No abstract

Urea is perhaps the most common denaturant used for studying proteins. However the mechanism of denaturation is still not well understood. Recent theoretical work suggests that van der Waals interactions between urea and non-polar amino acid residues are a major contributor to the protein denaturation process. However, there are few experimental data measuring the effect of urea on hydrophobic interactions. In this work we have determined how the addition of urea to the aqueous solvent affects the contact-pair formed between alkyl and phenyl groups in model compounds: the data indicate that for solutes having a radius smaller than 2.87 Å cavity formation energetics dominate, therefore the addition of urea promotes the formation of hydrophobic contact pairs; while for larger solutes, van der Waals interactions have the largest magnitude, causing urea to disrupt the formation of contact pairs. The influence of urea on hydrophobic interactions is shown to be continuous in the 1-8 M concentration range and is well-correlated with the predictions of scaled particle theory. This demonstrates that the effect of urea on hydrophobic contact pairs can be explained by the changes observed in the solvent packing density, without having to invoke changes in the hydrogen bonding network of water.

Given the very real harm posed by infusion of macroaggregates and cellular debris, and no clear disadvantage to filtration, routine filtration of stem cell products should be considered the standard of care.

Assessing potency of mesenchymal stem/stromal cells (MSC) used for immunologic applications such as the treatment of GVHD or other inflammatory disorders has been a challenge. Expression of PD-L1 or production of indoleamine-pyrrole 2,3-dioxygenase (IDO-1) has been proposed as potential potency markers. To screen for other pathways involved with suppression we undertook proteomic analysis of IFN-γ stimulated MSC. MSC isolated and expanded from normal healthy donors to 70-80% confluence were treated overnight with human rIFN-γ (30ng/ml). MSC were harvested using TrypLE Select and then immunologic and proteomic studies were performed. IFN-γ exposure increased a) MSC expression of IDO-1 and PD-L1, b) MSC suppression of 3rd party T lymphocyte proliferation, c) MSC inhibition of development of IFN-γ producing T lymphocytes, and d) MSC promotion of Treg expansion. Cellular proteomic changes that occur with IFN-γ exposures were studied in paired samples of control and IFN-γ treated MSC. Samples were prepared using a modified Filter Aided Sample Prep (FASP) and digests were separated using 2D liquid chromatography and analysed by tandem mass spectrometry. Data was processed with the Global Proteome Machine and only proteins with at least two confident peptides were reported. A total of 7621 proteins were identified of which 5575 were seen in all samples and 232 proteins were significantly upregulated in the IFN-γ treated cells relative to their controls. The proteomic analysis identified constitutive proteins seen in MSC. The upregulated proteins were significantly enriched (p<10-17) for GO processes such as "response to IFN-γ" and "cytokine mediated signaling pathway". Known inhibitory mediators (such as IDO-1, PD-L1, PGE2, galectin-9) were upregulated. Interestingly adhesion molecules (ICAM-1, VCAM-1, and CCL9) were increased. Other proteins with increased expression include Bone Marrow Stromal 2 (BST2). Conclusion: Proteomic analysis of response of MSC to IFN-γ has identified a signature of proteins upregulated with the activation of immune suppressive functions of MSC. Once confirmed these findings will support the development of a potency test for immunosuppressive potential of given MSC preparations - something that is sorely needed in the clinical manufacturing of MSC products. Acknowledgments: Q.D. is holding a postdoctoral fellowship from MS Society of Canada. This research was supported by The Bihlers' Professorship in Stem Cell Research to D.W. Disclosures No relevant conflicts of interest to declare.