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3. Markers of tumor-associated macrophages and microglia exhibit high intratumoral heterogeneity in human glioblastoma tissue.

Author

Ispirjan, Mikael, Marx, Sascha, Freund, Eric, Fleck, Steffen K., Baldauf, Joerg, Roessler, Karl, Schroeder, Henry W.S., and Bekeschus, Sander

Subjects
BIOMARKERS, TUMOR markers, GLIOBLASTOMA multiforme, BRAIN cancer, TUMOR microenvironment
Abstract

Background: Human glioblastoma multiforme (GBM) is a highly aggressive tumor with insufficient therapies available. Especially, novel concepts of immune therapies fail due to a complex immunosuppressive microenvironment, high mutational rates, and inter-patient variations. The intratumoral heterogeneity is currently not sufficiently investigated. Methods: Biopsies from six different locations were taken in a cohort of 16 GBM patients who underwent surgery. The tissue slides were analyzed utilizing high-content imaging microscopy and algorithm-based image quantification. Several immune markers for macrophage and microglia subpopulations were investigated. Flow cytometry was used to validate key results. Besides the surface marker, cytokines were measured and categorized based on their heterogenicity and overall expression. Results: M2-like antigens, including CD204, CD163, Arg1, and CSF1R, showed comparatively higher expression, with GFAP displaying the least intratumoral heterogeneity. In contrast, anti-tumor-macrophage-like antigens, such as PSGL-1, CD16, CD68, and MHC-II, exhibited low overall expression and concurrent high intratumoral heterogeneity. CD16 and PSGL-1 were the most heterogeneous antigens. High expression levels were observed for cytokines IL-6, VEGF, and CCL-2. VILIP-a was revealed to differentiate most in principle component analysis. Cytokines with the lowest overall expression, such as TGF-β1, β-NGF, TNF-α, and TREM1, showed low intratumoral heterogeneity, in contrast to βNGF, TNF-α, and IL-18, which displayed high heterogeneity despite low expression. Conclusion: The study showed high intratumoral heterogeneity in GBM, emphasizing the need for a more detailed understanding of the tumor microenvironment. The described findings could be essential for future personalized treatment strategies and the implementation of reliable diagnostics in GBM. [ABSTRACT FROM AUTHOR]

Published
2024
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5. Oxidized Melanoma Antigens Promote Activation and Proliferation of Cytotoxic T‐Cell Subpopulations.

Author

Clemen, Ramona, Miebach, Lea, Singer, Debora, Freund, Eric, von Woedtke, Thomas, Weltmann, Klaus‐Dieter, and Bekeschus, Sander

Subjects
REACTIVE oxygen species, LOW temperature plasmas, COLD gases, PLASMA gases, REACTIVE nitrogen species
Abstract

Increasing evidence suggests the role of reactive oxygen and nitrogen species (RONS) in regulating antitumor immune effects and immunosuppression. RONS modify biomolecules and induce oxidative post‐translational modifications (oxPTM) on proteins that can alarm phagocytes. However, it is unclear if and how protein oxidation by technical means could be a strategy to foster antitumor immunity and therapy. To this end, cold gas plasma technology producing various RONS simultaneously to oxidize the two melanoma‐associated antigens MART and PMEL is utilized. Cold plasma‐oxidized MART (oxMART) and PMEL (oxPMEL) are heavily decorated with oxPTMs as determined by mass spectrometry. Immunization with oxidized MART or PMEL vaccines prior to challenge with viable melanoma cells correlated with significant changes in cytokine secretion and altered T‐cell differentiation of tumor‐infiltrated leukocytes (TILs). oxMART promoted the activity of cytotoxic central memory T‐cells, while oxPMEL led to increased proliferation of cytotoxic effector T‐cells. Similar T‐cell results are observed after incubating splenocytes of tumor‐bearing mice with B16F10 melanoma cells. This study, for the first time, provides evidence of the importance of oxidative modifications of two melanoma‐associated antigens in eliciting anticancer immunity. [ABSTRACT FROM AUTHOR]

Published
2024
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13. Large volume spark discharge and plasma jet-technology for generating plasma-oxidized saline targeting colon cancer in vitro and in vivo.

Author

Freund, Eric, Miebach, Lea, Clemen, Ramona, Schmidt, Michael, Heidecke, Amanda, von Woedtke, Thomas, Weltmann, Klaus-Dieter, Kersting, Stephan, and Bekeschus, Sander

Subjects
*PLASMA flow, *COLON cancer, *PLASMA jets, *PERITONEAL cancer, *LOW temperature plasmas
Abstract

Cold physical plasma is a new anticancer agent. Various prototypes and a few certified devices have been developed during recent years. However, the large number of metastases disseminated into the peritoneal cavity is challenging to target directly with this emerging technology. Plasma-oxidized liquids containing tumor-toxic reactive species, in turn, are promising adjuvants in chemotherapeutic regimens. Yet, the generation of larger volumes of such liquid currently hinders its further exploration for practical application. In this study, the anticancer effect of oxidized saline generated using a spark discharge was investigated using CT26 colorectal cancer cells in vitro and in vivo and compared against oxidized saline generated by the well-investigated medical plasma jet kINPen. The reactive species deposition, as well as the storability of the liquid, was investigated. In a murine, syngeneic, orthotopic model of colorectal peritoneal carcinomatosis, the spark discharge-oxidized saline reduced the tumor burden to a lower extent when compared to the kINPen. However, and despite the lower therapeutic efficacy, the former had profound immunomodulatory effects on the levels of several peritoneal cytokines, as indicated by the marked decrease of interferon-γ, interleukin (IL)-2, IL-6, IL-10, and IL-17F. The effect observed with the kINPen on these mostly T-cell-related cytokines was less pronounced. This suggests that the spark discharge-oxidized saline unintendedly decreased the T-cell activity, presumably because of its low pH (2.7). Collectively, our results are vital in understanding the plasma-liquid-chemistry for use in oncology and will help design more optimal liquids for further exploration in research and future surgical applications. [ABSTRACT FROM AUTHOR]

Published
2021
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15. Immunophenotyping of Circulating and Intratumoral Myeloid and T Cells in Glioblastoma Patients

Author

Marx, Sascha, primary, Wilken, Fabian, additional, Miebach, Lea, additional, Ispirjan, Mikael, additional, Kinnen, Frederik, additional, Paul, Sebastian, additional, Bien-Möller, Sandra, additional, Freund, Eric, additional, Baldauf, Jörg, additional, Fleck, Steffen, additional, Siebert, Nikolai, additional, Lode, Holger, additional, Stahl, Andreas, additional, Rauch, Bernhard H., additional, Singer, Stephan, additional, Ritter, Christoph, additional, Schroeder, Henry W. S., additional, and Bekeschus, Sander, additional

Published
2022
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18. Cyclic Response of Shear Transfer Connections Between Shearwalls and Diaphragms in Woodframe Construction

Author

Ficcadenti, Seb J, Freund, Eric J, Pardoen, Gerard C, and Kazanjy, Robert P

Subjects
Blocked Diaphragm, FC, FN, TN, Unblocked Diaphragm, Toe Nailing, Framing Clamps, Flange Nailing
Abstract

The stiffness of a shearwall in woodframe construction is a function of its sheathing, the sliding and overturning anchorage at its base, and the shear transfer connection at the top of the wall. While one can find a number of studies conducted on the sheathing and anchorage components of shearwalls in resisting seismic forces, studies conducted on shear transfer components are notably scarce. Recent work on the connections between roof rafters and wall top plates has focused on the resistance to uplift loads induced by wind forces. The experimental program documented in this report focuses specifically on diaphragm to shearwall connections designed to resist seismic forces. Nine diaphragm to shearwall connection configurations were tested under cyclic loads using the shake table in the Structural Engineering Test Hall at the University of California, Irvine as a large actuator. Using the shake table to load the test specimens allowed the application of the load to be consistent with how it would be applied in an actual earthquake. Three shear transfer connection types were investigated, each in three different framing configurations. The three connection types were (TN) toe nailing of framing members to top plates, (FC) fastening framing clips to framing members and top plates, and (FN) flange nailing of framing members to top plates. The three framing configuration used with each connection type were (1) unblocked diaphragms supported by joist framing parallel to the shearwall, (2) unblocked diaphragms supported by joist framing perpendicular to the shearwall, and (3) blocked diaphragms supported by joist framing perpendicular to the shearwall. For each set of tests for a given connector type, two test specimens were tested for each unblocked diaphragm configuration and one additional test specimen was tested for the blocked diaphragm configuration. All together cyclic tests were conducted on 15 test specimens.

Published
2022
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20. Gas Plasma Exposure of Glioblastoma Is Cytotoxic and Immunomodulatory in Patient-Derived GBM Tissue

Author

Bekeschus, Sander, primary, Ispirjan, Mikael, additional, Freund, Eric, additional, Kinnen, Frederik, additional, Moritz, Juliane, additional, Saadati, Fariba, additional, Eckroth, Jacqueline, additional, Singer, Debora, additional, Stope, Matthias B., additional, Wende, Kristian, additional, Ritter, Christoph A., additional, Schroeder, Henry W. S., additional, and Marx, Sascha, additional

Published
2022
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23. The Anticancer Efficacy of Plasma-Oxidized Saline (POS) in the Ehrlich Ascites Carcinoma Model In Vitro and In Vivo

Author

Brito, Walison Augusto Silva, primary, Freund, Eric, additional, Nascimento, Thiago Daniel Henrique do, additional, Pasqual-Melo, Gabriella, additional, Sanches, Larissa Juliani, additional, Dionísio, Joyce Hellen Ribeiro, additional, Fumegali, William Capellari, additional, Miebach, Lea, additional, Cecchini, Alessandra Lourenço, additional, and Bekeschus, Sander, additional

Published
2021
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24. Inactivation of Cerebral Cavernous Malformation Genes Results in Accumulation of von Willebrand Factor and Redistribution of Weibel-Palade Bodies in Endothelial Cells

Author

Much, Christiane D., primary, Sendtner, Barbara S., additional, Schwefel, Konrad, additional, Freund, Eric, additional, Bekeschus, Sander, additional, Otto, Oliver, additional, Pagenstecher, Axel, additional, Felbor, Ute, additional, Rath, Matthias, additional, and Spiegler, Stefanie, additional

Published
2021
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25. Characterization of Antimicrobial Effects of Plasma‑Treated Water (PTW) Produced by Microwave‑Induced Plasma (MidiPLexc) on Pseudomonas fluorescens Biofilms

Author

Handorf, Oliver, Pauker, Viktoria Isabella, Schnabel, Uta, Weihe, Thomas, Freund, Eric, Bekeschus, Sander, Riedel, Katharina, and Ehlbeck, Jörg

Subjects
biology_other
Abstract

For the decontamination of surfaces in the food production industry, plasma generated compounds (PGCs) such as plasma-treated water (PTW) or plasma processed air (PPA) offer many promising possibilities for future applications. Therefore, the antimicrobial effect of water treated with microwave-induced plasma (MidiPLexc) on Pseudomonas fluorescens biofilms was investigated. 10 ml deionized water was treated with the MidiPLexc plasma source for 100 s, 300 s and 900 s (pre-treatment time) and the bacterial biofilms were exposed to the PTW for 1 min, 3 min and 5 min (post treatment time). To investigate the influence of PTW on P. fluorescens biofilms, microbiological assays (CFU, fluorescence and XTT assay) and imaging techniques (fluorescence microscopy, confocal laser scanning microscopy (CLSM), and atomic force microscopy (AFM)) were used. The CFU showed a maximum reduction of 6 log10 by using 300 s pre-treated PTW for 5 min. Additionally, a maximum reduction of 81 % for the viability of the cells and a 92 % reduction in the metabolic activity of the cells was achieved by using 900 s pre-treated PTW for 5 min. The microscopic images showed evident microbial inactivation within the biofilm even at the shortest pre treatment (100 s) and post-treatment (1 min) times. Moreover, reduction of the biofilm thickness and increased cluster formation within the biofilm was detected. Morphologically, the fusion of cell walls into a uniform dense cell mass. The findings correlated with a decrease in the pH value of the PTW, which forms the basis for the chemically active components of PTW and its antimicrobial effects. These results, provide valuable insights into the mechanisms of inactivation of biofilms by plasma generated compounds (PGCs) such as PTW and thus allow for further parameter adjustment for applications in food industry.

Published
2020

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

Author

Clemen, Ramona, Freund, Eric, Mrochen, Daniel, Miebach, Lea, Schmidt, Anke, Rauch, Bernhard H., Lackmann, Jan-Wilm, Martens, Ulrike, Wende, Kristian, Lalk, Michael, Delcea, Mihaela, Broker, Barbara M., Bekeschus, Sander, Clemen, Ramona, Freund, Eric, Mrochen, Daniel, Miebach, Lea, Schmidt, Anke, Rauch, Bernhard H., Lackmann, Jan-Wilm, Martens, Ulrike, Wende, Kristian, Lalk, Michael, Delcea, Mihaela, Broker, Barbara M., and Bekeschus, Sander

Abstract

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.

Published
2021

27. Non-thermal plasma modulates cellular markers associated with immunogenicity in a model of latent HIV-1 infection

Author

Mohamed, Hager, Clemen, Ramona, Freund, Eric, Lackmann, Jan-Wilm, Wende, Kristian, Connors, Jennifer, Haddad, Elias K., Dampier, Will, Wigdahl, Brian, Miller, Vandana, Bekeschus, Sander, Krebs, Fred C., Mohamed, Hager, Clemen, Ramona, Freund, Eric, Lackmann, Jan-Wilm, Wende, Kristian, Connors, Jennifer, Haddad, Elias K., Dampier, Will, Wigdahl, Brian, Miller, Vandana, Bekeschus, Sander, and Krebs, Fred C.

Abstract

Effective control of infection by human immunodeficiency virus type 1 (HIV-1), the causative agent of the acquired immunodeficiency syndrome (AIDS), requires continuous and life-long use of anti-retroviral therapy (ART) by people living with HIV-1 (PLWH). In the absence of ART, HIV-1 reemergence from latently infected cells is ineffectively suppressed due to suboptimal innate and cytotoxic T lymphocyte responses. However, ART-free control of HIV-1 infection may be possible if the inherent immunological deficiencies can be reversed or restored. Herein we present a novel approach for modulating the immune response to HIV-1 that involves the use of non-thermal plasma (NTP), which is an ionized gas containing various reactive oxygen and nitrogen species (RONS). J-Lat cells were used as a model of latent HIV-1 infection to assess the effects of NTP application on viral latency and the expression of pro-phagocytic and pro-chemotactic damage-associated molecular patterns (DAMPs). Exposure of J-Lat cells to NTP resulted in stimulation of HIV-1 gene expression, indicating a role in latency reversal, a necessary first step in inducing adaptive immune responses to viral antigens. This was accompanied by the release of pro-inflammatory cytokines and chemokines including interleukin-1 beta (IL-1 beta) and interferon-gamma (IFN-gamma); the display of pro-phagocytic markers calreticulin (CRT), heat shock proteins (HSP) 70 and 90; and a correlated increase in macrophage phagocytosis of NTP-exposed J-Lat cells. In addition, modulation of surface molecules that promote or inhibit antigen presentation was also observed, along with an altered array of displayed peptides on MHC I, further suggesting methods by which NTP may modify recognition and targeting of cells in latent HIV-1 infection. These studies represent early progress toward an effective NTP-based ex vivo immunotherapy to resolve the dysfunctions of the immune system that enable HIV-1 persistence in PLWH.

Published
2021

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

Author

Clemen, Ramona, primary, Freund, Eric, additional, Mrochen, Daniel, additional, Miebach, Lea, additional, Schmidt, Anke, additional, Rauch, Bernhard H., additional, Lackmann, Jan‐Wilm, additional, Martens, Ulrike, additional, Wende, Kristian, additional, Lalk, Michael, additional, Delcea, Mihaela, additional, Bröker, Barbara M., additional, and Bekeschus, Sander, additional

Published
2021
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34. Non-thermal plasma modulates cellular markers associated with immunogenicity in a model of latent HIV-1 infection

Author

Mohamed, Hager, primary, Clemen, Ramona, additional, Freund, Eric, additional, Lackmann, Jan-Wilm, additional, Wende, Kristian, additional, Connors, Jennifer, additional, Haddad, Elias K., additional, Dampier, Will, additional, Wigdahl, Brian, additional, Miller, Vandana, additional, Bekeschus, Sander, additional, and Krebs, Fred C., additional

Published
2021
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36. Effects of cold physical plasma on oral lichen planus: An in vitro study ( Effects of CAP on OLP )

Author

Seebauer, Christian, primary, Freund, Eric, additional, Hasse, Sybille, additional, Miller, Vandana, additional, Segebarth, Maria, additional, Lucas, Christian, additional, Kindler, Stefan, additional, Dieke, Tobias, additional, Metelmann, Hans‐Robert, additional, Daeschlein, Georg, additional, Jesse, Katja, additional, Weltmann, Klaus‐Dieter, additional, and Bekeschus, Sander, additional

Published
2020
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37. Risk Evaluation of EMT and Inflammation in Metastatic Pancreatic Cancer Cells Following Plasma Treatment

Author

Freund, Eric, primary, Spadola, Chiara, additional, Schmidt, Anke, additional, Privat-Maldonado, Angela, additional, Bogaerts, Annemie, additional, von Woedtke, Thomas, additional, Weltmann, Klaus-Dieter, additional, Heidecke, Claus-Dieter, additional, Partecke, Lars-Ivo, additional, Käding, André, additional, and Bekeschus, Sander, additional

Published
2020
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43. Expression of TGF-beta-S and their receptors during implantation and organogenesis of the mouse embryo

Author

Roelen, Bernard A.J., Lin, Herbert Y., Knezevic, Vladimir, Freund, Eric, and Mummery, Christine L.

Subjects
Transforming growth factors -- Research, Embryology -- Research, Biological sciences
Abstract

In site hybridization, reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemistry help study the temporal and spatial expression of the types I, II and III receptors of transforming growth factor beta (TGF-beta) and their TGF-beta1 and TGF-beta2 ligands in mouse growth after implantation. RT-PCR reveals the lack of signaling type II and ALK-5 type I receptors in the germ layers and the differential expression of the type III and Tsk 7L type I receptors in 6.0 to 7.5 days postcoitum (dpc) embryos. TGF-beta type II receptor is expressed during late developmental stages in all differentiating tissues and organs except the brain and neural tube.

Published
1994

44. Effects of cold physical plasma on oral lichen planus: An in vitro study (Effects of CAP on OLP).

Author

Seebauer, Christian, Freund, Eric, Hasse, Sybille, Miller, Vandana, Segebarth, Maria, Lucas, Christian, Kindler, Stefan, Dieke, Tobias, Metelmann, Hans‐Robert, Daeschlein, Georg, Jesse, Katja, Weltmann, Klaus‐Dieter, and Bekeschus, Sander

Subjects
*CYTOKINES, *INTERLEUKINS, *GRANULOCYTE-macrophage colony-stimulating factor, *IN vitro studies, *ORAL lichen planus, *MUCOUS membranes, *COMPARATIVE studies, *PLASMA gases, *T cells, *CHEMOKINES
Abstract

Objectives: In the search for more effective and safe treatment avenues, we investigated cold physical plasma as a new treatment modality for therapy of oral lichen planus (OLP). Material and Methods: Healthy and diseased human mucosal tissue samples with a size of 3 mm in diameter obtained from OLP patients were subjected to plasma treatment ex vivo or were left untreated. Tissue sections were quantified for immune‐infiltration of CD4+, CD8+, CD45RA+, and CD45R0+ T cells. Moreover, the tissues' inflammatory profile was assessed by analyzing 12 different cytokines in the surrounding media. Results: A significantly increased infiltrate of CD8+ and CD45‐R0+ T cells was detected in OLP tissue samples when compared to healthy tissue. A higher concentration of interleukin (IL) 1β, IL6, IL8, and granulocyte macrophage‐colony stimulating factor (GM‐CMF) was detected in OLP samples compared to healthy mucosal tissue. For all cytokines and chemokines investigated, 23 out of 24 comparisons showed a decrease in tendency (significant for IL1β, IL2, IL10, and GM‐CSF) in response to plasma treatment. In ex vivo‐treated tissue, a decrease of T‐cell infiltrate in OLP lesions compared with healthy tissue was observed. Conclusion: Our findings suggest cold physical plasma can be a promising therapeutic option for OLP that requires further validation in vivo. [ABSTRACT FROM AUTHOR]

Published
2021
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48. Killing melanoma cells with ROS provides immuno-protection against tumor growth in vivo

Author

Freund, Eric, Clemen, Ramona, Gandhirajan, Rajesh Kumar, and Bekeschus, Sander

Abstract

Purpose/Objectives: Immunogenic cancer cell death (ICD) delivers inflammatory stimuli to elicit anti-tumor immune responses. Release of adjuvant find-me (HMGB1, ATP) and eat-me (calreticulin; CRT) signals enables DC maturation and cross presentation of tumor antigen to T cells. Effective ICD-inducing therapies are drugs such as anthracyclines, ionizing irradiation, or photodynamic therapies. Intriguingly, these antitumor strategies show concomitant generation of reactive oxygen species (ROS) that can drive cell death and immunogenicity signaling responses through redox relays. In cancer cells, the importance of ROS are underappreciated in the onset of ICD. Materials/methods: A novel technology, cold physical plasma, was used to generate various ROS that can be effectively delivered to B16F10 melanoma cells. The oxidation and ATP levels as well as expression levels of MHC class I and CRT were measured. Plasma-treated tumor cells were injected into syngeneic mice with a subsequent re-challenge with live cells. Mitomycin C (MMC) and mitoxantrone (MTX) served as negative and positive control, respectively. Lymph node cells of sacrificed mice were co-cultured with melanoma cells, and T cell activation was assessed. Results: Cold physical plasma oxidized melanoma cells and led to an increase of ATP release as well as MHC class I and CRT expression. Mice “vaccinated” with plasma-treated melanoma cells showed less tumor growth compared to mice receiving MMC-treated cells but more tumor growth compared to positive control MTX. Activation of lymph node-derived T cells ex vivo with melanoma cells was best in MTX mice, followed by the cells from the plasma group, and was lowest in cells from the MMC group. Conclusion: Not only the event of cell death but also its auxiliary signals are important in the formation of anti-tumor immune responses. ROS play a significant role in this process. Deep phenotyping of ROS-treated melanoma cells will decipher the molecular machinery important in eliciting the responses observed., Proceedings on Cancer-Immuno-Oncology, Vol 1 No 1 (2018)

Published
2018
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50. Risk Assessment of kINPen Plasma Treatment of Four Human Pancreatic Cancer Cell Lines with Respect to Metastasis

Author

Bekeschus, Sander, primary, Freund, Eric, additional, Spadola, Chiara, additional, Privat-Maldonado, Angela, additional, Hackbarth, Christine, additional, Bogaerts, Annemie, additional, Schmidt, Anke, additional, Wende, Kristian, additional, Weltmann, Klaus-Dieter, additional, von Woedtke, Thomas, additional, Heidecke, Claus-Dieter, additional, Partecke, Lars-Ivo, additional, and Käding, André, additional

Published
2019
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