Chronic oxidative stress adaptation in head and neck cancer cells generates slow-cyclers with decreased tumour growth in vivo.

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.

Short- and long-term polystyrene nano- and microplastic exposure promotes oxidative stress and divergently affects skin cell architecture and Wnt/beta-catenin signaling.

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.

Consequences of nano and microplastic exposure in rodent models: the known and unknown.

The ubiquitous nature of micro- (MP) and nanoplastics (NP) is a growing environmental concern. However, their potential impact on human health remains unknown. Research increasingly focused on using rodent models to understand the effects of exposure to individual plastic polymers. In vivo data showed critical exposure effects depending on particle size, polymer, shape, charge, concentration, and exposure routes. Those effects included local inflammation, oxidative stress, and metabolic disruption, leading to gastrointestinal toxicity, hepatotoxicity, reproduction disorders, and neurotoxic effects. This review distillates the current knowledge regarding rodent models exposed to MP and NP with different experimental designs assessing biodistribution, bioaccumulation, and biological responses. Rodents exposed to MP and NP showed particle accumulation in several tissues. Critical responses included local inflammation and oxidative stress, leading to microbiota dysbiosis, metabolic, hepatic, and reproductive disorders, and diseases exacerbation. Most studies used MP and NP commercially provided and doses higher than found in environmental exposure. Hence, standardized sampling techniques and improved characterization of environmental MP and NP are needed and may help in toxicity assessments of relevant particle mixtures, filling knowledge gaps in the literature.

Gas Plasma-Augmented Wound Healing in Animal Models and Veterinary Medicine.

The loss of skin integrity is inevitable in life. Wound healing is a necessary sequence of events to reconstitute the body's integrity against potentially harmful environmental agents and restore homeostasis. Attempts to improve cutaneous wound healing are therefore as old as humanity itself. Furthermore, nowadays, targeting defective wound healing is of utmost importance in an aging society with underlying diseases such as diabetes and vascular insufficiencies being on the rise. Because chronic wounds' etiology and specific traits differ, there is widespread polypragmasia in targeting non-healing conditions. Reactive oxygen and nitrogen species (ROS/RNS) are an overarching theme accompanying wound healing and its biological stages. ROS are signaling agents generated by phagocytes to inactivate pathogens. Although ROS/RNS's central role in the biology of wound healing has long been appreciated, it was only until the recent decade that these agents were explicitly used to target defective wound healing using gas plasma technology. Gas plasma is a physical state of matter and is a partially ionized gas operated at body temperature which generates a plethora of ROS/RNS simultaneously in a spatiotemporally controlled manner. Animal models of wound healing have been vital in driving the development of these wound healing-promoting technologies, and this review summarizes the current knowledge and identifies open ends derived from in vivo wound models under gas plasma therapy. While gas plasma-assisted wound healing in humans has become well established in Europe, veterinary medicine is an emerging field with great potential to improve the lives of suffering animals.

Apical root resorption after orthodontic treatment in patients with unilateral cleft lip and palate.

OBJECTIVES: The aims of this retrospective longitudinal study were to present the incidence of external apical root resorption (EARR) in the maxillary anterior teeth of patients with complete unilateral cleft lip and palate (CUCLP) and to evaluate the influence of orthodontic treatment variables on the development of EARR. MATERIAL AND METHODS: Forty-one patients with CUCLP participated in the study. Orthopantomograms (OPGs), taken before (T2) treatment with multiband orthodontic appliances (MBA), and periapical radiographs (PAs) of the maxillary anterior teeth taken at the end (T3) of orthodontic treatment (OT) were assessed for EARR. RESULTS: The incidence of EARR at T3 (97.6%) was considerably higher than at T2 (51.2%). Central incisors and canines on the cleft side showed a significantly higher score (p < 0.01, p < 0.05 respectively) of EARR in comparison to the same group of teeth on the non-cleft side. Preexisting EARR and abnormal root morphology were identified as predisposing factors for EARR. CONCLUSIONS: Patients with CUCLP treated with MBA have higher incidence of EARR on the maxillary anterior teeth of the cleft side. Severe EARR is rather rare but more often seen on central incisors of the cleft side. CLINICAL RELEVANCE: As most of the patients with cleft lip and palate undergo a challenging and long-term OT with MBA, it is of importance to identify the predisposing factors related to the special anatomical features of the bone and teeth located in the cleft area, as well as the special OT needs of these patients.

Physical plasma and leukocytes - immune or reactive?

Leukocytes are professionals in recognizing and removing pathogenic or unwanted material. They are present in virtually all tissues, and highly motile to enter or leave specific sites throughout the body. Less than a decade ago, physical plasmas entered the field of medicine to deliver their delicate mix of reactive species and other physical agents for mainly dermatological or oncological therapy. Plasma treatment thus affects leukocytes via direct or indirect means: immune cells are either present in tissues during treatment, or infiltrate or exfiltrate plasma-treated areas. The immune system is crucial for human health and resolution of many types of diseases. It is therefore vital to study the response of leukocytes after plasma treatment in vitro and in vivo. This review gathers together the major themes in the plasma treatment of innate and adaptive immune cells, and puts these into the context of wound healing and oncology, the two major topics in plasma medicine.

A Neutrophil Proteomic Signature in Surgical Trauma Wounds.

Non-healing wounds continue to be a clinical challenge for patients and medical staff. These wounds have a heterogeneous etiology, including diabetes and surgical trauma wounds. It is therefore important to decipher molecular signatures that reflect the macroscopic process of wound healing. To this end, we collected wound sponge dressings routinely used in vacuum assisted therapy after surgical trauma to generate wound-derived protein profiles via global mass spectrometry. We confidently identified 311 proteins in exudates. Among them were expected targets belonging to the immunoglobulin superfamily, complement, and skin-derived proteins, such as keratins. Next to several S100 proteins, chaperones, heat shock proteins, and immune modulators, the exudates presented a number of redox proteins as well as a discrete neutrophil proteomic signature, including for example cathepsin G, elastase, myeloperoxidase, CD66c, and lipocalin 2. We mapped over 200 post-translational modifications (PTMs; cysteine/methionine oxidation, tyrosine nitration, cysteine trioxidation) to the proteomic profile, for example, in peroxiredoxin 1. Investigating manually collected exudates, we confirmed presence of neutrophils and their products, such as microparticles and fragments containing myeloperoxidase and DNA. These data confirmed known and identified less known wound proteins and their PTMs, which may serve as resource for future studies on human wound healing.

Role of Ambient Gas Composition on Cold Physical Plasma-Elicited Cell Signaling in Keratinocytes.

A particularly promising medical application of cold physical plasma is the support of wound healing. This is presumably achieved by modulating inflammation as well as skin cell signaling and migration. Plasma-derived reactive oxygen and nitrogen species (ROS/RNS) are assumed the central biologically active plasma components. We hypothesized that modulating the environmental plasma conditions from pure nitrogen (N2) to pure oxygen (O2) in an atmospheric pressure argon plasma jet (kINPen) will change type and concentration of ROS/RNS and effectively tune the behavior of human skin cells. To investigate this, HaCaT keratinocytes were studied in vitro with regard to cell metabolism, viability, growth, gene expression signature, and cytokine secretion. Flow cytometry demonstrated only slight effects on cytotoxicity. O2 shielding provided stronger apoptotic effects trough caspase-3 activation compared to N2 shielding. Gene array technology revealed induction of signaling and communication proteins such as immunomodulatory interleukin 6 as well as antioxidative and proproliferative molecules (HMOX1, VEGFA, HBEGF, CSF2, and MAPK) in response to different plasma shielding gas compositions. Cell response was correlated to reactive species: oxygen-shielding plasma induces a cell response more efficiently despite an apparent decrease of hydrogen peroxide (H2O2), which was previously shown to be a major player in plasma-cell regulation, emphasizing the role of non-H2O2 ROS like singlet oxygen. Our results suggest differential effects of ROS- and RNS-rich plasma, and may have a role in optimizing clinical plasma applications in chronic wounds.

A cold plasma jet accelerates wound healing in a murine model of full-thickness skin wounds.

Cold plasma has been successfully applied in several fields of medicine that require, for example, pathogen inactivation, implant functionalization or alteration of cellular activity. Previous studies have provided evidence that plasma supports the healing of wounds owing to its beneficial mixtures of reactive species and modulation of inflammation in cells and tissues. To investigate the wound healing activity of an atmospheric pressure plasma jet in vivo, we examined the cold plasma's efficacy on dermal regeneration in a murine model of dermal full-thickness ear wound. Over 14 days, female mice received daily plasma treatment. Quantitative analysis by transmitted light microscopy demonstrated a significantly accelerated wound re-epithelialization at days 3-9 in comparison with untreated controls. In vitro, cold plasma altered keratinocyte and fibroblast migration, while both cell types showed significant stimulation resulting in accelerated closure of gaps in scratch assays. This plasma effect correlated with the downregulation of the gap junctional protein connexin 43 which is thought to be important in the regulation of wound healing. In addition, plasma induced profound changes in adherence junctions and cytoskeletal dynamics as shown by downregulation of E-cadherin and several integrins as well as actin reorganization. Our results theorize cold plasma to be a beneficial treatment option supplementing existing wound therapies.

One Year Follow-Up Risk Assessment in SKH-1 Mice and Wounds Treated with an Argon Plasma Jet.

Multiple evidence in animal models and in humans suggest a beneficial role of cold physical plasma in wound treatment. Yet, risk assessment studies are important to further foster therapeutic advancement and acceptance of cold plasma in clinics. Accordingly, we investigated the longterm side effects of repetitive plasma treatment over 14 consecutive days in a rodent full-thickness ear wound model. Subsequently, animals were housed for 350 days and sacrificed thereafter. In blood, systemic changes of the proinflammatory cytokines interleukin 1ß and tumor necrosis factor α were absent. Similarly, tumor marker levels of α-fetoprotein and calcitonin remained unchanged. Using quantitative PCR, the expression levels of several cytokines and tumor markers in liver, lung, and skin were found to be similar in the control and treatment group as well. Likewise, histological and immunohistochemical analysis failed to detect abnormal morphological changes and the presence of tumor markers such as carcinoembryonic antigen, α-fetoprotein, or the neighbor of Punc11. Absence of neoplastic lesions was confirmed by non-invasive imaging methods such as anatomical magnetic resonance imaging and positron emission tomography-computed tomography. Our results suggest that the beneficial effects of cold plasma in wound healing come without apparent side effects including tumor formation or chronic inflammation.

Non-thermal plasma activates human keratinocytes by stimulation of antioxidant and phase II pathways.

Non-thermal atmospheric pressure plasma provides a novel therapeutic opportunity to control redox-based processes, e.g. wound healing, cancer, and inflammatory diseases. By spatial and time-resolved delivery of reactive oxygen and nitrogen species, it allows stimulation or inhibition of cellular processes in biological systems. Our data show that both gene and protein expression is highly affected by non-thermal plasma. Nuclear factor erythroid-related factor 2 (NRF2) and phase II enzyme pathway components were found to act as key controllers orchestrating the cellular response in keratinocytes. Additionally, glutathione metabolism, which is a marker for NRF2-related signaling events, was affected. Among the most robustly increased genes and proteins, heme oxygenase 1, NADPH-quinone oxidoreductase 1, and growth factors were found. The roles of NRF2 targets, investigated by siRNA silencing, revealed that NRF2 acts as an important switch for sensing oxidative stress events. Moreover, the influence of non-thermal plasma on the NRF2 pathway prepares cells against exogenic noxae and increases their resilience against oxidative species. Via paracrine mechanisms, distant cells benefit from cell-cell communication. The finding that non-thermal plasma triggers hormesis-like processes in keratinocytes facilitates the understanding of plasma-tissue interaction and its clinical application.

Gas Plasma Exposure Alters Microcirculation and Inflammation during Wound Healing in a Diabetic Mouse Model.

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.

Sonicated polyethylene terephthalate nano- and micro-plastic-induced inflammation, oxidative stress, and autophagy in vitro.

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.

Rotating spin and giant splitting: unoccupied surface electronic structure of Tl/Si(111).

We present a combined experimental and theoretical study on the unoccupied surface electronic structure of the Tl/Si(111) surface. Spin- and angle-resolved inverse-photoemission measurements with sensitivity to both the in-plane and the out-of-plane polarization direction detect a spin-orbit-split surface state, which is well described by theoretical calculations. We demonstrate that the spin polarization vector rotates from the classical in-plane Rashba polarization direction around Γ[over ¯] to the direction perpendicular to the surface at the K[over ¯](K[over ¯]') points-a direct consequence of the symmetry of the 2D hexagonal system. A giant splitting in energy of about 0.6 eV is observed and attributed to the strong localization of the unoccupied surface state close to the heavy Tl atoms. This leads to completely out-of-plane spin-polarized valleys in the vicinity of the Fermi level. As the valley polarization is oppositely oriented at the K[over ¯] and K[over ¯]' points, backscattering should be strongly suppressed in this system.

Immune and inflammatory responses of human macrophages, dendritic cells, and T-cells in presence of micro- and nanoplastic of different types and sizes.

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 amino-modified 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.

Antioxidant Defense in Primary Murine Lung Cells following Short- and Long-Term Exposure to Plastic Particles.

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 ß-catenin signaling changes. Our results serve as an important basis to point out the potential hazards of plastic contaminations and uptake in lung cells.

Comprehensive in vitro polymer type, concentration, and size correlation analysis to microplastic toxicity and inflammation.

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.

Medical gas plasma augments bladder cancer cell toxicity in preclinical models and patient-derived tumor tissues.

INTRODUCTION: Medical gas plasma therapy has been successfully applied to several types of cancer in preclinical 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 highlighted 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.

Challenges in the Implementation of Disruptive Innovations in Health Care Organizations.

Health organizations face barriers when seeking to deploy radical innovations, such as innovative telemonitoring approaches or AI based Clinical Decision Support Systems (CDSS) into their clinical workflow. However, these barriers are of various types and rarely known to organizations and their management. This study conducted a systematic literature review of 99 selected studies to identify the implementation barriers and factors encountered in this process. Using a hierarchical framework comprising of strategies, resources and capabilities, and processes, the study examined 16 barriers generated from the analysis of the individual studies. The findings highlight implementation barriers on all three levels of the proposed framework. By addressing these barriers comprehensively, health care organizations can successfully implement radical health innovations and enhance patient care outcomes and health care delivery.