New Flow Cytometric Methods for Monitoring STAT5 Signaling Reveal Responses to SARS-CoV-2 Antigen-Specific Stimulation in FOXP3+ Regulatory T Cells also in Patients with Advanced Chronic Lymphocytic Leukemia.

Increased frequency of CD4+CD25+ regulatory T-cells (Treg) has been associated with disease progression in chronic lymphocytic leukemia (CLL). Flow cytometric methods, which allow for the simultaneous analysis of their specific transcription factor Foxp3 and activated STAT proteins, together with proliferation can help to elucidate the signaling mechanisms driving Treg expansion and suppression of FOXP3- conventional CD4+T-cells (Tcon). Herein, we first report a novel approach in which STAT5 phosphorylation (pSTAT5) and proliferation (BrdU-FITC incorporation) could be analyzed specifically in FOXP3+ and FOXP3- responding cells after CD3/CD28 stimulation. The addition of magnetically purified CD4+CD25+ T-cells from healthy donors to cocultured autologous CD4+CD25- T-cells resulted in suppression of Tcon cell cycle progression accompanied by a decrease in pSTAT5. Next, a method using imaging flow cytometry is presented for the detection of cytokine-dependent pSTAT5 nuclear translocation in FOXP3-expressing cells. Finally, we discuss our experimental data obtained by combining Treg pSTAT5 analysis and antigen-specific stimulation with SARS-CoV-2 antigens. Applying these methods on samples from patients revealed Treg responses to antigen-specific stimulation and significantly higher basal pSTAT5 in CLL patients treated with immunochemotherapy. Thus, we speculate that through the use of this pharmacodynamic tool, the efficacy of immunosuppressive drugs and their possible off-target effects can be assessed.

Ruthenium complexes show potent inhibition of AKR1C1, AKR1C2, and AKR1C3 enzymes and anti-proliferative action against chemoresistant ovarian cancer cell line.

In this study, we present the synthesis, kinetic studies of inhibitory activity toward aldo-keto reductase 1C (AKR1C) enzymes, and anticancer potential toward chemoresistant ovarian cancer of 10 organoruthenium compounds bearing diketonate (1-6) and hydroxyquinolinate (7-10) chelating ligands with the general formula [(η6-p-cymene)Ru(chel)(X)]n+ where chel represents the chelating ligand and X the chlorido or pta ligand. Our studies show that these compounds are potent inhibitors of the AKR enzymes with an uncommon inhibitory mechanism, where two inhibitor molecules bind to the enzyme in a first fast and reversible step and a second slower and irreversible step. The binding potency of each step is dependent on the chemical structure of the monodentate ligands in the metalloinhibitors with the chlorido complexes generally acting as reversible inhibitors and pta complexes as irreversible inhibitors. Our study also shows that compounds 1-9 have a moderate yet better anti-proliferative and anti-migration action on the chemoresistant ovarian cancer cell line COV362 compared to carboplatin and similar effects to cisplatin.