Elisa Vintiñi; Marcela Medina
Abstract
Background: Polyinosinic:polycytidylic acid (Poly-IC) has been used as a viral stimulus to mimic in vivo and in vitro infection induced by some viruses. Objective: To determine whether non-viable Lactobacillus casei CRL431 (LcM) can modulate the immune response induced by Poly I:C in co-culture ...
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Background: Polyinosinic:polycytidylic acid (Poly-IC) has been used as a viral stimulus to mimic in vivo and in vitro infection induced by some viruses. Objective: To determine whether non-viable Lactobacillus casei CRL431 (LcM) can modulate the immune response induced by Poly I:C in co-culture models of peripheral blood mononuclear cells (PBMC) and A549 cells. Methods: T and NK cell activation was evaluated by flow cytometry and levels of TNF-α, IFN-γ, IL-10, IL-29, and IL-17 by ELISA. Cells in direct contact with A549 (PBMC-A549) and cells with no contact with it (PBMC//A549) were used for this purpose. PBMCs alone and both co-culture systems were stimulated for 24 h with the following stimuli: LPS (10 µg/ml), LcM (106 UFC/ml), Poly I:C (2 µg/ml), Poly I:C+LcM, and LcM (3 h)+Poly I:C. Moreover, unstimulated cells were used as a control. Results: Poly I:C and LcM (3 h)+Poly I:C in PBMC-A549 showed a significant increase in the percentage of CD8+ expression (p<0.05). All stimuli induced significant activation from T CD4+, CD8+ cells compared with unstimulated PBMCs in both co-culture cells system. However, activation percentages were higher in direct co-culture. Poly I:C induced a higher level of pro-inflammatory TNF-α and IFN-γ cytokines as well as IL-17 and IL-29 with lower IL-10 levels in both co-culture systems while LcM induced a beneficial pattern of cytokines that would regulate Poly I:C effect. Conclusion: This in vitro model allowed us to highlight the potential of LcM as a modulator of anti-viral immune response and suggest its potential use in formulations against RNA respiratory viruses.
Cheah Wen Yapp; Amal Widaad Mohaimin; Adi Idris
Abstract
Background: Cytosolic double-stranded RNA (dsRNA) is an important ‘molecular signature’ for the detection of intracellular viral infections. Although intracellular dsRNA is a known potent inducer of apoptosis, the optimal time and dose for the onset of dsRNA-mediated apoptosis have not been ...
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Background: Cytosolic double-stranded RNA (dsRNA) is an important ‘molecular signature’ for the detection of intracellular viral infections. Although intracellular dsRNA is a known potent inducer of apoptosis, the optimal time and dose for the onset of dsRNA-mediated apoptosis have not been studied in detail. Objective: To perform an accurate temporal assessment of the cell death responses in dsRNA-dependent cytotoxicity. Methods: Poly I:C (PIC), a synthetic dsRNA molecule was delivered intracellularly into J774.1 and RAW264.7 murine macrophages via electroporation. Cell viability was measured using the MTT assay and apoptosis was determined by sub-G0/G1 DNA content using flow cytometry. Results: Loss of cell viability was seen as early as 3h post-electroporation of macrophages. A significant increase in the sub-G0/G1 DNA content consistent with apoptosis was observed in PIC-electroporated macrophages as early as 3h post electroporation. Conclusion: Intracellular PIC delivery induces rapid macrophage cell death.
