Cardiac adherent proliferating cells reduce Coxsackievirus B3-induced HL-1 apoptosis in a nitric oxide- and interleukin-10-dependent manner and require interferon-γ priming.
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Bar graphs represent DiO+ Annexin V+/7AAD- HL-1 cells in cultures of uninfected (open bars) or CVB3-infected (closed bars) HL-1 with or without untreated or L-NAME treated CAPs or CAPs in the presence or absence of 1 µg/ml of anti-human IL-10 antibody (ab), or 1 µg/ml of anti-murine IFN–γ ab; n = 4/group.
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Phagocytosis assay
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To assess a potential role for, or IL-10 in phagocytosis of apoptotic cells by influenza-infected macrophages, mock- or Udorn-infected Mph1 and were incubated with an anti-Mer blocking antibody (& ; 5 µg/ml), (esearch HP3652L; 4 µg/ml) or recombinant human IL-10 (20 ng/ml) for 30 minutes before addition of the apoptotic neutrophils.
- The reagents were also present during the 1 h macrophage-neutrophil co-culture.
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Dendritic Cells and T Cells Function Assay
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Cytokines were quantified using specific ELISA kits, following the manufacturer’s instructions.
- IL-2, IL-12, and IL-10 kits were from , λ 1 (IL-29) and λ 2 (IL-28A, cross-reacting with IL-28B) were from& .
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Isolation of monocytes and cell culture
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Monocytes were obtained from buffy coats from healthy blood donors.
- Written informed consents were obtained from all donors in accordance with the ethical principles set out in the declaration of.
- This study was approved by the Medical Committee of the Academic Medical Center and the of the in , .
- Peripheral blood mononuclear cells (PBMCs) were isolated from buffy coats using Lymphoprep density gradient centrifugation.
- Monocytes were isolated by adherence to plastic and cultured in Iscove's medium'>modified medium'>Dulbecco's medium (IMDM;, , ) supplemented with 10% [v/v] heat-inactivated human pooled serum (HPS), penicillin (100 U/ml, , ), streptomycin (100 µg/ml) and ciproxin (5 μg/ml, , ) for 5 days in the presence of different cytokines: IFN-α (250 U/ml, , , ), IFN-β (250 U/ml), IFN-γ (250 U/ml and 50 U/ml) in combination with TNF-α (12.5 ng/ml, , , ), IL-4 (50 ng/ml), IL-10 (50 ng/ml), GM-CSF…
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Monocytes were obtained from buffy coats from healthy blood donors.
- Written informed consents were obtained from all donors in accordance with the ethical principles set out in the declaration of.
- This study was approved by the Medical Committee of the Academic Medical Center and the of the in , .
- Peripheral blood mononuclear cells (PBMCs) were isolated from buffy coats using Lymphoprep density gradient centrifugation.
- Monocytes were isolated by adherence to plastic and cultured in Iscove's medium'>modified medium'>Dulbecco's medium (IMDM;, , ) supplemented with 10% [v/v] heat-inactivated human pooled serum (HPS), penicillin (100 U/ml, , ), streptomycin (100 µg/ml) and ciproxin (5 μg/ml, , ) for 5 days in the presence of different cytokines: IFN-α (250 U/ml, , , ), IFN-β (250 U/ml), IFN-γ (250 U/ml and 50 U/ml) in combination with TNF-α (12.5 ng/ml, , , ), IL-4 (50 ng/ml), IL-10 (50 ng/ml), GM-CSF (50 ng/ml), M-CSF (50 ng/ml) or medium alone at 37°C in a humidified atmosphere supplemented with 5% CO2.
- At chosen concentrations, the cytokines showed a significant restriction of viral replication without affecting cell viability.
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A: Ex vivo IFNγ ELISpot responses in patients with acute DHF who developed shock and those who did not develop shock and also in healthy dengue seropositive donors and dengue seronegative donors.
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B: Spontaneously released IL-10 levels (pg/ml) in PBMC ELISpot supernatants in patients with shock and those without shock The mean spontaneous release of IL-10 from PBMC in patients with shock was 19.65 (SD±21.28 pg/ml), and the mean levels in those who did not develop shock was 11.28 (SD±24.5) pg/ml (p = 0.08).
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Characterization of a stable transfectant of PANC-1 cells expressing TGF-β1.
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Mean concentration of VEGF, IL-10, the active form of TGF-β1 derived from PANC/Mock or PANC/TGF-β was analyzed by ELISA.
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Ig Secretion by Human B Cells
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Human B cells were isolated from buffy coats of healthy donors obtained from the New York Blood Center by negative selection with B cell isolation kit II .
- The purity of the sorted B cells was more than 97%, as assessed by CD19 staining.
- Purified B cells (5×104) were plated in a 96-well U-bottom plate in 200 µl of complete medium'>RPMI 1640 medium containing 10% FBS, 2mM glutamine, 100U/ml streptomycin, 100U/ml penicillin, 1mM sodium pyruvate, and 10mM HEPES (all from, , ).
- The cells were treated with 20 µl of 293T cell supernatant transfected with Envwt or Env-fusion proteins in the presence of recombinant C40L (Enzo Life, , ) (200 ng/ml), interleukin-4 (IL-4) (10 ng/ml), and IL-10 (200 ng/ml) for 14 days.
- The amount of Env or Env-fusion proteins was normalized based on an anti-Env ELISA using the 2G12 MAb (data not shown) and adjusted using mock…
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Human B cells were isolated from buffy coats of healthy donors obtained from the New York Blood Center by negative selection with B cell isolation kit II .
- The purity of the sorted B cells was more than 97%, as assessed by CD19 staining.
- Purified B cells (5×104) were plated in a 96-well U-bottom plate in 200 µl of complete medium'>RPMI 1640 medium containing 10% FBS, 2mM glutamine, 100U/ml streptomycin, 100U/ml penicillin, 1mM sodium pyruvate, and 10mM HEPES (all from, , ).
- The cells were treated with 20 µl of 293T cell supernatant transfected with Envwt or Env-fusion proteins in the presence of recombinant C40L (Enzo Life, , ) (200 ng/ml), interleukin-4 (IL-4) (10 ng/ml), and IL-10 (200 ng/ml) for 14 days.
- The amount of Env or Env-fusion proteins was normalized based on an anti-Env ELISA using the 2G12 MAb (data not shown) and adjusted using mock transfection supernatant.
- Recombinant human IL-21 (rhIL-21) was used at 10 ng/ml concentration.
- Culture supernatants were collected for the analysis of immunoglobulin secretion by an enzyme-linked immunosorbent assay (ELISA) .
- For fold-change calculations, the background levels of IgG, IgA and IgM secretion induced by the stimulation cocktail without Env or fusion proteins were subtracted from the test values and the fold-change was calculated compared to Envwt.
- Note that B cells cultured with supernatant of cleaved EnvIL-21 co-transfected with a plasmid encoding furin were not viable; suggesting that residual furin in the supernatant had a toxic effect on these cells (data not shown).
- For this reason, the B cell experiments with cleavable Env and EnvIL-21 were performed with proteins prepared without furin co-transfection.
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Regulation of membrane-bound and sCD127.
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PBMCs were cultured for 24 h in PMI 1640 supplemented with 5% FCS with the following stimuli: anti-C3/C28–coated beads (1:100 bead/cell ratio), IL-2 (100 U/mL ), IL-7 (10 ng/mL& ), interferon-γ (IFN-γ; 10 ng/mL ), IL-4 (10 ng/mL), IL-17 (10 ng/mL& ), TNF-α (10 ng/mL& ), IL-10 (10 ng/mL& ), IL-12 (10 ng/mL), rapamycin (sirolimus, 10 ng/mL), FK506 (tacrolimus, 10 ng/mL ), mycophenolate mofetil (MMF, 1 μg/mLoche), daclizumab (1 μg/mLoche), high glucose (33.3 mmol/L), and insulin (100 U/mL).
- PBMCs were subsequently stained with anti-CD4 Pacific blue (clone RPA-T4 ), anti-CD8 allophycocyanin-Cy7 (clone ), and anti-CD127 phycoerythrin (clone ), and surface expression of CD127 in gated CD4+ and CD8+ T cells was analyzed by flow cytometry.
- Supernatant from PBMC cultures was taken to measure sCD127 concentration.
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Mitogen stimulation
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Differentiation of MBCs into ASCs in was initiated in bulk PBMC cultures based on a previously established protocol S.
- aureus Cowan I protein A (SAC) and CpG.
- IL-10 was added to the stimulation mix since a previous study showed that this enhanced the efficiency of MBC into ASC differentiation by more than 9 fold 6 cells/ml were added to 25 cm2 cell culture flasks .
- Culture medium was supplemented with 50 ng/ml PWM lectin derived from Phytolacca americana , 1∶5000 Protein A from aureus, Cowan Strain , 2.5 µg/ml ODN 2006 ( nucleotide-human ) and 25 ng/ml recombinant human IL-10 and incubated at 37°C, 5% CO2 for 5 days.
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