I have some questions regarding the results of my experiments. I need inspiration and ideas to discuss these results. Hopefully someone can help me to explain this.
A short introduction of my experiment:
I isolated human PBMC and stimulated those cells with anit-CD3-AK. Additionally i applied some subances to the cells: Terbutaline, Nadolol and Dexamethasone. After 24 h incubation i measured the IL-2 concentration.
I used different concentrations of TERB, NAD and DEX.
I don´t see a concentration-dependend effect by TERB and NAD and i don´t know why. I expected that after the treatment with TERB the IL-2 concentration decreases and by treatment with NAD increases.
Has someone an idea what the issue could be with the above mentioned scenario?
Hi Bianca,
Trying to address your point of not observing decreasing effect of TERB on the IL-2 concentration and a reverse (increasing) effect of NAD, I guess it might have resulted from a loss of a link between the β₂-AR (β₂-adrenergic receptor) and TCR signaling pathways since expression of IL-2 and IFN-c in activated cells largely depends on dephosphorylation of the transcription factor NFAT by calmodulin-dependent protein phosphatase calcineurin (CaN), a key element of the T cell receptor (TCR)-signaling pathway. Why so? Because disruption of the interaction between cAMP-dependent protein kinase A (PKA) and A-kinase anchoring protein fully blocked TERB-induced CaN inhibition, demonstrating that PKA-AKAP interaction is essential for the β₂-AR-mediated CaN inhibition. Thus please re-establish a link between the β₂-AR and TCR signaling pathways.
Best wishes,
Ilya
Hi Ilya,
Thank you. This is a very good point. But I repeated the experiments with five different test persons and nobody shows an effect of NAD or TERB on IL-2 concentration. Do you have an idea how the loss of the link between beta2-AR and TCR-signaling arises? Is it possible, that some other components of the PBMC disrupt this interaction and I should separate the T-cells?
Kind regards,
Bianca
Hi Bianca,
A very important point is suggested in the above conversation. However, You may also try for higher concentrations of TERB and NAD, dexamethasone to have the clear-cut effect and then try for lower concentrations. Try using a higher concentration of PBMC or you may use ConA to stimulate and go for your designed experiment. You have not mentioned the assay you are using for IL-2 measurement. you may try alternate one.
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