Single Cell Chemical Cytometry of Akt Activity in Rheumatoid Arthritis and Normal Fibroblast-like Synoviocytes in Response to Tumor Necrosis Factor α.

Published

Journal Article

The etiology of rheumatoid arthritis (RA) is poorly understood, and 30% of patients are unresponsive to established treatments targeting tumor necrosis factor α (TNFα). Akt kinase is implicated in TNFα signaling and may act as a barometer of patient responses to biologic therapies. Fluorescent peptide sensors and chemical cytometry were employed to directly measure Akt activity as well as proteolytic activity in individual fibroblast-like synoviocytes (FLS) from RA and normal subjects. The specificity of the peptide reporter was evaluated and shown to be a valid measure of Akt activity in single cells. The effect of TNFα treatment on Akt activity was highly heterogeneous between normal and RA subjects, which was not observable in bulk analyses. In 2 RA subjects, a bimodal distribution of Akt activity was observed, primarily due to a subpopulation (21.7%: RA Subject 5; 23.8%: RA Subject 6) of cells in which >60% of the reporter was phosphorylated. These subjects also possessed statistically elevated proteolytic cleavage of the reporter relative to normal subjects, suggesting heterogeneity in Akt and protease activity that may play a role in the RA-affected joint. We expect that chemical cytometry studies pairing peptide reporters with capillary electrophoresis will provide valuable data regarding aberrant kinase activity from small samples of clinical interest.

Full Text

Duke Authors

Cited Authors

  • Mainz, ER; Serafin, DS; Nguyen, TT; Tarrant, TK; Sims, CE; Allbritton, NL

Published Date

  • August 2, 2016

Published In

Volume / Issue

  • 88 / 15

Start / End Page

  • 7786 - 7792

PubMed ID

  • 27391352

Pubmed Central ID

  • 27391352

Electronic International Standard Serial Number (EISSN)

  • 1520-6882

Digital Object Identifier (DOI)

  • 10.1021/acs.analchem.6b01801

Language

  • eng

Conference Location

  • United States