Pandemics threaten societies, driving the need for new therapeutics. Medical gas plasma, a partially ionized gas, has been proposed as an antiviral agent, but its mechanisms of action are elusive. We employed a certified argon plasma jet and dynamic infection monitoring. Gas plasma exposure reduced adenovirus infectivity in lung cells in treatment time-dependently, with 90s yielding infectivity losses of 96%. The antioxidants N‑acetylcysteine and tyrosine, and the nitric oxide scavenger carboxy‑PTIO, abrogated gas plasma-mediated virus inactivation, implicating hydroxyl radicals and peroxynitrite chemistry (involving superoxide and nitric oxide) as key antiviral gas plasma agents. These did not induce virus DNA fragmentation or cross-linking but morphological and virus size alterations (16.5% decrease). High-resolution mass spectrometry identified oxidative hotspots on critical histidine and cysteine residues of penton, hexon, and fiber proteins, which are involved in cellular entry and stability. The dominant reactive pathways include oxidative and nitrosative chemistry caused by reactive oxygen and nitrogen species (e.g., •OH, 1O2, O3, H2O2, O2 -•, NO, and ONOO-) generated by gas plasma, which lead to viral protein oxidation, lysis, and nucleic acid base modifications. Together, these findings delineate the mechanisms of gas plasma-mediated viral inactivation and support its translational implementation.

Cold physical gas plasma generates various reactive oxygen and nitrogen species and has emerged as potential anticancer treatment. However, its effects on and into tumor tissue are poorly understood. Using three gas plasma‐treated tumor models (in ovo Panc1 pancreas carcinoma tissue, ex vivo CT26 colorectal carcinoma tissue, and in vivo SSC7 squamous carcinoma tissue), apoptosis and tissue penetration, along with stress markers, were analyzed. Across all models, a significant increase in TUNEL‐positive cells was revealed. The extent of apoptosis correlated with the intensity and duration of the gas plasma application, being most pronounced within the tumor periphery. Immunofluorescence quantification of oxidative and nitrosative stress markers, including 8‐hydroxy‐2′‐deoxyguanosine (8‐OHdG) and 3‐nitrotyrosine (3‐NT), showed elevated staining and significant intensity increases. This was particularly notable within the first 500 µm from the tissue surface. Finally, safety assessments were performed based on histopathological analyses of nontumor tissues in a healthy gas plasma‐treated murine skin. While apoptotic events were found to some degree, no significant adverse effects on adjacent healthy tissue, such as necrosis or off‐target tissue damage, were observed within the assessed zones following gas plasma exposure. These findings support a favorable safety profile under the tested conditions, which is a key consideration for future clinical translation.

Impaired diabetic wound healing is characterized by delayed tissue repair due to compromised immune function, reduced angiogenesis and blood flow, and decreased levels of essential growth factors. Gas plasma treatment is an emerging therapeutic approach in redox biology, characterized by its ability to modulate biological processes at different stages of tissue repair. This study examined the wound healing process in a preclinical mouse model of type 2 diabetes (T2DM). Repeated exposure to medical gas plasma has been demonstrated to improve microcirculatory parameters, including superficial tissue oxygenation. Consequently, an optimal wound environment is created. Regulating the interplay between proliferation, the balance of apoptotic pathways, and reactive species generated by gas plasma is critical for several cellular processes. This study revealed that the Hippo signaling pathway and yes-associated protein (YAP) activity play crucial roles in this process. Specifically, these mechanisms were observed to facilitate stage-specific cellular responses in diabetic wounds, significantly affecting the survival of skin cell types. Gas plasma stimulates various biological processes, including cell migration, granulation tissue formation, and collagen synthesis. This stimulation occurs through the remodeling of focal adhesions, the restoration of proper extracellular matrix (ECM) architecture, and the regulation of intercellular junctional structures (e.g., tight, adherens, and gap junctions). Mass spectrometry analysis of gas plasma-treated human wounds revealed that 6% of the identified proteins had undergone oxidative post-translational modifications, suggesting a link between gas plasma-accelerated healing responses and these modifications. These diverse effects highlight the potential of medical gas plasma as a versatile tool for improving wound management and tissue regeneration in diabetes.

The recent pandemic has highlighted the urgent need to elucidate the pathophysiological mechanisms underlying viral effects in humans and is driving the search for innovative antiviral therapies. Several studies have investigated the ability of gas plasma, a partially ionized gas that simultaneously generates several reactive species, to be a new antiviral tool. However, several aspects of the mechanisms of antiviral action of gas plasma remained elusive. In this study, we, for the first time, used a gas plasma device approved for medical purposes and routinely applied in the clinics, especially for wound healing, to test its antiviral activity against a murine corona-virus in vitro (MHV-GFP), a research model analogous to human coronaviruses such as SARS-CoV-2. For this, we established a novel high-content imaging assay that gave quantitative and kinetic information about infection and reduced viral activity in murine fibroblasts (17Cl-1) host cells. Gas plasma treatment delayed viral infectivity and reduced overall infection and toxicity in 17Cl1 cells. Various antioxidants at different concentrations were screened to identify ROS relevant to antiviral effects. Catalase provided no virus protection, and DMSO, mannitol, histidine, Trolox, and ascorbic acid only modestly reduced gas plasma virucidal efficacy. By contrast, glutathione, tyrosine, and cysteine showed profound but not complete protection of MHV from gas plasma-derived reactive species, suggesting pivotal roles of superoxide radicals and peroxynitrite gas in plasma-driven viral inactivation. At extended gas plasma exposure times, fewer intact MHV RNA were detected, indicative of reactive species-driven RNA modifications or degradation as an additional mechanism of action. Virus particle size changes measured by electron microscopy were moderate. Collectively, we identified the potent antiviral activity of a clinically approved argon plasma jet along with potential mechanisms of action.

Introduction Diabetes mellitus is a chronic disease that can disrupt physiologic wound healing. Medical gas plasma technology produces therapeutic reactive species that support wound healing. Objective Previous studies have shown that increasing the transcriptional activity of the redox regulator nuclear factor erythroid 2-related factor 2 (Nrf2) in diabetic models can improve insulin sensitivity, reduce blood glucose levels, and ameliorate diabetic complications. However, the therapeutic potential and mechanisms of action of gas plasma have not been addressed in this context. Methods Full-thickness dermal ear wounds were created in a preclinical mouse model of type II diabetes and compared with a native wild-type strain of C57BL/6 mice. First, the formation of reactive species in the plasma gas phase was determined by optical emission spectroscopy. Second, qPCR, protein expression, and inflammation analysis by cytokine secretion were performed to confirm the transcriptional results. Finally, qPCR and cytokine profiling were conducted to measure the effects of gas plasma in patient wound samples. Results Repeated in vivo treatment with medical gas plasma supported wound healing, e.g., re-epithelialization, in both sexes. Gas plasma-stimulated changes in Nrf2 signaling associated with downstream targets were supported by the evidence of impaired wound healing in Nrf2 knockout mice. In addition, gas plasma treatment significantly affected inflammation by modulating local and systemic cytokine levels. In vivo, treatment of human diabetic wounds underscored the involvement of Nrf2 signaling in protecting against oxidative stress, as assessed by qPCR. The cytokine signature of human diabetic wounds outlined different response patterns among patients after a single exposure, while inflammatory mediators were consistently reduced after repeated plasma treatment. Conclusions The present finding of accelerated wound healing by the Nrf2 activator underlines the high potential of medical gas plasma therapy in non-diabetic and diabetic wound healing.

Diabetes can disrupt physiological wound healing, caused by decreased levels or impaired activity of angiogenic factors. This can contribute to chronic inflammation, poor formation of new blood vessels, and delayed re-epithelialization. The present study describes the preclinical application of medical gas plasma to treat a dermal, full-thickness ear wound in streptozotocin (STZ)-induced diabetic mice. Gas plasma-mediated effects occurred in both sexes but with gender-specific differences. Hyperspectral imaging demonstrated gas plasma therapy changing microcirculatory parameters, particularly oxygen saturation levels during wound healing, presumably due to the gas plasma's tissue delivery of reactive species and other bioactive components. In addition, gas plasma treatment significantly affected cell adhesion by regulating focal adhesion kinase and vinculin, which is important in maintaining skin barrier function by regulating syndecan expression and increasing re-epithelialization. An anticipated stimulation of blood vessel formation was detected via transcriptional and translational increase of angiogenic factors in gas plasma-exposed wound tissue. Moreover, gas plasma treatment significantly affected inflammation by modulating systemic growth factors and cytokine levels. The presented findings may help explain the mode of action of successful clinical plasma therapy of wounds of diabetic patients.

Introduction Hippo is a signaling pathway that is evolutionarily conserved and plays critical roles in wound healing and tissue regeneration. Disruption of the transcriptional activity of both Hippo-associated factors, the yes-associated protein (YAP), and the transcriptional co-activator with PDZ binding motif (TAZ) has been associated with cardiovascular diseases, fibrosis, and cancer. This makes the Hippo pathway an appealing target for therapeutic interventions. Objectives Prior research has indicated that medical gas plasma promotes wound healing by delivering a combination of reactive species directly to the affected areas. However, the involvement of YAP/TAZ and other signaling pathways in diabetic wound healing remains unexplored. Methods To this extent, ear wounds were generated and treated with gas plasma in streptozotocin (STZ)-induced diabetic mice. Transcriptome profiling at two wound healing stages (days 9 and 20 post-wounding) was performed in female and male mice. Additionally, we employed gene and protein expression analyses, utilizing immunohistological and −chemical staining of various targets as well as quantitative PCR and Western blot analysis. Results Gas plasma treatment accelerated healing by increasing re-epithelialization and modifying extracellular matrix components. Transcriptomic profiling charting the major alterations in gene expression following plasma treatment was followed by a validation of several targets using transcriptional and translational quantification as well as localization analyses. Conclusion Our study evaluated the cellular regulation of essential targets of the Hippo and related pathways such as YAP/TAZ, β-catenin, tumor growth factor β, and oxidative stress signaling after plasma treatment. The activation of genes, pathways, and their regulators is an attractive therapeutic aim for a therapeutic intervention in dermal skin repair in diabetic diseases using medical gas plasmas.

The environmental presence of nano- and micro-plastic particles (NMPs) is suspected to have a negative impact on human health. Environmental NMPs are difficult to sample and use in life science research, while commercially available plastic particles are too morphologically uniform. Additionally, this NMPs exposure exhibited biological effects, including cell internalization, oxidative stress, inflammation, cellular adaptation, and genotoxicity. Therefore, developing new methods for producing heterogenous NMPs as observed in the environment is important as reference materials for research. Thus, we aimed to generate and characterize NMPs suspensions using a modified ultrasonic protocol and to investigate their biological effects after exposure to different human cell lines. To this end, we produced polyethylene terephthalate (PET) NMPs suspensions and characterized the particles by dynamic light scattering and scanning electron microscopy. Ultrasound treatment induced polymer degradation into smaller and heterogeneous PET NMPs shape fragments with similar surface chemistry before and after treatment. A polydisperse suspension of PET NMPs with 781 nm in average size and negative surface charge was generated. Then, the PET NMPs were cultured with two human cell lines, A549 (lung) and HaCaT (skin), addressing inhalation and topical exposure routes. Both cell lines interacted with and have taken up PET NMPs as quantified via cellular granularity assay. A549 but not HaCaT cell metabolism, viability, and cell death were affected by PET NMPs. In HaCaT keratinocytes, large PET NMPs provoked genotoxic effects. In both cell lines, PET NMPs exposure affected oxidative stress, cytokine release, and cell morphology, independently of concentration, which we could relate mechanistically to Nrf2 and autophagy activation. Collectively, we present a new PET NMP generation model suitable for studying the environmental and biological consequences of exposure to this polymer.

Reactive oxygen species (ROS) play roles in physiological processes and pathological conditions. Higher ROS levels induce oxidative distress and cytotoxic responses, such as chronic inflammatory conditions and cancer. While the cellular responses of various cell types to cytotoxic pro-oxidative conditions have been well studied in the past decades, much less is known about the cellular gene and expression profiles that are a priori associated with subsequent cellular demise to oxidative stress. To this end, we used 25 human cancer cell lines of different origins and established the inhibitory concentration (IC25) of hypochlorous acid (HOCl), an oxidant readily produced by neutrophils frequently present in many inflamed tissues, including cancer. The HOCl sensitivity varied throughout the 25 cell lines investigated, showing a more than 5-fold difference between the most sensitive and resistant types. In parallel, we investigated untreated cells and their basal gene expression using transcriptomic microarray and performed correlation analyses to HOCl IC25 values of all cell lines. Transcriptomic analyses and functional classification of significant correlating genes revealed reduced expression of tumor protein p53 signaling network members, including BAX, CDKN1A (p21), and BTG2, as well as the p53 gene (TP53) itself to associate with cell line sensitivity to HOCl toxicity. Further, baseline surface membrane expression analysis of 33 inflammation- and redox-related molecules identified nitric oxide (NO) synthase 2 and the ER-stress-associated chaperone calreticulin to correlate significantly with HOCl resistance. We identified targets associated with HOCl sensitivity. Nevertheless, further studies are needed to map gene and protein expression patterns associated with oxidative stress-induced cytotoxicity.

Environmental pollution by microplastics (MPs) is a growing concern regarding their impact on aquatic and terrestrial systems and human health. Typical exposure routes of MPs are dermal contact, digestion, and inhalation. Recent in vitro and in vivo studies observed alterations in immunity after MPs exposure, but systemic studies using primary human immune cells are scarce. In our investigation, we addressed the effect of polystyrene (PS) and poly methyl methacrylate (PMMA) in three different sizes (50-1100 nm) as well as aminomodified PS (PS-NH2; 50 nm) on cells of the adaptive and innate immune system. T-cells isolated from human peripheral blood mononuclear cells (PBMCs) were least affected regarding the cytotoxicity but displayed increased activation marker expression after 72 h, and strongly modulated cytokine secretion patterns. Conversely, phagocytic dendritic cells and macrophages derived from isolated monocytes were highly sensitive to pristine MPs. Their marker expression suggested a downregulation of the inflammatory phenotypes indicative of M2 macrophage induction after MPs exposure for 24 h. Our results showed that even pristine MPs affected immune cell function and inflammatory phenotype dependent on MPs polymers, size, and immune cell type.

The ubiquitous nature of microplastic particles (MP) is a growing environmental and ecological concern due to their impact on aquatic and terrestrial systems and potentially on human health. The potential impact on human health may be due to MP daily exposure by several routes, but little is known about the cellular effects. Previous in vitro and in vivo studies have described inflammation, oxidative stress, and metabolic disruption upon plastic exposure, while the effect of individual plastic parameters is not fully unraveled. To this end, we investigated plastic exposure to different polymer types, sizes, and concentrations in three human cell lines (A549, HEK293, and HeLa). Particles were polystyrene (PS) or polymethylmethacrylate (PMMA) in three sizes and concentrations, and amine-modified PS served as positive control. After MP size validation using dynamic light scattering, a high-throughput high-content imaging-based and algorithm-driven multi-z-stack analysis was established to quantify intracellular fluorescent particle accumulation in 3D objects and cell maximum intensity projections. MP uptake correlated with concentration and for PS with size (1.040 μm), while for PMMA it was maximal for 400 nm MP. Uptake increased in HEK cells independent of MP parameters. Except for positive controls, no major effect on metabolic activity, viability, and cell cycle was observed, while intracellular thiol content and cytokine secretion were affected to a considerable extent. Interestingly, particle uptake was correlated significantly with particle size and concentration, underlining the dependence of MP parameters on biological effects.

BACKGROUND:Reactive oxygen species (ROS) are implicated in cancer therapy and as drivers of microenvironmental tumour cell adaptations. Medical gas plasma is a multi-ROS generating technology that has been shown effective for palliative tumour control in head and neck cancer (HNC) patients before tumour cells adapted to the oxidative stress and growth regressed fatally.METHODS:In a bedside-to-bench approach, we sought to explore the oxidative stress adaptation in two human squamous cell carcinoma cell lines. Gas plasma was utilised as a putative therapeutic agent and chronic oxidative stress inducer.RESULTS:Cellular responses of single and multiple treated cells were compared regarding sensitivity, cellular senescence, redox state and cytokine release. Whole transcriptome analysis revealed a strong correlation of cancer cell adaption with increased interleukin 1 receptor type 2 (IL1R2) expression. Using magnetic resonance imaging, tumour growth and gas plasma treatment responses of wild-type (WT) and repeatedly exposed (RE) A431 cells were further investigated in a xenograft model in vivo. RE cells generated significantly smaller tumours with suppressed inflammatory secretion profiles and increased epidermal growth factor receptor (EGFR) activity showing significantly lower gas plasma sensitivity until day 8.CONCLUSIONS:Clinically, combination treatments together with cetuximab, an EGFR inhibitor, may overcome acquired oxidative stress resistance in HNC.

Polystyrene nano- and micro-sized plastic particles (NMP) are one of the common plastic materials produced that dramatically pollute the environment, water, and oceanic habitats worldwide. NMP are continuously absorbed by the body through a number of routes, especially via intestinal ingestion, dermal uptake, and inhalation into the lung. Several studies provided evidence of NMP provoking oxidative stress and affecting cellular responses. Yet, the NMP effects on primary lung cells have not been studied. To this end, we isolated and cultured murine lung cells and exposed them short-term or long-term to polystyrene 0.2–6.0 µm-sized NMP. We studied cellular consequences regarding oxidative stress, morphology, and secretion profiling. Visualization, distribution, and expression analyses confirmed lung cells accumulating NMP and showed several significant correlations with particle size. Moreover, we found substantial evidence of biological consequences of small-scale NMP uptake in lung cells. Besides alterations of cytokine secretion profiles resulting in inflammatory responses, indicators of oxidative stress were identified that were accompanied by Nrf2 and β-catenin signaling changes. Our results serve as an important basis to point out the potential hazards of plastic contaminations and uptake in lung cells.

Introduction: Medical gas plasma therapy has been successfully applied to several types of cancer in pre -clinical models. First palliative tumor patients suffering from advanced head and neck cancer benefited from this novel therapeutic modality. The gas plasma-induced biological effects of reactive oxygen and nitrogen species (ROS/RNS) generated in the plasma gas phase result in oxidation-induced lethal damage to tumor cells.Objectives: This study aimed to verify these anti-tumor effects of gas plasma exposure on urinary bladder cancer. Methods: 2D cell culture models, 3D tumor spheroids, 3D vascularized tumors grown on the chicken chorion-allantois-membrane (CAM) in ovo , and patient-derived primary cancer tissue gas plasma -treated ex vivo were used.Results: Gas plasma treatment led to oxidation, growth retardation, motility inhibition, and cell death in 2D and 3D tumor models. A marked decline in tumor growth was also observed in the tumors grown in ovo. In addition, results of gas plasma treatment on primary urothelial carcinoma tissues ex vivo high-lighted the selective tumor-toxic effects as non-malignant tissue exposed to gas plasma was less affected. Whole-transcriptome gene expression analysis revealed downregulation of tumor-promoting fibroblast growth factor receptor 3 (FGFR3) accompanied by upregulation of apoptosis-inducing factor 2 (AIFm2), which plays a central role in caspase-independent cell death signaling.Conclusion: Gas plasma treatment induced cytotoxicity in patient-derived cancer tissue and slowed tumor growth in an organoid model of urinary bladder carcinoma, along with less severe effects in non-malignant tissues. Studies on the potential clinical benefits of this local and safe ROS therapy are awaited.(c) 2023 The Authors. Published by Elsevier B.V. on behalf of Cairo University. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Nano- 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.

Pancreatic cancer is associated with a highly abundant stroma and low-grade inflammation. In the local tumour microenvironment, elevated glucose levels, the presence of tumour-associated stellate cells and macrophages are hypothesised to promote the tumour progression and invasion. The present study investigated the influence by the microenvironment on pancreatic cancer cell invasion in vitro. After co-culture with tumour-associated pancreatic stellate cells (TPSCs), pancreatic cancer cells displayed up to 8-fold reduction in levels of epithelial-mesenchymal transition (EMT) markers E-cadherin and ZO-1, while β-catenin and vimentin levels were increased. A 3D organotypic model showed that TPSCs stimulated pancreatic cancer cell invasion, both as single cell (PANC-1) and cohort (MIAPaCa-2) invasion. The combined presence of TPSCs and M2-like macrophages induced invasion of the non-invasive BxPC-3 cells. High glucose conditions further enhanced changes in EMT markers as well as the cancer cell invasion. In summary, co-culture with TPSCs induced molecular changes associated with EMT in pancreatic cancer cells, regardless of differentiation status, and the organotypic model demonstrated the influence of microenvironmental factors, such as glucose, stellate cells and macrophages, on pancreatic cancer cell invasion.