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Cell stimulation versus cell death induced by sequential treatments with pulsed electric fields and cold atmospheric pressure plasma
Pulsed electric fields (PEFs) and cold atmospheric pressure plasma (CAP) are currently both investigated for medical applications. The exposure of cells to PEFs can induce the formation of pores in cell membranes and consequently facilitate the uptake of molecules. In contrast, CAP mainly acts through reactive species that are generated in the liquid environment. The objective of this study was to determine, if PEFs combined with plasma-treated cell culture medium can mutually reinforce effects on viability of mammalian cells. Experiments were conducted with rat liver epithelial WB-F344 cells and their tumorigenic counterpart WB-ras for a direct comparison of non-tumorigenic and tumorigenic cells from the same origin. Viability after treatments strongly depended on cell type and applied field strength. Notably, tumorigenic WB-ras cells responded more sensitive to the respective treatments than non-tumorigenic WB-F344 cells. More cells were killed when plasma-treated medium was applied first in combination with treatments with 100-μs PEFs. For the reversed treatment order, i.e. application of PEFs first, the combination with 100-ns PEFs resulted in a stimulating effect for non-tumorigenic but not for tumorigenic cells. The results suggest that other mechanisms, besides simple pore formation, contributed to the mutually reinforcing effects of the two methods.
Side effects by oral application of atmospheric pressure plasma on the mucosa in mice
Cold atmospheric pressure plasma (CAP) has been investigated with promising results for peri-implant diseases treatment. However, prior to in-vivo applications of CAP sources in humans, short-term harmful mucosal damage or other unwanted side effects have to be reviewed. 180 male mice (B6C3F1) were divided into twelve treatment groups (n = 15). The right buccal cheek mucosa was treated with CAP. The first and second group each received continuous 10 sec irradiation with 2 different plasma sources (kINPen09, PS-MWM). The third group was treated with the kINPen09 for one minute. Control groups were treated with a corresponding dose of ultraviolet light for 8 seconds or 48 seconds and the other one was left untreated. The animals were weighed before and after treatment. The animals were sacrificed one day or one week after exposure. Stained tissue samples were histologically examined for tissue damage independently by two experienced pathologists. One day after CAP treatment histological analysis showed focal mucosal erosion with superficial ulceration and necrosis accompanied by a mild inflammatory reaction. One week after CAP treatment, the mucosal defects were completely re-epithelialized, associated with remnants of granulation tissue in the stroma irrespective of treatment duration. Furthermore, no cytological atypia was found and no severe weight loss occurred. The control groups did not show any alterations at all. CAP treatment led to a superficial mucosal damage that healed within few days. Nonetheless, further long-term experiments are necessary to exclude undesirable side effects after longer observation time. Particularly, potential carcinogenic effects must be ruled out prior to the application of CAP treatment in daily dental practice.
Nitrosylation vs. oxidation – How to modulate cold physical plasmas for biological applications
Thiol moieties are major targets for cold plasma-derived nitrogen and oxygen species, making CAPs convenient tools to modulate redox-signaling pathways in cells and tissues. The underlying biochemical pathways are currently under investigation but especially the role of CAP derived RNS is barely understood. Their potential role in protein thiol nitrosylation would be relevant in inflammatory processes such as wound healing and improving their specific production by CAP would allow for enhanced treatment options beyond the current application. The impact of a modified kINPen 09 argon plasma jet with nitrogen shielding on cysteine as a thiol-carrying model substance was investigated by FTIR spectroscopy and high-resolution mass spectrometry. The deposition of short-lived radical species was measured by electron paramagnetic resonance spectroscopy, long-lived species were quantified by ion chromatography (NO2-, NO3-) and xylenol orange assay (H2O2). Product profiles were compared to samples treated with the so-called COST jet, being introduced by a European COST initiative as a reference device, using both reference conditions as well as conditions adjusted to kINPen gas mixtures. While thiol oxidation was dominant under all tested conditions, an Ar + N2/O2 gas compositions combined with a nitrogen curtain fostered nitric oxide deposition and the desired generation of S-nitrosocysteine. Interestingly, the COST-jet revealed significant differences in its chemical properties in comparison to the kINPen by showing a more stable production of RNS with different gas admixtures, indicating a different •NO production pathway. Taken together, results indicate various chemical properties of kINPen and COST-jet as well as highlight the potential of plasma tuning not only by gas admixtures alone but by adjusting the surrounding atmosphere as well.
Non-thermal plasma modulates cellular markers associated with immunogenicity in a model of latent HIV-1 infection
Effective control of infection by human immunodeficiency virus type 1 (HIV-1), the causative agent of the acquired immunodeficiency syndrome (AIDS), requires continuous and life-long use of anti-retroviral therapy (ART) by people living with HIV-1 (PLWH). In the absence of ART, HIV-1 reemergence from latently infected cells is ineffectively suppressed due to suboptimal innate and cytotoxic T lymphocyte responses. However, ART-free control of HIV-1 infection may be possible if the inherent immunological deficiencies can be reversed or restored. Herein we present a novel approach for modulating the immune response to HIV-1 that involves the use of non-thermal plasma (NTP), which is an ionized gas containing various reactive oxygen and nitrogen species (RONS). J-Lat cells were used as a model of latent HIV-1 infection to assess the effects of NTP application on viral latency and the expression of pro-phagocytic and pro-chemotactic damage-associated molecular patterns (DAMPs). Exposure of J-Lat cells to NTP resulted in stimulation of HIV-1 gene expression, indicating a role in latency reversal, a necessary first step in inducing adaptive immune responses to viral antigens. This was accompanied by the release of pro-inflammatory cytokines and chemokines including interleukin-1β (IL-1β) and interferon-γ (IFN-γ); the display of pro-phagocytic markers calreticulin (CRT), heat shock proteins (HSP) 70 and 90; and a correlated increase in macrophage phagocytosis of NTP-exposed J-Lat cells. In addition, modulation of surface molecules that promote or inhibit antigen presentation was also observed, along with an altered array of displayed peptides on MHC I, further suggesting methods by which NTP may modify recognition and targeting of cells in latent HIV-1 infection. These studies represent early progress toward an effective NTP-based ex vivo immunotherapy to resolve the dysfunctions of the immune system that enable HIV-1 persistence in PLWH.