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Our new paper is now online in @ISEV_JEV!
Collaboration between @UtrechtUni, @amsterdamumc and @sanquin lead by @EstherNolteHoen and me.
https://www.tandfonline.com/doi/full/10.1080/20013078.2020.1764213
What are our main findings? A thread:
Collaboration between @UtrechtUni, @amsterdamumc and @sanquin lead by @EstherNolteHoen and me.
https://www.tandfonline.com/doi/full/10.1080/20013078.2020.1764213
What are our main findings? A thread:
We previously found that TLR-triggering of murine dendritic cells affected the amount of Y-RNA in EV.
https://link.springer.com/article/10.1007/s00018-018-2842-8
Y-RNA is also one of the most abundant RNA types in plasma. Combining these two bits; could Y-RNA have potential as inflammatory biomarker?
https://link.springer.com/article/10.1007/s00018-018-2842-8
Y-RNA is also one of the most abundant RNA types in plasma. Combining these two bits; could Y-RNA have potential as inflammatory biomarker?
Plasma is a heterogeneous fluid that contains various RNA-containing structures, such as (lipo)protein complexes. We made use of size-exclusion chromatography, density gradient centrifugation and RNase protection assays to address which structures in plasma contain Y-RNA.
To detect the four Y-RNA subtypes separately, we designed primers against the most variable loop region. Note: this region is not part of fragmented Y-RNA, therefore we specifically detect full-length Y-RNA.
Y-RNA elutes in early SEC fractions, from which the majority is pelleted at 100,000g and floats to EV-densities. Y-RNA is protected from degradation by RNase / protease, which shows that Y-RNA is predominantly EV associated in healthy plasma.
Which blood cells release Y-RNA containing EV? We compared red blood cells, platelets, neutrophils, PBMC and HUVEC endothelial cells on EV release and Y-RNA content. These cells release different amounts of EV, each with detectable amounts of Y-RNA.
Strikingly, EV from different blood cells have distinct ratio's between Y-RNA subtypes. Especially neurophils have a unique Y4/Y3 ratio which makes it possible to distinguish neutrophil-EV from other EV.
What happens with these cells during inflammation? We cultured these cells for 2-4h in presence of LPS. In this timeframe, neutrophils released high amounts of EV and thus high amounts of Y-RNA. Interestingly, the Y-RNA ratio's remained stable during activation.
We then tested what happens in human plasma during systemic inflammation. We tested plasma from 16 individuals before, 2h and 6h after LPS injection. Within 2h, an increase of neutrophil-specific Y4/Y3 ratio was observed in total plasma, which correlated with neutrophil numbers.
The Y4/Y3 ratio remained elevated after the peak of inflammation had faded. Also, this ratio showed good discriminatory power in ROC analysis.
This is a proof-of-concept that Y-RNA subtype ratios correlate w/ circulating immune cells during inflammation. We propose that Y-RNA ratios may be of value in diagnosing inflammatory conditions. Future research: test applicability for specific diseases, local inflammation, etc.
Thanks to everybody who helped me complete this research project. It was great fun to work together with so many different people, and to be able to connect the dots in the end!
And thanks to YOU for making it all the way to the end of this thread!
And thanks to YOU for making it all the way to the end of this thread!
