UVC als Alternative zur 8-MOP / UVA-Behandlung in der extrakorporalen Photopherese
UVC as an Alternative to 8-MOP / UVA-Treatment in Extracorporeal Photopheresis
by Carla Barrios-Bussmann
Date of Examination:2020-03-10
Date of issue:2020-02-07
Advisor:Prof. Dr. Tobias Legler
Referee:Prof. Dr. Tobias Legler
Referee:Dr. Ivan Prof Bogeski
Referee:Prof. Dr. Martin Oppermann
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Abstract
English
Background Graft-versus host complications and other (auto-)immune diseases are successfully treated with Extracorporeal Photopheresis (ECP), a widely accepted cellular therapy. The international standard for the ex vivo treatment of the leukapheresis product is the application of 8-Methoxypsoralen (8-MOP) and subsequent irradiation with UV-A light. However, the injection of 8-MOP to the illumination bag is associated with a potential risk of bacterial contamination. Aims The fundamental principle of the ECP is the induction of apoptosis in the irradiated leukocytes. In search of an alternative to the conventional method, this project focused on inducing apoptosis without introducing external substances into the closed system. To this end, the study investigates the apoptosis levels and kinetics in leukocytes after treatment with conventional 8-MOP+UV-A compared to UV-C treatment without additional 8-MOP. Methods We used an in vitro 72 hour cell culture approach with human mononuclear cells from healthy blood donors. Untreated control cells were compared with cells treated with 0,2 µg/ml 8-MOP plus 2J/cm2 UV-A on the one hand and cells treated with 2J/cm2 (effective dose) UV-C on the other hand. The apoptosis level in several leukocyte sub-populations was detected daily with Annexin V and 7-AAD flow cytometry standings. Results The apoptosis analysis of CD3 CD4 T-helper cells, CD3 CD8 cytotoxic T-cells, CD19 B-cells, CD14 Monocytes, CD3neg CD56 NK-cells, CD3 CD56 NKT cells and CD4 CD39 regulatory T-cells revealed no statistical differences in almost all of these cell types after treatment with 8-MOP/UV-A or UV-C light. The apoptosis kinetic as well as the final apoptosis after 72 hours were similar in both treatment groups. Conclusion The addition of 8-MOP to the photopheresis irradiation bag during ECP-treatment is a risk for potential infections. The main effect of the 8-MOP/UV-A treatment is most probably the induction of apoptosis in the irradiated leukocytes. Our project provides information that a satisfactory level of apoptosis can also be achieved with exclusive UV-C irradiation without the need of 8-MOP addition. The apoptosis patterns in most tested leukocyte subpopulations are very similar after treatment with UV-C compared with 8-MOP/UV-A treatment. Follow-up studies with in vivo material are needed to prove the therapeutic effect of UV-C treated cells in the ECP setting.
Keywords: Extracorporeal Photopheresis; Graft-versus-host disease; UVC; 8-Methoxypsoralen