Journal: PLoS Pathogens

Article Title: Monocytes Regulate the Mechanism of T-cell Death by Inducing Fas-Mediated Apoptosis during Bacterial Infection

doi: 10.1371/journal.ppat.1002814

Figure Lengend Snippet: A) Peripheral blood mononuclear cells (PBMC), purified CD3 + T-cells (CD3 + CD14 − ) or co-cultures of purified CD3 + T-cells with 10% purified CD14 + monocytes (CD3+ CD14+) were mock-infected (MOI = 0, white bars) or challenged with D39 Streptococcus pneumoniae (MOI = 50, black bars) for 16 h and accumulation of hypodiploid DNA (% Sub G0/1) measured in CD3 + T-cells, n = 4. B) caspase 3 activation in CD3 + T-cells under the same condition as A), n = 4. C) Representative western blot probed for Bid, Bim and actin from CD3 + T-cells isolated from PBMC or CD3 enriched cultures following bacterial challenge as in A), D) densitometry summarizes the fold change in Bid compared to the mock infected (MI), derived from three separate experiments. E) % Sub G0/1 purified CD3 + T-cells in co-cultures containing 10% or 20% of purified CD14 + monocytes under the same conditions as A), n = 4 or in F) co-cultures of CD3 + T-cells and purified CD14 + monocytes with (CD14 + /CD16 − ) or without (CD14 + /CD16 + ) CD16 + monocyte depletion, under the same conditions as A), n = 4, ns (not significant) * p<0.05, ** p<0.01, *** p<0.001; statistical analysis by ANOVA.

Article Snippet: Cell surface marker expression was with 1 μg/ml mouse anti-human anti- CD14 (61D3) phycoerythrin (PE), (eBioscience), anti-CD3 (SK7) fluorescein isothiocyanate (FITC), (BD Pharmingen), anti-CD161 (HP-3G10) allophycocyanin (APC), (eBioscience), anti-CD4 (CSK3) peridinin chlorophyll protein (PerCP) and anti-CD19 (HIB19) FITC (BD Pharmingen) with appropriate isotype controls.

Techniques: Purification, Infection, Activation Assay, Western Blot, Isolation, Derivative Assay

Journal: PLoS Pathogens

Article Title: Monocytes Regulate the Mechanism of T-cell Death by Inducing Fas-Mediated Apoptosis during Bacterial Infection

doi: 10.1371/journal.ppat.1002814

Figure Lengend Snippet: A) Mean caspase 8 relative luminescence units (Caspase 8 RLU) in CD3 + T-cells from peripheral blood mononuclear cells (PBMC), purified CD3 + T-cells (CD3+ CD14−) or co-cultures of purified CD3 + T-cells with 10% purified CD14 + monocytes (CD3+ CD14+) 6 h following mock-infection (multiplicity of infection (MOI) = 0) or challenge with D39 Streptococcus pneumoniae (MOI = 50), n = 5. B) Representative western blot probed for Bid, Bim and actin from CD3 + T-cells purified from PBMC 16 h following mock-infection (D39−) or challenge with D39 at MOI = 0.1 (D39+) in the presence of isotype control (Isotype+) or ZB4 neutralizing anti-Fas antibody (Anti-Fas+) added at 1 µg/ml. C) Densitometry summarises the fold change in Bid compared to the mock infected (MI) cells from three separate experiments with different donors. D) Cytosolic and membrane fractions were also probed for cytochrome c, actin and Cox-4, and E) densitometry was performed, n = 4. F) Hypodiploid DNA accumulation (Sub G0/1) was measured in CD3 + T-cells in PBMC cultured under the same conditions as B), n = 4. G) The percentage of Annexin V + CD3 + T-cells in PBMC cultured under the same condition as in B), n = 3, * p<0.05; statistical analysis by ANOVA or t-test.

Article Snippet: Cell surface marker expression was with 1 μg/ml mouse anti-human anti- CD14 (61D3) phycoerythrin (PE), (eBioscience), anti-CD3 (SK7) fluorescein isothiocyanate (FITC), (BD Pharmingen), anti-CD161 (HP-3G10) allophycocyanin (APC), (eBioscience), anti-CD4 (CSK3) peridinin chlorophyll protein (PerCP) and anti-CD19 (HIB19) FITC (BD Pharmingen) with appropriate isotype controls.

Techniques: Purification, Infection, Western Blot, Control, Membrane, Cell Culture

Journal: Nature communications

Article Title: Thymosin β4-sulfoxide attenuates inflammatory cell infiltration and promotes cardiac wound healing

doi: 10.1038/ncomms3081

Figure Lengend Snippet: Co-cultures of human CD14+ monocytes and CD4+ T-cells treated with either Tβ4 or Tβ4-SO resulted in significantly decreased levels of IFNγ; n=4 co-cultures per treatment; error bars are standard error of the mean (SEM; a ). In both single CD14+ monocyte cultures and co-cultures with CD4+ T-cells, Tβ4-SO significantly reduced the adherence of the cells in the presence of IFNγ; n=3 cultures per treatment; error bars are standard error of the mean (SEM; b, c ); presence of cells in the right FACS quadrant reflects non-adherent monocytes when treated with Tβ4-SO ( c ). Vital dye staining (trypan blue) of CD14+ cultures demonstrated Tβ4-SO significantly increased monocyte cell death, which was further increased by the additive effect of IFNγ; n=3 cultures per treatment; error bars are standard error of the mean (SEM; d ). In isolated murine peritoneal macrophages Tβ4-SO resulted in a significant fold increase in reactive oxygen species (ROS); n=3 cultures per treatment; error bars are standard error of the mean (SEM; e ) which was reflected by increased dihydroethidium (DE) fluorescence ( f ). Statistics: Wilcoxon matched-pairs signed rank test ( a ) and Student’s t-test ( b , d , e ); * p ≤ 0.05.

Article Snippet: For FACS experiments with human cells, cells were isolated as above, washed once in PBS, fixed using 2% paraformaldehyde and stained for flow cytometry with the following antibodies: Anti-CD4-PerCP-Cy5.5 (BD Bioscience), Anti-CD14-APC (Serotec), Anti-CD163-FITC (Santa Cruz), Anti-CD206-PE (Immunotec), Anti-CD25-PE (Miltanyi Biotech), Anti-CD28-FITC (Serotec), Anti-CD69-FITC (Serotec) and Anti-Ox40-PE (Biolegend).

Techniques: Staining, Isolation, Fluorescence