Publications by authors named "Anke Schmidt"

61 Publications

Gas Plasma Technology Augments Ovalbumin Immunogenicity and OT-II T Cell Activation Conferring Tumor Protection in Mice.

Adv Sci (Weinh) 2021 05 8;8(10):2003395. Epub 2021 Mar 8.

ZIK plasmatis Leibniz Institute for Plasma Science and Technology (INP) Felix-Hausdorff-Str. 2 Greifswald 17489 Germany.

Reactive oxygen species (ROS/RNS) are produced during inflammation and elicit protein modifications, but the immunological consequences are largely unknown. Gas plasma technology capable of generating an unmatched variety of ROS/RNS is deployed to mimic inflammation and study the significance of ROS/RNS modifications using the model protein chicken ovalbumin (Ova vs oxOva). Dynamic light scattering and circular dichroism spectroscopy reveal structural modifications in oxOva compared to Ova. T cells from Ova-specific OT-II but not from C57BL/6 or SKH-1 wild type mice presents enhanced activation after Ova addition. OxOva exacerbates this activation when administered ex vivo or in vivo, along with an increased interferon-gamma production, a known anti-melanoma agent. OxOva vaccination of wild type mice followed by inoculation of syngeneic B16F10 Ova-expressing melanoma cells shows enhanced T cell number and activation, decreased tumor burden, and elevated numbers of antigen-presenting cells when compared to their Ova-vaccinated counterparts. Analysis of oxOva using mass spectrometry identifies three hot spots regions rich in oxidative modifications that are associated with the increased T cell activation. Using Ova as a model protein, the findings suggest an immunomodulating role of multi-ROS/RNS modifications that may spur novel research lines in inflammation research and for vaccination strategies in oncology.
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http://dx.doi.org/10.1002/advs.202003395DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8132054PMC
May 2021

Tumor cell metabolism correlates with resistance to gas plasma treatment: The evaluation of three dogmas.

Free Radic Biol Med 2021 May 9;167:12-28. Epub 2021 Mar 9.

ZIK plasmatis, Leibniz Institute for Plasma Science and Technology (INP), Felix-Hausdorff-Str. 2, 17489, Greifswald, Germany.

Gas plasma is a partially ionized gas increasingly recognized for targeting cancer. Several hypotheses attempt to explain the link between plasma treatment and cytotoxicity in cancer cells, all focusing on cellular membranes that are the first to be exposed to plasma-generated reactive oxygen species (ROS). One proposes high levels of aquaporins, membrane transporters of water and hydrogen peroxide, to mark tumor cell line sensitivity to plasma treatment. A second focuses on membrane-expression of redox-related enzymes such as NADPH oxidases (NOX) that may modify or amplify the effects of plasma-derived ROS, fueling plasma-induced cancer cell death. Another hypothesis is that the decreased cholesterol content of tumor cell membranes sensitizes these to plasma-mediated oxidation and subsequently, cytotoxicity. Screening 33 surface molecules in 36 tumor cell lines in correlation to their sensitivity to plasma treatment, the expression of aquaporins or NOX members could not explain the sensitivity but were rather associated with treatment resistance. Correlation with transporter or enzyme activity was not tested. Analysis of cholesterol content confirmed the proposed positive correlation with treatment resistance. Strikingly, the strongest correlation was found for baseline metabolic activity (Spearman r = 0.76). Altogether, these data suggest tumor cell metabolism as a novel testable hypothesis to explain cancer cell resistance to gas plasma treatment for further elucidating this innovative field's chances and limitations in oncology.
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http://dx.doi.org/10.1016/j.freeradbiomed.2021.02.035DOI Listing
May 2021

Tumor cytotoxicity and immunogenicity of a novel V-jet neon plasma source compared to the kINPen.

Sci Rep 2021 01 8;11(1):136. Epub 2021 Jan 8.

ZIK plasmatis, Leibniz Institute for Plasma Science and Technology (INP), Felix-Hausdorff-Str. 2, 17489, Greifswald, Germany.

Recent research indicated the potential of cold physical plasma in cancer therapy. The plethora of plasma-derived reactive oxygen and nitrogen species (ROS/RNS) mediate diverse antitumor effects after eliciting oxidative stress in cancer cells. We aimed at exploiting this principle using a newly designed dual-jet neon plasma source (jet) to treat colorectal cancer cells. A treatment time-dependent ROS/RNS generation induced oxidation, growth retardation, and cell death within 3D tumor spheroids were found. In TUM-CAM, a semi in vivo model, the jet markedly reduced vascularized tumors' growth, but an increase of tumor cell immunogenicity or uptake by dendritic cells was not observed. By comparison, the argon-driven single jet kINPen, known to mediate anticancer effects in vitro, in vivo, and in patients, generated less ROS/RNS and terminal cell death in spheroids. In the TUM-CAM model, however, the kINPen was equivalently effective and induced a stronger expression of immunogenic cancer cell death (ICD) markers, leading to increased phagocytosis of kINPen but not jet plasma-treated tumor cells by dendritic cells. Moreover, the jet was characterized according to the requirements of the DIN-SPEC 91315. Our results highlight the plasma device-specific action on cancer cells for evaluating optimal discharges for plasma cancer treatment.
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http://dx.doi.org/10.1038/s41598-020-80512-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7794240PMC
January 2021

Unexpected Gene-Flow in Urban Environments: The Example of the European Hedgehog.

Animals (Basel) 2020 Dec 7;10(12). Epub 2020 Dec 7.

Department of Evolutionary Genetics, Leibniz Institute for Zoo and Wildlife Research (IZW), Alfred-Kowalke-Straße 17, 10315 Berlin, Germany.

We use the European hedgehog (), a mammal with limited mobility, as a model species to study whether the structural matrix of the urban environment has an influence on population genetic structure of such species in the city of Berlin (Germany). Using ten established microsatellite loci we genotyped 143 hedgehogs from numerous sites throughout Berlin. Inclusion of all individuals in the cluster analysis yielded three genetic clusters, likely reflecting spatial associations of kin (larger family groups, known as gamodemes). To examine the potential bias in the cluster analysis caused by closely related individuals, we determined all pairwise relationships and excluded close relatives before repeating the cluster analysis. For this data subset ( = 65) both clustering algorithms applied (Structure, Baps) indicated the presence of a single genetic cluster. These results suggest that the high proportion of green patches in the city of Berlin provides numerous steppingstone habitats potentially linking local subpopulations. Alternatively, translocation of individuals across the city by hedgehog rescue facilities may also explain the existence of only a single cluster. We therefore propose that information about management activities such as releases by animal rescue centres should include location data (as exactly as possible) regarding both the collection and the release site, which can then be used in population genetic studies.
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http://dx.doi.org/10.3390/ani10122315DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7762246PMC
December 2020

Gas plasma-spurred wound healing is accompanied by regulation of focal adhesion, matrix remodeling, and tissue oxygenation.

Redox Biol 2021 01 25;38:101809. Epub 2020 Nov 25.

ZIK Plasmatis, Leibniz Institute for Plasma Science and Technology (INP), Felix-Hausdorff-Str. 2, 17489, Greifswald, Germany. Electronic address:

In response to injury, efficient migration of skin cells to rapidly close the wound and restore barrier function requires a range of coordinated processes in cell spreading and migration. Gas plasma technology produces therapeutic reactive species that promote skin regeneration by driving proliferation and angiogenesis. However, the underlying molecular mechanisms regulating gas plasma-aided cell adhesion and matrix remodeling essential for wound closure remain elusive. Here, we combined in vitro analyses in primary dermal fibroblasts isolated from murine skin with in vivo studies in a murine wound model to demonstrate that gas plasma treatment changed phosphorylation of signaling molecules such as focal adhesion kinase and paxillin α in adhesion-associated complexes. In addition to cell spreading and migration, gas plasma exposure affected cell surface adhesion receptors (e.g., integrinα5β1, syndecan 4), structural proteins (e.g., vinculin, talin, actin), and transcription of genes associated with differentiation markers of fibroblasts-to-myofibroblasts and epithelial-to-mesenchymal transition, cellular protrusions, fibronectin fibrillogenesis, matrix metabolism, and matrix metalloproteinase activity. Finally, we documented that gas plasma exposure increased tissue oxygenation and skin perfusion during ROS-driven wound healing. Altogether, these results provide critical insights into the molecular machinery of gas plasma-assisted wound healing mechanisms.
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http://dx.doi.org/10.1016/j.redox.2020.101809DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7710641PMC
January 2021

The molecular and physiological consequences of cold plasma treatment in murine skin and its barrier function.

Free Radic Biol Med 2020 12 1;161:32-49. Epub 2020 Oct 1.

Plasma Life Science and ZIK Plasmatis, Leibniz Institute for Plasma Science and Technology (INP), Felix-Hausdorff-Str. 2, 17489, Greifswald, Germany. Electronic address:

Cold 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.
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http://dx.doi.org/10.1016/j.freeradbiomed.2020.09.026DOI Listing
December 2020

Plasma Treatment Limits Cutaneous Squamous Cell Carcinoma Development In Vitro and In Vivo.

Cancers (Basel) 2020 Jul 21;12(7). Epub 2020 Jul 21.

ZIK plasmatis, Leibniz Institute for Plasma Science and Technology (INP), Felix-Hausdorff-Str. 2, 17489 Greifswald, Germany.

Cutaneous squamous cell carcinoma (SCC) is the most prevalent cancer worldwide, increasing the cost of healthcare services and with a high rate of morbidity. Its etiology is linked to chronic ultraviolet (UV) exposure that leads to malignant transformation of keratinocytes. Invasive growth and metastasis are severe consequences of this process. Therapy-resistant and highly aggressive SCC is frequently fatal, exemplifying the need for novel treatment strategies. Cold physical plasma is a partially ionized gas, expelling therapeutic doses of reactive oxygen and nitrogen species that were investigated for their anticancer capacity against SCC in vitro and SCC-like lesions in vivo. Using the kINPen argon plasma jet, a selective growth-reducing action of plasma treatment was identified in two SCC cell lines in 2D and 3D cultures. In vivo, plasma treatment limited the progression of UVB-induced SSC-like skin lesions and dermal degeneration without compromising lesional or non-lesional skin. In lesional tissue, this was associated with a decrease in cell proliferation and the antioxidant transcription factor Nrf2 following plasma treatment, while catalase expression was increased. Analysis of skin adjacent to the lesions and determination of global antioxidant parameters confirmed the local but not systemic action of the plasma anticancer therapy in vivo.
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http://dx.doi.org/10.3390/cancers12071993DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7409328PMC
July 2020

Medical Gas Plasma Jet Technology Targets Murine Melanoma in an Immunogenic Fashion.

Adv Sci (Weinh) 2020 May 30;7(10):1903438. Epub 2020 Mar 30.

ZIK plasmatis Leibniz Institute for Plasma Science and Technology (INP Greifswald) Felix-Hausdorff-Str. 3 Greifswald 17489 Germany.

Medical technologies from physics are imperative in the diagnosis and therapy of many types of diseases. In 2013, a novel cold physical plasma treatment concept was accredited for clinical therapy. This gas plasma jet technology generates large amounts of different reactive oxygen and nitrogen species (ROS). Using a melanoma model, gas plasma technology is tested as a novel anticancer agent. Plasma technology derived ROS diminish tumor growth in vitro and in vivo. Varying the feed gas mixture modifies the composition of ROS. Conditions rich in atomic oxygen correlate with killing activity and elevate intratumoral immune-infiltrates of CD8 cytotoxic T-cells and dendritic cells. T-cells from secondary lymphoid organs of these mice stimulated with B16 melanoma cells ex vivo show higher activation levels as well. This correlates with immunogenic cancer cell death and higher calreticulin and heat-shock protein 90 expressions induced by gas plasma treatment in melanoma cells. To test the immunogenicity of gas plasma treated melanoma cells, 50% of mice vaccinated with these cells are protected from tumor growth compared to 1/6 and 5/6 mice negative control (mitomycin C) and positive control (mitoxantrone), respectively. Gas plasma jet technology is concluded to provide immunoprotection against malignant melanoma both in vitro and in vivo.
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http://dx.doi.org/10.1002/advs.201903438DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7237847PMC
May 2020

Pulmonary vein isolation and beyond: Predictive value of vagal reactions in second-generation cryoballoon ablation for the outcome of persistent atrial fibrillation.

Heart Rhythm 2020 04 11;17(4):600-606. Epub 2019 Dec 11.

Clinic for Electrophysiology, Herz- und Diabeteszentrum NRW, Ruhr-Universität Bochum, Bad Oeynhausen, Germany.

Background: Cryoballoon ablation (CBA) is gaining increasing acceptance in the treatment of persistent (per) atrial fibrillation (AF). The cardiac autonomic nervous system plays a pivotal role in the regulation of AF.

Objective: We evaluated the impact of vagal reactions (VRs), as a surrogate marker for autonomic nervous system modulation, on the outcome of CBA in patients (pts) with perAF.

Methods: A total of 250 consecutive pts (mean age 63.9 ± 10.0 years; 175 pts, 70% male) undergoing primary second-generation CBA for perAF were studied. VRs were defined as bradycardia <40 beats/min, asystole, or higher-degree atrioventricular block. Follow-up visits at 3, 6, and 12 months included 7-day Holter electrocardiograms.

Results: VRs were recorded in 61 pts (24%). These pts showed a significantly reduced recurrence rate of AF (5%) than did those without VRs (log-rank, P < .01). Univariate Cox regression analyses confirmed VRs as a strong predictor of AF-free survival (hazard ratio [HR] 0.10; P < .01). Female sex (HR 1.71; P = .02), preprocedural tachycardia (HR 1.01; P = .01), and AF (HR 1.75; P = .01) before CBA at admission were revealed as predictors of AF recurrence. Multivariate regression model calculation solely identified VRs (HR 0.11; 95% confidence interval 0.03-0.34; P < .01) and male sex (HR 0.57; 95% confidence interval 0.36-0.89; P = .01) as independent predictors of AF-free survival.

Conclusion: VR is an independent predictor of AF-free survival after CBA for perAF.
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http://dx.doi.org/10.1016/j.hrthm.2019.12.006DOI Listing
April 2020

ROS from Physical Plasmas: Redox Chemistry for Biomedical Therapy.

Oxid Med Cell Longev 2019 8;2019:9062098. Epub 2019 Oct 8.

ZIK plasmatis, Leibniz Institute for Plasma Science and Technology (INP Greifswald), Greifswald, Germany.

Physical 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 analysis dissected potential reaction pathways of plasma-derived reactive species with biological membranes, and and 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.
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http://dx.doi.org/10.1155/2019/9062098DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6800937PMC
March 2020

Elevated H2AX Phosphorylation Observed with kINPen Plasma Treatment Is Not Caused by ROS-Mediated DNA Damage but Is the Consequence of Apoptosis.

Oxid Med Cell Longev 2019 19;2019:8535163. Epub 2019 Sep 19.

Department of Urology, Greifswald University Medical Center, Greifswald, Germany.

Phosphorylated histone 2AX (H2AX) 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 H2AX 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). , increase of H2AX has often been observed with plasma treatment, leading to the conclusion that DNA damage is a direct consequence of plasma exposure. However, increase in H2AX 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 H2AX induction in a lymphocyte cell line upon ROS exposure (plasma, hydrogen peroxide, or hypochlorous acid) or UV-B light. Cytotoxicity and H2AX 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 H2AX phosphorylation for each cell cycle phase. SB202190 (p38-MAPK inhibitor) and Z-VAD-FMK (pan-caspase inhibitor) significantly inhibited H2AX induction upon ROS but not UV treatment. Finally, and despite H2AX 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 H2AX 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.
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http://dx.doi.org/10.1155/2019/8535163DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6770374PMC
March 2020

Risk Assessment of kINPen Plasma Treatment of Four Human Pancreatic Cancer Cell Lines with Respect to Metastasis.

Cancers (Basel) 2019 Aug 23;11(9). Epub 2019 Aug 23.

Department of General, Visceral, Thoracic, and Vascular Surgery, Greifswald University Medical Center, Ferdinand-Sauerbruch-Str., 17475 Greifswald, Germany.

Cold physical plasma has limited tumor growth in many preclinical models and is, therefore, suggested as a putative therapeutic option against cancer. Yet, studies investigating the cells' metastatic behavior following plasma treatment are scarce, although being of prime importance to evaluate the safety of this technology. Therefore, we investigated four human pancreatic cancer cell lines for their metastatic behavior in vitro and in chicken embryos (in ovo). Pancreatic cancer was chosen as it is particularly metastatic to the peritoneum and systemically, which is most predictive for outcome. In vitro, treatment with the kINPen plasma jet reduced pancreatic cancer cell activity and viability, along with unchanged or decreased motility. Additionally, the expression of adhesion markers relevant for metastasis was down-regulated, except for increased CD49d. Analysis of 3D tumor spheroid outgrowth showed a lack of plasma-spurred metastatic behavior. Finally, analysis of tumor tissue grown on chicken embryos validated the absence of an increase of metabolically active cells physically or chemically detached with plasma treatment. We conclude that plasma treatment is a safe and promising therapeutic option and that it does not promote metastatic behavior in pancreatic cancer cells in vitro and in ovo.
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http://dx.doi.org/10.3390/cancers11091237DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6769931PMC
August 2019

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

Clin Oral Investig 2020 May 13;24(5):1807-1819. Epub 2019 Aug 13.

Charité - Universitätsmedizin Berlin, CC03 Department of Orthodontics, Dentofacial Orthopedics and Pedodontics, Berlin, Germany.

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.
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http://dx.doi.org/10.1007/s00784-019-03044-2DOI Listing
May 2020

Plasma Medicine: A Field of Applied Redox Biology.

In Vivo 2019 Jul-Aug;33(4):1011-1026

Leibniz Institute for Plasma Science and Technology, INP Greifswald, Greifswald, Germany.

Plasma medicine comprises the application of physical plasma directly on or in the human body for therapeutic purposes. Three most important basic plasma effects are relevant for medical applications: i) inactivation of a broad spectrum of microorganisms, including multidrug-resistant pathogens, ii) stimulation of cell proliferation and angiogenesis with lower plasma treatment intensity, and iii) inactivation of cells by initialization of cell death with higher plasma treatment intensity, above all in cancer cells. Based on own published results as well as on monitoring of relevant literature the aim of this topical review is to summarize the state of the art in plasma medicine and connect it to redox biology. One of the most important results of basic research in plasma medicine is the insight that biological plasma effects are mainly mediated via reactive oxygen and nitrogen species influencing cellular redox-regulated processes. Plasma medicine can be considered a field of applied redox biology.
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http://dx.doi.org/10.21873/invivo.11570DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6689367PMC
December 2019

Nrf2 signaling and inflammation are key events in physical plasma-spurred wound healing.

Theranostics 2019 30;9(4):1066-1084. Epub 2019 Jan 30.

Leibniz-Institute for Plasma Science and Technology (INP Greifswald), Plasma Life Science and ZIK plasmatis, Felix-Hausdorff-Str. 2, 17489 Greifswald, Germany.

Wound healing is strongly associated with the presence of a balanced content of reactive species in which oxygen-dependent, redox-sensitive signaling represents an essential step in the healing cascade. Numerous studies have demonstrated that cold physical plasma supports wound healing due to its ability to deliver a beneficial mixture of reactive species directly to the cells. : We described a preclinical proof-of-principle-concept of cold plasma use in a dermal, full-thickness wound model in immunocompetent SKH1 mice. Quantitative PCR, Western blot analysis, immunohistochemistry and immunofluorescence were perfomed to evaluate the expression and cellular translocation of essential targets of Nrf2 and p53 signaling as well as immunomodulatory and angiogenetic factors. Apoptosis and proliferation were detected using TUNEL assay and Ki67 staining, respectively. Cytokine levels in serum were measured using bead-based multiplex cytokine analysis. Epidermal keratinocytes and dermal fibroblasts were isolated from mouse skin to perform functional knockdown experiments. Intravital fluorescence analysis was used to illustrate and quantified microvascular features. : Plasma exerted significant effects on wound healing in mice, including the promotion of granulation and reepithelialization as a consequence of the migration of skin cells, the balance of antioxidant and inflammatory response, and the early induction of macrophage and neutrophil recruitment to the wound sites. Moreover, through an early and local plasma-induced p53 inhibition with a concomitant stimulation of proliferation, the upregulation of angiogenetic factors, and an increased outgrowth of new vessels, our findings explain why dermal skin repair is accelerated. The cellular redox homeostasis was maintained and cells were defended from damage by a strong modulation of the nuclear E2-related factor (Nrf2) pathway and redox-sensitive p53 signaling. : Although acute wound healing is non-problematic, the pathways highlighted that mainly the activation of Nrf2 signaling is a promising strategy for the clinical use of cold plasma in chronic wound healing.
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http://dx.doi.org/10.7150/thno.29754DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6401410PMC
January 2020

Cold Physical Plasma Modulates p53 and Mitogen-Activated Protein Kinase Signaling in Keratinocytes.

Oxid Med Cell Longev 2019 13;2019:7017363. Epub 2019 Jan 13.

Leibniz-Institute for Plasma Science and Technology e. V., Felix-Hausdorff-Str. 2, 17489 Greifswald, Germany.

Small 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.
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http://dx.doi.org/10.1155/2019/7017363DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6348845PMC
March 2019

Highlight Issue: Plasma Medicine.

Biol Chem 2018 12;400(1):1-2

Leibniz Institute for Plasma Science and Technology (INP Greifswald), Felix-Hausdorff-Straße 2, D-17489 Greifswald, Germany.

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http://dx.doi.org/10.1515/hsz-2018-0410DOI Listing
December 2018

Upregulation Is a Mutual Marker in Human Tumor Cells Exposed to Physical Plasma-Derived Oxidants.

Antioxidants (Basel) 2018 Oct 27;7(11). Epub 2018 Oct 27.

ZIK plasmatis, Leibniz Institute for Plasma Science and Technology (INP), Felix-Hausdorff-Str. 2, 17489 Greifswald, Germany.

Increasing numbers of cancer deaths worldwide demand for new treatment avenues. Cold physical plasma is a partially ionized gas expelling a variety of reactive oxygen and nitrogen species, which can be harnesses therapeutically. Plasmas and plasma-treated liquids have antitumor properties in vitro and in vivo. Yet, global response signatures to plasma treatment have not yet been identified. To this end, we screened eight human cancer cell lines to investigate effects of low-dose, tumor-static plasma-treated medium (PTM) on cellular activity, immune-modulatory properties, and transcriptional levels of 22 redox-related genes. With PTM, a moderate reduction of metabolic activity and modest modulation of chemokine/cytokine pattern and markers of immunogenic cell death was observed. Strikingly, the Nuclear factor (erythroid-derived 2)-like 2 () target heme oxygenase 1 () was upregulated in all cell lines 4 h post PTM-treatment. was not changed, but its baseline expression inversely and significantly correlated with expression after exposure to PTM. Besides awarding a central role with plasma-derived oxidants, we present a transcriptional redox map of 22 targets and chemokine/cytokine secretion map of 13 targets across eight different human tumor cell lines of four tumor entities at baseline activity that are useful for future studies in this field.
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http://dx.doi.org/10.3390/antiox7110151DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6262576PMC
October 2018

Redox for Repair: Cold Physical Plasmas and Nrf2 Signaling Promoting Wound Healing.

Antioxidants (Basel) 2018 Oct 19;7(10). Epub 2018 Oct 19.

ZIK-PRE, Leibniz Institute for Plasma Science and Technology (INP Greifswald), Felix-Hausdorff-Str. 2, 17489 Greifswald, Germany.

Chronic wounds and ulcers are major public health threats. Being a substantial burden for patients and health care systems alike, better understanding of wound pathophysiology and new avenues in the therapy of chronic wounds are urgently needed. Cold physical plasmas are particularly effective in promoting wound closure, irrespective of its etiology. These partially ionized gases deliver a therapeutic cocktail of reactive oxygen and nitrogen species safely at body temperature and without genotoxic side effects. This field of plasma medicine reanimates the idea of redox repair in physiological healing. This review compiles previous findings of plasma effects in wound healing. It discusses new links between plasma treatment of cells and tissues, and the perception and intracellular translation of plasma-derived reactive species via redox signaling pathways. Specifically, (i) molecular switches governing redox-mediated tissue response; (ii) the activation of the nuclear E2-related factor (Nrf2) signaling, together with antioxidative and immunomodulatory responses; and (iii) the stabilization of the scaffolding function and actin network in dermal fibroblasts are emphasized in the light of wound healing.
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http://dx.doi.org/10.3390/antiox7100146DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6210784PMC
October 2018

Physical plasma and leukocytes - immune or reactive?

Biol Chem 2018 12;400(1):63-75

Leibniz-Institute for Plasma Science and Technology (INP Greifswald), ZIK plasmatis, Felix-Hausdorff-Str. 2, D-17489 Greifswald, Germany.

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.
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http://dx.doi.org/10.1515/hsz-2018-0224DOI Listing
December 2018

A Neutrophil Proteomic Signature in Surgical Trauma Wounds.

Int J Mol Sci 2018 Mar 7;19(3). Epub 2018 Mar 7.

Leibniz-Institute for Plasma Science and Technology (INP Greifswald), ZIK Plasmatis, Felix-Hausdorff-Str. 2, 17489 Greifswald, Germany.

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.
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http://dx.doi.org/10.3390/ijms19030761DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5877622PMC
March 2018

Human-mediated introduction of introgressed deer across Wallace's line: Historical biogeography of and .

Ecol Evol 2018 02 27;8(3):1465-1479. Epub 2017 Dec 27.

Department of Evolutionary Genetics Leibniz Institute for Zoo and Wildlife Research Berlin Germany.

In this study we compared the phylogeographic patterns of two Rusa species, and , in order to understand what drove and maintained differentiation between these two geographically and genetically close species and investigated the route of introduction of individuals to the islands outside of the Sunda Shelf. We analyzed full mitogenomes from 56 archival samples from the distribution areas of the two species and 18 microsatellite loci in a subset of 16 individuals to generate the phylogeographic patterns of both species. Bayesian inference with fossil calibration was used to estimate the age of each species and major divergence events. Our results indicated that the split between the two species took place during the Pleistocene, ~1.8 Mya, possibly driven by adaptations of to the drier climate found on Java compared to the other islands of Sundaland. Although both markers identified two well-differentiated clades, there was a largely discrepant pattern between mitochondrial and nuclear markers. While nDNA separated the individuals into the two species, largely in agreement with their museum label, mtDNA revealed that all sampled to the east of the Sunda shelf carried haplotypes from and one from South Sumatra carried a haplotype. Our results show that hybridization occurred between these two sister species in Sundaland during the Late Pleistocene and resulted in human-mediated introduction of hybrid descendants in all islands outside Sundaland.
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http://dx.doi.org/10.1002/ece3.3754DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5792523PMC
February 2018

High throughput image cytometry micronucleus assay to investigate the presence or absence of mutagenic effects of cold physical plasma.

Environ Mol Mutagen 2018 05 8;59(4):268-277. Epub 2018 Feb 8.

ZIK plasmatis, Leibniz-Institute for Plasma Science and Technology (INP Greifswald), Greifswald, Germany.

Promising cold physical plasma sources have been developed in the field of plasma medicine. An important prerequisite to their clinical use is lack of genotoxic effects in cells. During optimization of one or even different plasma sources for a specific application, large numbers of samples need to be analyzed. There are soft and easy-to-assess markers for genotoxic stress such as phosphorylation of histone H2AX (γH2AX) but only few tests are accredited by the OECD with regard to mutagenicity detection. The micronucleus (MN) assay is among them but often requires manual counting of many thousands of cells per sample under the microscope. A high-throughput MN assay is presented using image flow cytometry and image analysis software. A human lymphocyte cell line was treated with plasma generated with ten different feed gas conditions corresponding to distinct reactive species patterns that were investigated for their genotoxic potential. Several millions of cells were automatically analyzed by a MN quantification strategy outlined in detail in this work. Our data demonstrates the absence of newly formed MN in any feed gas condition using the atmospheric pressure plasma jet kINPen. As positive control, ionizing radiation gave a significant 5-fold increase in micronucleus frequency. Thus, this assay is suitable to assess the genotoxic potential in large sample sets of cells exposed chemical or physical agents including plasmas in an efficient, reliable, and semiautomated manner. Environ. Mol. Mutagen. 59:268-277, 2018. © 2018 Wiley Periodicals, Inc.
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http://dx.doi.org/10.1002/em.22172DOI Listing
May 2018

Basic Research in Plasma Medicine - A Throughput Approach from Liquids to Cells.

J Vis Exp 2017 11 17(129). Epub 2017 Nov 17.

ZIK plasmatis, Leibniz-Institute for Plasma Science and Technology.

In plasma medicine, ionized gases with temperatures close to that of vertebrate systems are applied to cells and tissues. Cold plasmas generate reactive species known to redox regulate biological processes in health and disease. Pre-clinical and clinical evidence points to beneficial effects of plasma treatment in the healing of chronic ulcer of the skin. Other emerging topics, such as plasma cancer treatment, are receiving increasing attention. Plasma medical research requires interdisciplinary expertise in physics, chemistry, and biomedicine. One goal of plasma research is to characterize plasma-treated cells in a variety of specific applications. This includes, for example, cell count and viability, cellular oxidation, mitochondrial activity, cytotoxicity and mode of cell death, cell cycle analysis, cell surface marker expression, and cytokine release. This study describes the essential equipment and workflows required for such research in plasma biomedicine. It describes the proper operation of an atmospheric pressure argon plasma jet, specifically monitoring its basic emission spectra and feed gas settings to modulate reactive species output. Using a high-precision xyz-table and computer software, the jet is hovered in millisecond-precision over the cavities of 96-well plates in micrometer-precision for maximal reproducibility. Downstream assays for liquid analysis of redox-active molecules are shown, and target cells are plasma-treated. Specifically, melanoma cells are analyzed in an efficient sequence of different consecutive assays but using the same cells: measurement of metabolic activity, total cell area, and surface marker expression of calreticulin, a molecule important for the immunogenic cell death of cancer cells. These assays retrieve content-rich biological information about plasma effects from a single plate. Altogether, this study describes the essential steps and protocols for plasma medical research.
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http://dx.doi.org/10.3791/56331DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5755427PMC
November 2017

Toxicity and Immunogenicity in Murine Melanoma following Exposure to Physical Plasma-Derived Oxidants.

Oxid Med Cell Longev 2017 27;2017:4396467. Epub 2017 Jun 27.

ZIK plasmatis, Leibniz Institute for Plasma Science and Technology (INP Greifswald), Felix-Hausdorff-Str. 2, 17489 Greifswald, Germany.

Metastatic 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.
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http://dx.doi.org/10.1155/2017/4396467DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5518506PMC
April 2018

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

Biophys J 2017 Jun;112(11):2397-2407

Center for Innovation Competence (ZIK) Plasmatis, Greifswald, Germany.

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 (N) to pure oxygen (O) 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. O shielding provided stronger apoptotic effects trough caspase-3 activation compared to N 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 (HO), which was previously shown to be a major player in plasma-cell regulation, emphasizing the role of non-HO 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.
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http://dx.doi.org/10.1016/j.bpj.2017.04.030DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5474686PMC
June 2017

Oxygen atoms are critical in rendering THP-1 leukaemia cells susceptible to cold physical plasma-induced apoptosis.

Sci Rep 2017 06 5;7(1):2791. Epub 2017 Jun 5.

Coupled Plasma-Solid State Systems, Faculty of Physics and Astronomy, Ruhr University Bochum, Bochum, Germany.

Cold physical plasma has been suggested as a powerful new tool in oncology. However, some cancer cells such as THP-1 leukaemia cells have been shown to be resistant towards plasma-induced cell death, thereby serving as a good model for optimizing plasmas in order to foster pro-apoptotic anticancer effects. A helium/oxygen radio frequency driven atmospheric plasma profoundly induced apoptosis in THP-1 cells whereas helium, humidified helium, and humidified helium/oxygen plasmas were inefficient. Hydrogen peroxide - previously shown as central plasma-derived agent - did not participate in the killing reaction but our results suggest hypochlorous acid to be responsible for the effect observed. Proteomic analysis of THP-1 cells exposed to He/O plasma emphasized a prominent growth retardation, cell stress, apoptosis, and a pro-immunogenic profile. Altogether, a plasma setting that inactivates previously unresponsive leukaemia cells is presented. Crucial reactive species in the plasma and liquid environment were identified and discussed, deciphering the complexity of plasma from the gas phase into the liquid down to the cellular response mechanism. These results may help tailoring plasmas for clinical applications such as oxidation-insensitive types of cancer.
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http://dx.doi.org/10.1038/s41598-017-03131-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5459849PMC
June 2017

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

Int J Mol Sci 2017 Apr 19;18(4). Epub 2017 Apr 19.

Leibniz-Institute for Plasma Science and Technology (INP Greifswald), Departments of Plasma Life Science and ZIK Plasmatis, Felix-Hausdorff-Str. 2, 17489 Greifswald, Germany.

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 1β 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 . 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.
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http://dx.doi.org/10.3390/ijms18040868DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5412449PMC
April 2017

Air-liquid interface enhances oxidative phosphorylation in intestinal epithelial cell line IPEC-J2.

Cell Death Discov 2017 27;3:17001. Epub 2017 Feb 27.

Institute of Anatomy , Otto-von-Guericke University, Magdeburg 39120, Germany.

The intestinal porcine epithelial cell line IPEC-J2, cultured under the air-liquid interface (ALI) conditions, develops remarkable morphological characteristics close to intestinal epithelial cells . Improved oxygen availability has been hypothesised to be the leading cause of this morphological differentiation. We assessed oxygen availability in ALI cultures and examined the influence of this cell culture method on glycolysis and oxidative phosphorylation in IPEC-J2 using the submerged membrane culture (SMC) and ALI cultures. Furthermore, the role of HIF-1 as mediator of oxygen availability was analysed. Measurements of oxygen tension confirmed increased oxygen availability at the medium-cell interface and demonstrated reduced oxygen extraction at the basal compartment in ALI. Microarray analysis to determine changes in the genetic profile of IPEC-J2 in ALI identified 2751 modified transcripts. Further examinations of candidate genes revealed reduced levels of glycolytic enzymes hexokinase II and GAPDH, as well as lactate transporting monocarboxylate transporter 1 in ALI, whereas expression of the glucose transporter GLUT1 remained unchanged. Cytochrome oxidase (COX) subunit 5B protein analysis was increased in ALI, although mRNA level remained at constant level. COX activity was assessed using photometric quantification and a three-fold increase was found in ALI. Quantification of glucose and lactate concentrations in cell culture medium revealed significantly reduced glucose levels and decreased lactate production in ALI. In order to evaluate energy metabolism, we measured cellular adenosine triphosphate (ATP) aggregation in homogenised cell suspensions showing similar levels. However, application of the uncoupling agent FCCP reduced ATP levels in ALI but not in SMC. In addition, HIF showed reduced mRNA levels in ALI. Furthermore, HIF-1 protein was reduced in the nuclear compartment of ALI when compared to SCM as confirmed by confocal microscopy. These results indicate a metabolic switch in IPEC-J2 cultured under ALI conditions enhancing oxidative phosphorylation and suppressing glycolysis. ALI-induced improvement of oxygen supply reduced nuclear HIF-1, demonstrating a major change in the transcriptional response.
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http://dx.doi.org/10.1038/cddiscovery.2017.1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5327501PMC
February 2017

Distinct cytokine and chemokine patterns in chronic diabetic ulcers and acute wounds.

Exp Dermatol 2017 02 21;26(2):145-147. Epub 2016 Dec 21.

Leibniz-Institute for Plasma Science and Technology (INP Greifswald), ZIK plasmatis, Greifswald, Germany.

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http://dx.doi.org/10.1111/exd.13215DOI Listing
February 2017