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: Pharmaceutics

Article Title: Bacterial Cellulose as Drug Delivery System for Optimizing Release of Immune Checkpoint Blocking Antibodies

doi: 10.3390/pharmaceutics14071351

Figure Lengend Snippet: Human IgG loading and in vitro release were performed with the ‘injection method’. ( a ) A total of 50 µg IgG in a volume of 25 µL PBS was injected into the center of a BC fleece. To visualize the injection depot, the IgG solution was supplied with Trypan blue. The loaded fleeces were then put in blocking buffer (1% BSA/0.05% Tween-20 in PBS) and IgG release was followed. At several time-points, the fleeces were taken out, and the Trypan blue injection spots were photographed. Graph ( b ) shows the cumulative IgG release in %, with the inset of the first 24 h shown in ( c ). Release data are shown as cumulative release, which are displayed as mean ± SD for a triplicate measurement.

Article Snippet: BC fleeces were loaded with human IgG antibody (50 µg) or anti-CTLA-4 (Syrian hamster IgG, clone 9H10; Bioxcell, Lebanon, NH, USA; 100 µg) according to .

Techniques: In Vitro, Injection, Blocking Assay

Journal: Pharmaceutics

Article Title: Bacterial Cellulose as Drug Delivery System for Optimizing Release of Immune Checkpoint Blocking Antibodies

doi: 10.3390/pharmaceutics14071351

Figure Lengend Snippet: Cytotoxicity evaluation of BC extracts in MC38 tumor cells with 7-AAD staining and MTS. BC extracts were prepared by dissolving 1 g of BC in 50 mL culture medium, which yielded an extraction ratio of 20 mg/mL. Empty extracts or extracts supplied with human IgG or anti-CTLA-4 (both starting at a concentration of 50 µg/mL) were tested on MC38 cells. DMSO was used as positive cell killing control. The primary measure was cell viability, which was assessed by 7-AAD staining (live-dead cells exclusion marker; figures a – c ). Higher 7-AAD MFI signal is a hallmark of dying cells with leaky cell membranes. Cytotoxicity was also assessed using a cell metabolism assay (MTS), in which higher cell metabolism is indicative of higher cell viability ( d – f ). Cell metabolism was measured 48 h after incubation. All data are shown as mean ± SD for triplicates. Only the DMSO treatment significantly decreased cell viability, which was assessed with a Student’s t test, with **** denoting p < 0.0001.

Article Snippet: BC fleeces were loaded with human IgG antibody (50 µg) or anti-CTLA-4 (Syrian hamster IgG, clone 9H10; Bioxcell, Lebanon, NH, USA; 100 µg) according to .

Techniques: Staining, Concentration Assay, Marker, Incubation

Journal: Pharmaceutics

Article Title: Bacterial Cellulose as Drug Delivery System for Optimizing Release of Immune Checkpoint Blocking Antibodies

doi: 10.3390/pharmaceutics14071351

Figure Lengend Snippet: BC reduces serum antibody levels in vivo. Photographs in ( a ) depict in chronological order the procedures of the BC implantation, starting with loading, placing the BC sample underneath the skin, wound closure, visual inspection of the skin and removing the BC implant at D21 (after the mice were killed). Body weight measurements are depicted in ( b ), for the BC-treated mice, the body weight differences at various time-points were compared with the initial body weight at D0. Body weight differences were assessed with a paired Student’s t -test, with NS denoting no statistical differences compared to the body weights at D0. In ( c ) and ( d ), the serum IgG and anti-CTLA-4 levels are shown for several time-points after treatment, respectively. Statistical differences between the BC and PBS group were assessed with an unpaired Student’s t -test and are denoted as * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001.

Article Snippet: BC fleeces were loaded with human IgG antibody (50 µg) or anti-CTLA-4 (Syrian hamster IgG, clone 9H10; Bioxcell, Lebanon, NH, USA; 100 µg) according to .

Techniques: In Vivo