adult WB shows a high degree of inter-individual variation. WB from term cord blood (term, N=11-17) and healthy adults (adult, N=11-18) was incubated with (A, B) LPS, Pam3Cys4K or R848 or with (C, D) E. coli , S. agalactiae (GBS), S. aureus or S. epidermidis (CNS) bacteria for 5h and the production of TNF-α and IL-6 was measured. (E) The inter-individual variation in production of TNF-α and IL-6 between individuals was determined for WB from term cord blood and healthy adults (N=11-18) for LPS (100 ng/mL), Pam3Cys4K (10 μg/mL) or R848 (10 μM) or bacteria (10 5 CFU/well). Cytokine concentrations are shown in pg/mL. CFU, colony-forming units.

Article Snippet: Biotin Mouse anti-Human TNF-α detection antibody (BD PharmingenTM), Biotin Mouse Anti-Human IL-6 detection antibody (eBioscienceTM) or Biotin mouse anti-Human IL-1β detection antibody (R&D Systems) was added as secondary antibody.

Techniques: Incubation, Bacteria

Journal: Frontiers in Immunology

Article Title: The monocyte-derived cytokine response in whole blood from preterm newborns against sepsis-related bacteria is similar to term newborns and adults

doi: 10.3389/fimmu.2024.1353039

Figure Lengend Snippet: Preterm neonatal WB produces lower levels of IL-6 and similar levels of TNF-α compared to term neonatal WB. Whole blood from preterm cord blood (preterm, N=8-10), term cord blood (term, N=17) and healthy adults (adult, N=17-21) was incubated with TLR ligands LPS (100 ng/mL), Pam3Cys4K (10 μg/mL) or R848 (10 μM) or with E. coli , S. agalactiae (GBS), S. aureus or S. epidermidis (CNS) bacteria (10 5 CFU/well) for 5h and the production of (A, B) TNF-α, (C, D) IL-6 and (E, F) IL-1β was measured. (G-I) The inter-individual variation in production of (G) TNF-α, (H) IL-6 and (I) IL-1β between individuals was determined for preterm cord blood (N=8-10). Cytokine concentrations are shown in log-transformed pg/mL. The dotted line indicates the limit of detection. CV = coefficiency of variation. * = P<0.05. ** = P<0.01. *** = P<0.001.

Article Snippet: Biotin Mouse anti-Human TNF-α detection antibody (BD PharmingenTM), Biotin Mouse Anti-Human IL-6 detection antibody (eBioscienceTM) or Biotin mouse anti-Human IL-1β detection antibody (R&D Systems) was added as secondary antibody.

Techniques: Incubation, Bacteria, Transformation Assay

Journal: medRxiv

Article Title: Crypt-top and crypt-bottom colonic epithelial cell microRNA profiling reveals cell type-specific response in active and quiescent ulcerative colitis

doi: 10.1101/2022.09.25.22280336

Figure Lengend Snippet: (a) FACS of colonic epithelial cells and distribution of crypt-top CD44 - CD66 + and crypt-bottom CD44 + CD66 - epithelial cell types in inflammatory (aUC) (n=16) and non-inflammatory (qUC and HC) (n=15 and 17) colon tissues. Each dot represents a sample from the patients of the second study group. Mean ±SD of each group is represented by vertical lines. To compare the groups a nonparametric Mann–Whitney U test was performed, p* < 0.05. (b) MDS plot showing the similarity structure of the miRNA transcriptomes in crypt-top (CD66a + ) and crypt-bottom (CD44 + ) colonic epithelial cell populations in active (aUC) (n=16), quiescent UC (qUC) (n=15), and in HC (n=17) based on normalized expression values. The dots represent samples shaped by cell population. Dot colours represent condition. The centroid of ellipses corresponds to the condition group mean, the shapes are defined by covariance within the group. (c, d) Volcano plots of differentially expressed miRNAs in crypt-top (CD66a + ) and crypt-bottom (CD44 + ) colonic epithelial cell populations in active (aUC) (n=16), quiescent UC (qUC) (n=15), and HC (n=17). Colours indicate significantly (FDR < 0.05) differentially expressed miRNAs with an absolute value of log 2 FC > 1 between compared groups. (e-g) Venn diagrams representing the numbers of commonly and uniquely differentially expressed miRNAs in (e) crypt-bottom (CD44 + ) and (f) crypt-top (CD66a + ) epithelial cell populations in different UC activity, and (g) between crypt-bottom and crypt-top cells in the same condition.

Article Snippet: Antibodies used in this study were selected based on previous study and included: mouse anti-human CD326/EpCAM-FITC (clone VU-1D9, RRID: AB_2534535m Life Technologies, USA), mouse anti-human CD44-APC (clone G44-26, RRID: AB_395868, BD Biosciences, USA), mouse anti-human CD66a-PE (clone 283340, R&D Systems, USA).

Techniques: MANN-WHITNEY, Expressing, Activity Assay

Journal: medRxiv

Article Title: Crypt-top and crypt-bottom colonic epithelial cell microRNA profiling reveals cell type-specific response in active and quiescent ulcerative colitis

doi: 10.1101/2022.09.25.22280336

Figure Lengend Snippet: Top 10 overrepresented pathways within (a) and between (b) crypt-top (CD66a + ) and crypt-bottom (CD44 + ) colonic epithelial cell populations during active (aUC) (n=16), quiescent UC (qUC) (n=15) and in controls (HC) (n=17) identified by miRNA-target gene set enrichment analysis. Dot size represents the number of miRNA gene-target count in the significantly enriched (FDR < 0.05) Reactome pathways (a) and GO biological process (BP) categories (b) ; (c) a heatmap showing correlations between miRNA expression and endoscopic Mayo subscore in crypt-bottom (CD44 + ) (n=48) and crypt-top (CD66a + ) (n=48) colonic epithelial cell populations. Colour of the box represent the value of Spearman’s correlation coefficient (rho). Dots mark significant correlations (FDR < 0.05) in each cell population.

Article Snippet: Antibodies used in this study were selected based on previous study and included: mouse anti-human CD326/EpCAM-FITC (clone VU-1D9, RRID: AB_2534535m Life Technologies, USA), mouse anti-human CD44-APC (clone G44-26, RRID: AB_395868, BD Biosciences, USA), mouse anti-human CD66a-PE (clone 283340, R&D Systems, USA).

Techniques: Expressing