human tumour necrosis factor alpha  (R&D Systems)

Journal: Memórias do Instituto Oswaldo Cruz

Article Title: Aedes aegypti salivary gland extract enhances Zika virus replication through immune modulation

doi: 10.1590/0074-02760250272

Figure Lengend Snippet: Aedes aegypti salivary gland extract (SGE) modulates cytokine production by antigen-presenting cell lines during Zika virus (ZIKV) infection. JAWS II dendritic cells and RAW 264.7 macrophages were cultured for 72 h under the indicated conditions (Only Media, SGE, ZIKV, or ZIKV+SGE at different concentrations). Cytokine concentrations [tumour necrosis factor-alpha (TNF-α), interleukin-1beta (IL-1β), IL-10, IL-12] were quantified in culture supernatants by enzyme-linked immunosorbent assay (ELISA). In JAWS II cells (A-D), significant differences were observed for TNF-α, IL-1β, and IL-12, while IL-10 levels remained stable. In RAW 264.7 cells (E-H), SGE enhanced TNF-α, IL-1β, and IL-10 secretion, whereas IL-12 showed no significant modulation. Data represent individual values from independent experiments with bars indicating mean ± standard error of the mean (SEM). Statistical analysis was performed using Kruskal-Wallis with Dunn’s post-hoc test; p < 0.05 was considered significant.

Article Snippet: The supernatants were separated after 72 h of infection and used to quantify IL-1β, tumour necrosis factor-alpha (TNF-α), IL-12, and IL-10 using commercial enzyme-linked immunosorbent assay (ELISA) following the manufacturer’s instructions (Thermo Fisher).

Techniques: Virus, Infection, Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: iScience

Article Title: Plasma extracellular vesicle modulate immune cell transcriptional responses following acute myocardial infarction

doi: 10.1016/j.isci.2026.114665

Figure Lengend Snippet: Enrichment of miRNA-320b in endothelial cell-derived EV following inflammatory stimulation HUVECs were cultured under control (black) or TNF-α-stimulated (red) conditions to model endothelial inflammation. (A) Total and viable cell counts at study endpoint confirm similar cell numbers across conditions. (B) Soluble VCAM-1 levels in supernatants were significantly increased following TNF-α stimulation, confirming proinflammatory activation. (C and D) Single-particle interferometric reflectance imaging (NanoView) detected CD9, CD63, and CD81-positive EVs in both conditions. Scale bars, 10 μm. (D) Total captured EV concentrations were significantly higher in TNF-α-stimulated cultures. (E) Average particle diameters of captured EVs did not differ significantly. (F) NTA showed elevated particle concentrations in EV preparations from TNF-α-treated HUVECs. (G) Size distribution profiles of EVs from control and TNF-α conditions. (H) EV counts were normalized to total and live cell numbers, confirming increased release under inflammatory stimulation. (I) Western blot analysis of HUVEC-derived EVs confirmed enrichment of canonical EV markers (TSG101, Syntenin-1, and CD9), absence of cellular contaminant GM130, and presence of VCAM-1 specifically in TNF-α-derived EVs. (J) TEM images showed characteristic EV morphology; scale bars, 1 μm or 500 nm. (K) RT-qPCR showed no change in HUVEC cellular miRNA-320b levels between conditions. (L) Significant enrichment of miRNA-320b was detected in EVs from TNF-α-stimulated HUVECs. miRNA expression was normalized to UniSp6 spike-in control, because endogenous controls (e.g., miR-103a-3p) were undetectable in EV samples. Data are presented as group means ± SD. N = 4 biological replicates per group. Statistical analysis: (B, F, L) unpaired Student’s t test; (D, H) two-way ANOVA with Tukey’s post hoc test. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001.

Article Snippet: Subsequently, 15 mL of EV-depleted medium, with or without 10 ng / mL recombinant human tumour necrosis factor-α (TNF-α) (R&D Systems) was exposed to cells at the specified time points.

Techniques: Derivative Assay, Cell Culture, Control, Activation Assay, Single Particle, Imaging, Western Blot, Quantitative RT-PCR, Expressing

Journal: iScience

Article Title: Plasma extracellular vesicle modulate immune cell transcriptional responses following acute myocardial infarction

doi: 10.1016/j.isci.2026.114665

Figure Lengend Snippet: Identification of miRNA-320b mRNA targets and pathway analysis (A) Venn diagram showing the overlap of predicted miRNA-320b targets identified using miRWalk, TargetScan, and miRDB databases. (B) GO biological process analysis of overlapping miRNA-320b targets revealed significant enrichment of pathways related to homophilic cell adhesion via plasma membrane adhesion molecules and cell-cell adhesion. (C) Functional validation of miRNA-320b EV activity in a monocyte adhesion assay. HUVEC-derived EVs (from TNF-α-stimulated cells) enriched in miRNA-320b significantly enhanced THP-1 monocyte adhesion to TNF-α-activated endothelial cell monolayers compared with control EVs. Representative fluorescence microscopy images are shown. Scale bars, 20 μm. Data are presented as group means ± SD. N = 4–5 biological replicates per group. Statistical analysis: (C) one-way ANOVA with Tukey’s post hoc test. ∗∗∗∗ p < 0.0001.

Article Snippet: Subsequently, 15 mL of EV-depleted medium, with or without 10 ng / mL recombinant human tumour necrosis factor-α (TNF-α) (R&D Systems) was exposed to cells at the specified time points.

Techniques: Clinical Proteomics, Membrane, Functional Assay, Biomarker Discovery, Activity Assay, Cell Adhesion Assay, Derivative Assay, Control, Fluorescence, Microscopy

Journal: iScience

Article Title: Plasma extracellular vesicle modulate immune cell transcriptional responses following acute myocardial infarction

doi: 10.1016/j.isci.2026.114665

Figure Lengend Snippet: Impact of inflammatory endothelial cell-derived extracellular vesicles (EC-EVs) on monocyte-derived macrophage cytokine production Human CD14 + monocytes were differentiated into macrophages, pretreated with EC-EVs from control or TNF-α-stimulated HUVECs, and subsequently activated under M1 polarizing conditions (LPS and IFN-γ). (A and B) RT-qPCR analysis showed significantly increased IL6 and TNF mRNA expression in macrophages treated with inflammatory EC-EVs (red) compared to untreated controls (black) ( N = 4 biological donors per group). (C and D) In contrast, ELISA of cell culture supernatants revealed reduced secretion of IL-6 and TNF-α protein in TNF-α-EC-EV-treated macrophages under the same M1 conditions ( N = 6 biological donors per group). This discrepancy suggests post-transcriptional modulation of cytokine production by inflammatory EC-EVs. Data are presented as group means ± SD. Statistical analysis: two-way ANOVA with Tukey’s post hoc test. ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001.

Article Snippet: Subsequently, 15 mL of EV-depleted medium, with or without 10 ng / mL recombinant human tumour necrosis factor-α (TNF-α) (R&D Systems) was exposed to cells at the specified time points.

Techniques: Derivative Assay, Control, Quantitative RT-PCR, Expressing, Enzyme-linked Immunosorbent Assay, Cell Culture