Journal: Journal of Neuroinflammation

Article Title: Anti-tat Hutat2:Fc mediated protection against tat-induced neurotoxicity and HIV-1 replication in human monocyte-derived macrophages

doi: 10.1186/s12974-014-0195-2

Figure Lengend Snippet: Evaluation of the biological binding function of Hutat2:Fc and protective effects of Hutat2:Fc against HIV-1 Tat 86 -mediated toxicity in HTB-11 cells. (A) Specific binding of Hutat2:Fc to HIV-1 Tat. HIV-1 Tat 86 (Clade B) loaded nitrocellular membranes (NCM) were incubated with cell culture supernatants collected from HR-Hutat2-transduced HTB-11 (HTB-Hutat2), U937 (U937-Hutat2), or hMDM (hMDM-Hutat2) at 4°C overnight followed by incubation with rabbit anti-human IgG (H+L) and goat anti-rabbit IgG HRP conjugated antibodies. Specific binding was visualized by the color deposition on the NCM. The Tat 86 -loaded membrane incubated with rabbit anti-Tat serum served as a positive control (Pos Ctl) while incubated with cell culture supernatant from HR-A3H5 transduced HTB-11 served as a negative control (HTB-A3H5). The NCM loaded with Tat dilution buffer was used as a blank control (BLK Ctl). (B) Functional antagonization of Hutat2:Fc against HIV-1 Tat 86 -induced toxicity in HTB-11 cells by an MTT assay. The OD 570 value of untreated HTB-11 cells was arbitrarily defined as 100% cell viability. The relative cell viability (%) was expressed as a percentage relative to the untreated control cells. The cell viability was significantly higher for the cells treated with the conditioned mediums from transduced cells releasing Hutat:Fc when compared to the cultures that received Tat 86 (500 nM) alone (* P <0.01 for HTB-Hutat2 medium; # P <0.05 for U937-Hutat2 medium, and hMDM-Hutat2 medium). (C) Protection of HR-Hutat2 transduction against Tat 86 -induced toxicity by an MTT assay. No significant difference of cell viability was detected between normal and vector HR-Hutat2 transduced HTB-11 cells (HTB-Hutat2) ( P >0.05). However, the cell viability of HTB-11 transduced with the vector HR-Hutat2 was significantly higher than that of HTB-A3H5 in the presence of HIV-1 Tat 86 (500 nM) (* P <0.01). All experiments were performed in quadruplicate. Error bars denote the s.e.m.

Article Snippet: After washing three times with TBST, the plate was incubated with diluted Hutat2:Fc containing supernatant samples for 1 hour and then incubated with a goat anti-human IgG Fc biotin-conjugated detection antibody (Rockland) for 1 hour.

Techniques: Binding Assay, Incubation, Cell Culture, Membrane, Positive Control, Negative Control, Control, Functional Assay, MTT Assay, Transduction, Plasmid Preparation

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