T-cell death, phosphatidylserine exposure and reduced proliferation rate to validate extracorporeal photochemotherapy.

BACKGROUND AND OBJECTIVES: Extracorporeal photochemotherapy (ECP) is an established therapy in various diseases, such as cutaneous T-cell lymphoma and graft-versus-host disease. This study was performed to investigate the practicability of a flow cytometric T-cell evaluation after ECP as a tool to validate the quality of ECP procedures and to enable the comparability of treatments with different ECP devices. MATERIALS AND METHODS: Peripheral blood mononuclear cells (PBMNCs) of healthy volunteer blood donors were treated by offline ECP. To quantify the effect of ECP on T cells in vitro, phosphatidylserine exposure and 7-aminoactinomycin D (7-AAD) reactivity as well as the proliferative activity of phytohaemagglutinin-induced, viable CD3(+) lymphocytes were analysed by flow cytometry. RESULTS: The expected T-cell death after ECP was confirmed by 7-AAD measurements. Phosphatidylserine exposure gradually increased between 20 and 70 h after ECP. Treatment-related inhibition of T-cell proliferation was 92.6 ± 1.4%. CONCLUSION: The combination of viability, phosphatidylserine exposure and T-cell division analyses by flow cytometry in a single-platform system provides a valuable tool to validate ECP procedures.

Extracts from Leonurus sibiricus L. increase insulin secretion and proliferation of rat INS-1E insulinoma cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Traditional Mongolian medicine (TMM) uses preparations from herbs as one form of medication for the treatment of a diversity of diseases including diabetes mellitus (DM). We evaluated the effect of extracts from the plant Leonurus sibiricus L. (LS), used in TMM to treat typical symptoms of type 2 DM, on insulin secretion, electrophysiological properties, intracellular calcium concentration and cell proliferation of INS-1E insulinoma cells under standard cell culture conditions (SCC; 11.1mM glucose). MATERIALS AND METHODS: Insulin secretion was measured by ELISA, electrical properties were assessed by whole cell patch clamping, intracellular calcium concentration (Cai) by Fluo-4 time lapse imaging, insulin receptor expression was verified by RT-PCR and cell proliferation assessed by CellTiter-Glo® cell viability assay. RESULTS: Insulin released from INS-1E cells into the culture medium over 24h was significantly increased in presence of 500 mg/L aqueous LS extract (LS OWE) as well as methanolic LS extract (LS MeOH/H2O) but not in the presence of the butanol-soluble extract (LS MeOH/BuOH). Acute application of LS OWE resulted in a depolarization of the cell membrane potential paralleled by an initial increase and subsequent decline and silencing of action potential frequency, by KATP channel inhibition, persisting depolarization and an increase in Cai. The electrophysiological effects were comparable to those of 100 µM tolbutamide, which, however failed to elevate insulin secretion under SCC. Furthermore all LS extracts stimulated INS-1E cell proliferation. CONCLUSIONS: The finding that extracts from Leonurus sibiricus L. enhance insulin secretion and/or foster cell proliferation may provide possible explanations for the underlying therapeutic principles in the empirical use of LS-containing formulations in DM and DM-related disorders as applied in TMM.