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- ItemNon-thermal plasma modulates cellular markers associated with immunogenicity in a model of latent HIV-1 infection(San Francisco, California, US : PLOS, 2021) Mohamed, Hager; Clemen, Ramona; Freund, Eric; Lackmann, Jan-Wilm; Wende, Kristian; Connors, Jennifer; Haddad, Elias K.; Dampier, Will; Wigdahl, Brian; Miller, Vandana; Bekeschus, Sander; Krebs, Fred C.; Kashanchi, FatahEffective 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.
- ItemShort- and long-term polystyrene nano- and microplastic exposure promotes oxidative stress and divergently affects skin cell architecture and Wnt/beta-catenin signaling(London : BioMed Central, 2023) Schmidt, Anke; da Silva Brito, Walison Augusto; Singer, Debora; Mühl, Melissa; Berner, Julia; Saadati, Fariba; Wolff, Christina; Miebach, Lea; Wende, Kristian; Bekeschus, SanderNano- and microplastic particles (NMP) are strong environmental contaminants affecting marine ecosystems and human health. The negligible use of biodegradable plastics and the lack of knowledge about plastic uptake, accumulation, and functional consequences led us to investigate the short- and long-term effects in freshly isolated skin cells from mice. Using fluorescent NMP of several sizes (200 nm to 6 µm), efficient cellular uptake was observed, causing, however, only minor acute toxicity as metabolic activity and apoptosis data suggested, albeit changes in intracellular reactive species and thiol levels were observed. The internalized NMP induced an altered expression of various targets of the nuclear factor-2-related transcription factor 2 pathway and were accompanied by changed antioxidant and oxidative stress signaling responses, as suggested by altered heme oxygenase 1 and glutathione peroxide 2 levels. A highly increased beta-catenin expression under acute but not chronic NMP exposure was concomitant with a strong translocation from membrane to the nucleus and subsequent transcription activation of Wnt signaling target genes after both single-dose and chronic long-term NMP exposure. Moreover, fibroblast-to-myofibroblast transdifferentiation accompanied by an increase of α smooth muscle actin and collagen expression was observed. Together with several NMP-induced changes in junctional and adherence protein expression, our study for the first time elucidates the acute and chronic effects of NMP of different sizes in primary skin cells' signaling and functional biology, contributing to a better understanding of nano- and microplastic to health risks in higher vertebrates.
- ItemToxicity and Immunogenicity in Murine Melanoma following Exposure to Physical Plasma-Derived Oxidants(Austin, Tex. : Landes Bioscience, 2017) Bekeschus, Sander; Rödder, Katrin; Fregin, Bob; Otto, Oliver; Lippert, Maxi; Weltmann, Klaus-Dieter; Wende, Kristian; Schmidt, Anke; Gandhirajan, Rajesh KumarMetastatic melanoma is an aggressive and deadly disease. Therapeutic advance has been achieved by antitumor chemo- and radiotherapy. These modalities involve the generation of reactive oxygen and nitrogen species, affecting cellular viability, migration, and immunogenicity. Such species are also created by cold physical plasma, an ionized gas capable of redox modulating cells and tissues without thermal damage. Cold plasma has been suggested for anticancer therapy. Here, melanoma cell toxicity, motility, and immunogenicity of murine metastatic melanoma cells were investigated following plasma exposure in vitro. Cells were oxidized by plasma, leading to decreased metabolic activity and cell death. Moreover, plasma decelerated melanoma cell growth, viability, and cell cycling. This was accompanied by increased cellular stiffness and upregulation of zonula occludens 1 protein in the cell membrane. Importantly, expression levels of immunogenic cell surface molecules such as major histocompatibility complex I, calreticulin, and melanocortin receptor 1 were significantly increased in response to plasma. Finally, plasma treatment significantly decreased the release of vascular endothelial growth factor, a molecule with importance in angiogenesis. Altogether, these results suggest beneficial toxicity of cold plasma in murine melanomas with a concomitant immunogenicity of potential interest in oncology.
- ItemRedox Stimulation of Human THP-1 Monocytes in Response to Cold Physical Plasma(Austin, Tex. : Landes Bioscience, 2015) Bekeschus, Sander; Schmidt, Anke; Bethge, Lydia; Masur, Kai; von Woedtke, Thomas; Hasse, Sybille; Wende, KristianIn plasma medicine, cold physical plasma delivers a delicate mixture of reactive components to cells and tissues. Recent studies suggested a beneficial role of cold plasma in wound healing. Yet, the biological processes related to the redox modulation via plasma are not fully understood. We here used the monocytic cell line THP-1 as a model to test their response to cold plasma in vitro. Intriguingly, short term plasma treatment stimulated cell growth. Longer exposure only modestly compromised cell viability but apparently supported the growth of cells that were enlarged in size and that showed enhanced metabolic activity. A significantly increased mitochondrial content in plasma treated cells supported this notion. On THP-1 cell proteome level, we identified an increase of protein translation with key regulatory proteins being involved in redox regulation (hypoxia inducible factor 2α), differentiation (retinoic acid signaling and interferon inducible factors), and cell growth (Yin Yang 1). Regulation of inflammation is a key element in many chronic diseases, and we found a significantly increased expression of the anti-inflammatory heme oxygenase 1 (HMOX1) and of the neutrophil attractant chemokine interleukin-8 (IL-8). Together, these results foster the view that cold physical plasma modulates the redox balance and inflammatory processes in wound related cells.
- ItemROS from Physical Plasmas: Redox Chemistry for Biomedical Therapy(London: Hindawi, 2019) Privat-Maldonado, Angela; Schmidt, Anke; Lin, Abraham; Weltmann, Klaus-Dieter; Wende, Kristian; Bogaerts, Annemie; Bekeschus, SanderPhysical plasmas generate unique mixes of reactive oxygen and nitrogen species (RONS or ROS). Only a bit more than a decade ago, these plasmas, operating at body temperature, started to be considered for medical therapy with considerably little mechanistic redox chemistry or biomedical research existing on that topic at that time. Today, a vast body of evidence is available on physical plasma-derived ROS, from their spatiotemporal resolution in the plasma gas phase to sophisticated chemical and biochemical analysis of these species once dissolved in liquids. Data from in silico analysis dissected potential reaction pathways of plasma-derived reactive species with biological membranes, and in vitro and in vivo experiments in cell and animal disease models identified molecular mechanisms and potential therapeutic benefits of physical plasmas. In 2013, the first medical plasma systems entered the European market as class IIa devices and have proven to be a valuable resource in dermatology, especially for supporting the healing of chronic wounds. The first results in cancer patients treated with plasma are promising, too. Due to the many potentials of this blooming new field ahead, there is a need to highlight the main concepts distilled from plasma research in chemistry and biology that serve as a mechanistic link between plasma physics (how and which plasma-derived ROS are produced) and therapy (what is the medical benefit). This inevitably puts cellular membranes in focus, as these are the natural interphase between ROS produced by plasmas and translation of their chemical reactivity into distinct biological responses. © 2019 Angela Privat-Maldonado et al.
- ItemNitrosylation vs. oxidation – How to modulate cold physical plasmas for biological applications(San Francisco, California, US : PLOS, 2019) Lackmann, Jan-Wilm; Bruno, Giuliana; Jablonowski, Helena; Kogelheide, Friederike; Offerhaus, Björn; Held, Julian; Schulz-von der Gathen, Volker; Stapelmann, Katharina; von Woedtke, Thomas; Wende, KristianThiol 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.
- ItemElevated H2AX Phosphorylation Observed with kINPen Plasma Treatment Is Not Caused by ROS-Mediated DNA Damage but Is the Consequence of Apoptosis(London: Hindawi, 2019) Bekeschus, Sander; Schütz, Clarissa S.; Nießner, Felix; Wende, Kristian; Weltmann, Klaus-Dieter; Gelbrich, Nadine; von Woedtke, Thomas; Schmidt, Anke; Stope, Matthias B.Phosphorylated histone 2AX (γH2AX) is a long-standing marker for DNA double-strand breaks (DSBs) from ionizing radiation in the field of radiobiology. This led to the perception of γH2AX being a general marker of direct DNA damage with the treatment of other agents such as low-dose exogenous ROS that unlikely act on cellular DNA directly. Cold physical plasma confers biomedical effects majorly via release of reactive oxygen and nitrogen species (ROS). In vitro, increase of γH2AX has often been observed with plasma treatment, leading to the conclusion that DNA damage is a direct consequence of plasma exposure. However, increase in γH2AX also occurs during apoptosis, which is often observed with plasma treatment as well. Moreover, it must be questioned if plasma-derived ROS can reach into the nucleus and still be reactive enough to damage DNA directly. We investigated γH2AX induction in a lymphocyte cell line upon ROS exposure (plasma, hydrogen peroxide, or hypochlorous acid) or UV-B light. Cytotoxicity and γH2AX induction was abrogated by the use of antioxidants with all types of ROS treatment but not UV radiation. H2AX phosphorylation levels were overall independent of analyzing either all nucleated cells or segmenting γH2AX phosphorylation for each cell cycle phase. SB202190 (p38-MAPK inhibitor) and Z-VAD-FMK (pan-caspase inhibitor) significantly inhibited γH2AX induction upon ROS but not UV treatment. Finally, and despite γH2AX induction, UV but not plasma treatment led to significantly increased micronucleus formation, which is a functional read-out of genotoxic DNA DSBs. We conclude that plasma-mediated and low-ROS γH2AX induction depends on caspase activation and hence is not the cause but consequence of apoptosis induction. Moreover, we could not identify lasting mutagenic effects with plasma treatment despite phosphorylation of H2AX.
- ItemThe molecular and physiological consequences of cold plasma treatment in murine skin and its barrier function(New York, NY [u.a.] : Elsevier, 2020) Schmidt, Anke; Liebelt, Grit; Striesow, Johanna; Freund, Eric; Woedtke, Thomas von; Wende, Kristian; Bekeschus, SanderCold plasma technology is an emerging tool facilitating the spatially controlled delivery of a multitude of reactive species (ROS) to the skin. While the therapeutic efficacy of plasma treatment has been observed in several types of diseases, the fundamental consequences of plasma-derived ROS on skin physiology remain unknown. We aimed to bridge this gap since the epidermal skin barrier and perfusion plays a vital role in health and disease by maintaining homeostasis and protecting from environmental damage. The intact skin of SKH1 mice was plasma-treated in vivo. Gene and protein expression was analyzed utilizing transcriptomics, qPCR, and Western blot. Immunofluorescence aided the analysis of percutaneous skin penetration of curcumin. Tissue oxygenation, perfusion, hemoglobin, and water index was investigated using hyperspectral imaging. Reversed-phase liquid-chromatography/mass spectrometry was performed for the identification of changes in the lipid composition and oxidation. Transcriptomic analysis of plasma-treated skin revealed modulation of genes involved in regulating the junctional network (tight, adherence, and gap junctions), which was confirmed using qPCR, Western blot, and immunofluorescence imaging. Plasma treatment increased the disaggregation of cells in the stratum corneum (SC) concomitant with increased tissue oxygenation, gap junctional intercellular communication, and penetration of the model drug curcumin into the SC preceded by altered oxidation of skin lipids and their composition in vivo. In summary, plasma-derived ROS modify the junctional network, which promoted tissue oxygenation, oxidation of SC-lipids, and restricted penetration of the model drug curcumin, implicating that plasma may provide a novel and sensitive tool of skin barrier regulation. © 2020 The Author(s)
- ItemOxidants and Redox Signaling: Perspectives in Cancer Therapy, Inflammation, and Plasma Medicine(Austin, Tex. : Landes Bioscience, 2017) Bekeschus, Sander; Bräutigam, Lars; Wende, Kristian; Hanschmann, Eva-Maria[No abstract available]
- ItemCold Physical Plasma Modulates p53 and Mitogen-Activated Protein Kinase Signaling in Keratinocytes(London: Hindawi, 2019) Schmidt, Anke; Bekeschus, Sander; Jarick, Katja; Hasse, Sybille; von Woedtke, Thomas; Wende, KristianSmall reactive oxygen and nitrogen species (ROS/RNS) driven signaling plays a significant role in wound healing processes by controlling cell functionality and wound phase transitions. The application of cold atmospheric pressure plasma (CAP), a partially ionized gas expelling a variety of ROS and RNS, was shown to be effective in chronic wound management and contrastingly also in malignant diseases. The underlying molecular mechanisms are not well understood but redox signaling events are involved. As a central player, the cellular tumor antigen p53 governs regulatory networks controlling proliferation, death, or metabolism, all of which are grossly modulated by anti- and prooxidant signals. Using a human skin cell model, a transient phosphorylation and nuclear translocation of p53, preceded by the phosphorylation of upstream serine- (ATM) and serine/threonine-protein kinase (ATR), was detected after CAP treatment. Results indicate that ATM acts as a direct redox sensor without relevant contribution of phosphorylation of the histone A2X, a marker of DNA damage. Downstream events are the activation of checkpoint kinases Chk1/2 and several mitogen-activated (MAP) kinases. Subsequently, the expression of MAP kinase signaling effectors (e.g., heat shock protein Hsp27), epithelium derived growth factors, and cytokines (Interleukins 6 + 8) was increased. A number of p53 downstream effectors pointed at a decrease of cell growth due to DNA repair processes. In summary, CAP treatment led to an activation of cell repair and defense mechanisms including a modulation of paracrine inflammatory signals emphasizing the role of prooxidant species in CAP-related cell signaling. © 2019 Anke Schmidt et al.