Pulsed Electric Fields Induce STING Palmitoylation and Polymerization Independently of Plasmid DNA Electrotransfer.

Gene therapy approaches may target skeletal muscle due to its high protein-expressing nature and vascularization. Intramuscular plasmid DNA (pDNA) delivery via pulsed electric fields (PEFs) can be termed electroporation or electrotransfer. Nonviral delivery of plasmids to cells and tissues activates DNA-sensing pathways. The central signaling complex in cytosolic DNA sensing is the cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING). The effects of pDNA electrotransfer on the signaling of STING, a key adapter protein, remain incompletely characterized. STING undergoes several post-translational modifications which modulate its function, including palmitoylation. This study demonstrated that in mouse skeletal muscle, STING was constitutively palmitoylated at two sites, while an additional site was modified following electroporation independent of the presence of pDNA. This third palmitoylation site correlated with STING polymerization but not with STING activation. Expression of several palmitoyl acyltransferases, including zinc finger and DHHC motif containing 1 (zDHHC1), coincided with STING activation. Expression of several depalmitoylases, including palmitoyl protein thioesterase 2 (PPT2), was diminished in all PEF application groups. Therefore, STING may not be regulated by active modification by palmitate after electroporation but inversely by the downregulation of palmitate removal. These findings unveil intricate molecular changes induced by PEF application.

Transcriptomic Analysis of the Acute Skeletal Muscle Effects after Intramuscular DNA Electroporation Reveals Inflammatory Signaling.

Skeletal muscle is a promising tissue for therapeutic gene delivery because it is highly vascularized, accessible, and capable of synthesizing protein for therapies or vaccines. The application of electric pulses (electroporation) enhances plasmid DNA delivery and expression by increasing membrane permeability. Four hours after plasmid electroporation, we evaluated acute gene and protein expression changes in mouse skeletal muscle to identify regulated genes and genetic pathways. RNA sequencing followed by functional annotation was used to evaluate differentially expressed mRNAs. Our data highlighted immune signaling pathways that may influence the effectiveness of DNA electroporation. Cytokine and chemokine protein levels in muscle lysates revealed the upregulation of a subset of inflammatory proteins and confirmed the RNA sequencing analysis. Several regulated DNA-specific pattern recognition receptor mRNAs were also detected. Identifying unique molecular changes in the muscle will facilitate a better understanding of the underlying molecular mechanisms and the development of safety biomarkers and novel strategies to improve skeletal muscle targeted gene therapy.

Acute Effects of Intratumor DNA Electrotransfer.

Intratumor therapeutic DNA electroporation or electrotransfer is in clinical trials in the United States and is under development in many other countries. Acute changes in endogenous gene expression in response to DNA or to pulse application may significantly modulate the therapeutic efficacy of the expressed proteins. Oligonucleotide arrays were used in this study to quantify changes in mRNA expression in B16-F10 mouse melanoma tumors four hours after DNA electrotransfer. The data were subjected to the DAVID v6.8 web server for functional annotation to reveal regulated genes and genetic pathways. Gene ontology analysis revealed several molecular functions related to cytoskeletal remodeling and inflammatory signaling. In B16-F10 cells, F-actin remodeling was confirmed by phalloidin staining in cells that received pulse application alone or in the presence of DNA. Chemokine secretion was confirmed in cells receiving DNA electrotransfer. These results indicate that pulse application alone or in the presence of DNA may modulate the therapeutic efficacy of therapeutic DNA electrotransfer.

Monopolar gene electrotransfer enhances plasmid DNA delivery to skin.

A novel monopolar electroporation system and methodologies were developed for in vivo electroporation intended for potential clinical applications such as gene therapy. We hypothesized that an asymmetric anode/cathode electrode applicator geometry could produce favorable electric fields for electroporation, without the typical drawback associated with traditional needle and parallel plate geometries. Three monopolar electrode applicator prototypes were built and tested for gene delivery of reporter genes to the skin in a guinea pig model. Gene expression was evaluated in terms of kinetics over time and expression distribution within the treatment site. Different pulsing parameters, including pulse amplitude, pulse duration, and pulse number were evaluated. Monopolar gene electrotransfer significantly enhanced gene expression compared to controls over the course of 21 days. Gene expression distribution was observed throughout the full thickness of the epidermis, as well as notable expression in the deeper layers of the skin, including the dermis, and the underlying striated muscle without any damage at the treatment site, which is a substantial improvement over previously reported expression confined to the epidermis only. Expression distribution observed is consistent with the electric field distribution model, indicating that our novel electrode geometry results in targeted electroporation and gene transfer. This is important, as it may facilitate translation of many electroporation-based clinical therapies including gene therapies, IRE, and ECT.

Delivery and expression of plasmid DNA into cells by a novel non-thermal plasma source.

Medical applications such as plasma assisted gene transfer is a minimally invasive approach that can substantially reduce potential discomfort of treated area. Atmospheric pressure plasma discharge is an effective approach to deliver plasmid DNA for in vitro and in vivo applications. We investigated plasma assisted delivery in vitro in mouse melanoma cells (B16F10) using a novel surface plasma device, which is operated in air. We evaluated the influence of applied voltage and distance between the surface device and cell monolayer. We found no significant effect on the viability of cells. Highest expression following delivery of a plasmid encoding green fluorescent protein was achieved with an applied voltage of 11.25 kV at a 2 mm distance and 5 s exposure time. To better understand the influence of oxidative damages and stress on cells after plasma delivery, a mRNA expression study was performed. Our results indicated that TNFα mRNA was significantly upregulated. The mRNA response may be attributed to the RONS generated by plasma; however, this mRNA upregulation was not adequate to be reflected in a coordinate protein upregulation. From the results reported here, it is clear that this novel plasma device could be used for plasmid delivery.

Radiation Induced Upregulation of DNA Sensing Pathways is Cell-Type Dependent and Can Mediate the Off-Target Effects.

Irradiation of tumors generates danger signals and inflammatory cytokines that promote the off-target bystander and abscopal effects, evident especially when radiotherapy is administered in combination with the immune checkpoint inhibitors (ICI). The underlying mechanisms are not fully understood; however, cGAS-STING pathway was recognized as the main mediator. In our study, we demonstrate by immunofluorescent staining that tumor cells as well as macrophages, cell types abundant in the tumor microenvironmeent (TME) accumulate DNA in their cytosol soon after irradiation. This accumulation activated several distinct DNA sensing pathways, most prominently activated DNA sensors being DDX60, DAI, and p204 in tumor cells and DDX60, DAI, p204, and RIG-I in macrophages as determined by PCR and immunofluorescence imaging studies. This was accompanied by increased expression of cytokines evaluated by flow cytometry, TNFα, and IFNß in tumor cells and IL1ß and IFNß in macrophages, which can alter the TME and mediate off-target effects (bystander or abscopal effects). These results give insight into the mechanisms involved in the stimulation of antitumor immunity by radiation.

Secretion of proteins and antibody fragments from transiently transfected endothelial progenitor cells.

In neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, multiple sclerosis and amyotrophic lateral sclerosis, neuroinflammation can lead to blood-brain barrier (BBB) breakdown. After intravenous or intra-arterial injection into mice, endothelial progenitor cells (EPCs) home to the damaged BBB to promote neurovascular repair. Autologous EPCs transfected to express specific therapeutic proteins offer an innovative therapeutic option. Here, we demonstrate that EPC transfection by electroporation with plasmids encoding the reporter protein GFP or an anti-ß-amyloid antibody fragment (Fab) leads to secretion of each protein. We also demonstrate the secreted anti-ß-amyloid Fab protein functions in ß-amyloid aggregate solubilization.

Multiple cytosolic DNA sensors bind plasmid DNA after transfection.

Mammalian cells express a variety of nucleic acid sensors as one of the first lines of defense against infection. Despite extensive progress in the study of sensor signaling pathways during the last decade, the detailed mechanisms remain unclear. In our previous studies, we reported increased type I interferon expression and the upregulation of several proposed cytosolic DNA sensors after transfection of several tumor cell types with plasmid DNA (pDNA). In the present study, we sought to reveal the early events in the cytosolic sensing of this nucleic acid in a myoblast cell line. We demonstrated that DNA-dependent activator of interferon regulatory factors/Z-DNA binding protein 1 (DAI/ZBP1) bound plasmid DNA in the cytosol within 15 minutes of transfection and at consistent levels for 4 h. Interferon activated gene 204 protein (p204) and DEAH box helicase 9 (DHX9) also bound pDNA, peaking 15 and 30 min respectively. Plasmid DNA was not detectably bound by DEAD box helicase 60 (DDX60) protein, despite a similar level of mRNA upregulation to DAI/ZBP1, or by cyclic GMP-AMP synthase (cGAS), despite its presence in the cell cytosol. Taken together, these results indicate several DNA sensors may participate and cooperate in the complex process of cytosolic DNA sensing.

Upregulation of DNA Sensors in B16.F10 Melanoma Spheroid Cells After Electrotransfer of pDNA.

Increased expression of cytosolic DNA sensors, a category of pattern recognition receptor, after control plasmid DNA electrotransfer was observed in our previous studies on B16.F10 murine melanoma cells. This expression was correlated with the upregulation of proinflammatory cytokines and chemokines and was associated with cell death. Here, we expanded our research to include the influence of features of cells in a 3-dimensional environment, which better represents the tumors' organization in vivo. Our results show that lower number of cells were transfected in spheroids compared to 2-dimensional cultures, that growth was delayed after electroporation alone or after electrotransfer of plasmid DNA, and that DNA sensors DDX60, DAI/ZBP1, and p204 were upregulated 4 hours and 24 hours after electrotransfer of plasmid DNA. Moreover, the cytokines interferon ß and tumor necrosis factor α were also upregulated but only 4 hours after electrotransfer of plasmid DNA. Thus, our results confirm the results obtained in 2-dimensional cell cultures demonstrating that electrotransfer of plasmid DNA to tumor cells in spheroids also upregulated cytosolic DNA sensors and cytokines.

Tumor cell death after electrotransfer of plasmid DNA is associated with cytosolic DNA sensor upregulation.

Cytosolic DNA sensors are a subgroup of pattern recognition receptors (PRRs) and are activated by the abnormal presence of the DNA in the cytosol. Their activation leads to the upregulation of pro-inflammatory cytokines and chemokines and can also induce cell death. The presence of cytosolic DNA sensors and inflammatory cytokines in TS/A murine mammary adenocarcinoma and WEHI 164 fibrosarcoma cells was demonstrated using real time reverse transcription polymerase chain reaction (RT-PCR), western blotting and enzyme-linked immunosorbent assay (ELISA). After electrotransfer of plasmid DNA (pDNA) using two pulse protocols, the upregulation of DNA-depended activator of interferon regulatory factor or Z-DNA binding protein 1 (DAI/ZBP1), DEAD (Asp-Glu-Ala-Asp) box polypeptide 60 (DDX60) and interferon-inducible protein 204 (p204) mRNAs was observed in both tumor cell lines, but their expression was pulse protocol dependent. A decrease in cell survival was also observed; it was cell type, DNA concentration and pulse protocol dependent. Furthermore, the different protocols of electrotransfer led to different cell death outcomes, necrosis and apoptosis, as indicated by an annexin V and 7AAD assays. The obtained data provide new insights on the presence of cytosolic DNA sensors in tumor cells and the activation of different types of cells death after electrotransfer of pDNA. These observations have important implications on the planning of gene therapy or DNA vaccination protocols.

Electrotransfer of Different Control Plasmids Elicits Different Antitumor Effectiveness in B16.F10 Melanoma.

Several studies have shown that different control plasmids may cause antitumor action in different murine tumor models after gene electrotransfer (GET). Due to the differences in GET protocols, plasmid vectors, and experimental models, the observed antitumor effects were incomparable. Therefore, the current study was conducted comparing antitumor effectiveness of three different control plasmids using the same GET parameters. We followed cytotoxicity in vitro and the antitumor effect in vivo after GET of control plasmids pControl, pENTR/U6 scr and pVAX1 in B16.F10 murine melanoma cells and tumors. Types of cell death and upregulation of selected cytosolic DNA sensors and cytokines were determined. GET of all three plasmids caused significant growth delay in melanoma tumors; nevertheless, the effect of pVAX1 was significantly greater than pControl. While DNA sensors in vivo were not upregulated significantly, cytokines IFN ß and TNF α were upregulated after GET of pVAX1. In vitro, the mRNAs of some cytosolic DNA sensors were overexpressed after GET; however, with no significant difference among the three plasmids. In summary, although differences in antitumor effects were observed among control plasmids in vivo, no differences in cellular responses to plasmid GET were detected in tumor cells in vitro. Thus, the tumor microenvironment as well as some plasmid properties are most probably responsible for the antitumor effectiveness.

Long-term metabolic persistence of gram-positive bacteria on health care-relevant plastic.

BACKGROUND: Health care-associated opportunistic pathogens Staphylococcus aureus and Enterococcus faecium persist on dry environments and can contribute to organism transmission through contact. These organisms can be monitored on surfaces by culture, molecular methods, or metabolic assays. This study was designed to determine the kinetics of bacterial persistence on acrylonitrile butadiene styrene, a plastic commonly used in the manufacture of bedrails. MATERIALS AND METHODS: Polymerase chain reaction for genomic DNA was used to confirm the presence of bacteria cells on this plastic irrespective of viability. Bacterial viability was measured by culture, ATP quantification, and a metabolic assay at time points up to and longer than 1 year. RESULTS: Polymerase chain reaction confirmed the presence of bacteria on the plastic for the entire time period studied. However, S aureus culturability was reduced after 3 and 7 weeks; neither organism was culturable after 1 year. At 7 weeks, ATP levels were reduced for both organisms, paralleling S aureus culturability but indicating that ATP quantification did not predict E faecium culturability. S aureus-reducing potential was reduced after 7 weeks, whereas E faecium-reducing potential reached the level of fresh inoculum after 12 hours in broth culture. Low but significant levels of metabolic activity were detected for each organism after 1 year. CONCLUSIONS: S aureus and E faecium cells may retain viability on plastic for longer than 1 year.

Coordination of different ligands to copper(II) and cobalt(III) metal centers enhances Zika virus and dengue virus loads in both arthropod cells and human keratinocytes.

Trace elements such as copper and cobalt have been associated with virus-host interactions. However, studies to show the effect of conjugation of copper(II) or cobalt(III) metal centers to thiosemicarbazone ligand(s) derived from either food additives or mosquito repellent such as 2-acetylethiazole or citral, respectively, on Zika virus (ZIKV) or dengue virus (serotype 2; DENV2) infections have not been explored. In this study, we show that four compounds comprising of thiosemicarbazone ligand derived from 2-acetylethiazole viz., (E)-N-ethyl-2-[1-(thiazol-2-yl)ethylidene]hydrazinecarbothioamide (acetylethTSC) (compound 1), a copper(II) complex with acetylethTSC as a ligand (compound 2), a thiosemicarbazone ligand-derived from citral (compound 3) and a cobalt(III) complex with a citral-thiosemicarbazone ligand (compound 4) increased DENV2 and ZIKV replication in both mosquito C6/36 cells and human keratinocytes (HaCaT cells). Treatment of both cell lines with compounds 2 or 4 showed increased dengue viral titers at all three tested doses. Enhanced dengue viral plaque formation was also noted at the tested dose of 100µM, suggesting higher production of infectious viral particles. Treatment with the compounds 2 or 4 enhanced ZIKV and DENV2 RNA levels in HeLa cell line and primary cultures of mouse bone marrow derived dendritic cells. Also, pre- or post treatments with conjugated compounds 2 or 4 showed higher loads of ZIKV or DENV2 envelope (E) protein in HaCaT cells. No changes in loads of E-protein were found in ZIKV-infected C6/36 cells, when compounds were treated after infection. In addition, we tested bis(1,10-phenanthroline)copper(II) chloride ([Cu(phen)2]Cl2, (compound 5) and tris(1,10-phenanthroline)cobalt(III) chloride ([Co(phen)3]Cl3, (compound 6) that also showed enhanced DENV2 loads. Also, we found that copper(II) chloride dehydrate (CuCl2·2H2O) or cobalt(II) chloride hexahydrate (CoCl2·6H2O) alone had no effects as "free" cations. Taken together, these findings suggest that use of Cu(II) or Co(III) conjugation to organic compounds, in insect repellents and/or food additives could enhance DENV2/ZIKV loads in human cells and perhaps induce pathogenesis in infected individuals or individuals pre-exposed to such conjugated complexes. IMPORTANCE: Mosquito-borne diseases are of great concern to the mankind. Use of chemicals/repellents against mosquito bites and transmission of microbes has been the topic of interest for many years. Here, we show that thiosemicarbazone ligand(s) derived from 2-acetylethiazole or citral or 1,10-phenanthroline upon conjugation with copper(II) or cobalt(III) metal centers enhances dengue virus (serotype 2; DENV2) and/or Zika virus (ZIKV) infections in mosquito, mouse and human cells. Enhanced ZIKV/DENV2 capsid mRNA or envelope protein loads were evident in mosquito cells and human keratinocytes, when treated with compounds before/after infections. Also, treatment with copper(II) or cobalt(III) conjugated compounds increased viral titers and number of plaque formations. These studies suggest that conjugation of compounds in repellents/essential oils/natural products/food additives with copper(II) or cobalt(III) metal centers may not be safe, especially in tropical and subtropical places, where several dengue infection cases and deaths are reported annually or in places with increased ZIKV caused microcephaly.

Electrosensitization Increases Antitumor Effectiveness of Nanosecond Pulsed Electric Fields In Vivo.

Nanosecond pulsed electric fields are emerging as a new modality for tissue and tumor ablation. We previously reported that cells exposed to pulsed electric fields develop hypersensitivity to subsequent pulsed electric field applications. This phenomenon, named electrosensitization, is evoked by splitting the pulsed electric field treatment in fractions (split-dose treatments) and causes in vitro a 2- to 3-fold increase in cytotoxicity. The aim of this study was to show the benefit of split-dose treatments for in vivo tumor ablation by nanosecond pulsed electric field. KLN 205 squamous carcinoma cells were embedded in an agarose gel or grown subcutaneously as tumors in mice. Nanosecond pulsed electric field ablations were produced using a 2-needle probe with a 6.5-mm interelectrode distance. In agarose gel, splitting a pulsed electric field dose of 300, 300-ns pulses (20 Hz, 4.4-6.4 kV) in 2 equal fractions increased cell death up to 3-fold compared to single-train treatments. We then compared the antitumor effectiveness of these treatments in vivo. At 24 hours after treatment, sensitizing tumors by a split-dose pulsed electric field exposure (150 + 150, 300-ns pulses, 20 Hz, 6.4 kV) caused a 4- and 2-fold tumor volume reduction as compared to sham and single-train treatments, respectively. Tumor volume reduction that exceeds 75% was 43% for split-dose-treated animals compared to only 12% for single-dose treatments. The difference between the 2 experimental groups remained statistically significant for at least 1 week after the treatment. The results show that electrosensitization occurs in vivo and can be exploited to assist in vivo cancer ablation.

Cytosolic DNA Sensor Upregulation Accompanies DNA Electrotransfer in B16.F10 Melanoma Cells.

In several preclinical tumor models, antitumor effects occur after intratumoral electroporation, also known as electrotransfer, of plasmid DNA devoid of a therapeutic gene. In mouse melanomas, these effects are preceded by significant elevation of several proinflammatory cytokines. These observations implicate the binding and activation of intracellular DNA-specific pattern recognition receptors or DNA sensors in response to DNA electrotransfer. In tumors, IFNß mRNA and protein levels significantly increased. The mRNAs of several DNA sensors were detected, and DAI, DDX60, and p204 tended to be upregulated. These effects were accompanied with reduced tumor growth and increased tumor necrosis. In B16.F10 cells in culture, IFNß mRNA and protein levels were significantly upregulated. The mRNAs for several DNA sensors were present in these cells; DNA-dependent activator of interferon regulatory factor (DAI), DEAD (Asp-Glu-Ala-Asp) box polypeptide 60 (DDX60), and p204 were significantly upregulated while DDX60 protein levels were coordinately upregulated. Upregulation of DNA sensors in tumors could be masked by the lower transfection efficiency compared to in vitro or to dilution by other tumor cell types. Mirroring the observation of tumor necrosis, cells underwent a significant DNA concentration-dependent decrease in proliferation and survival. Taken together, these results indicate that DNA electrotransfer may cause the upregulation of several intracellular DNA sensors in B16.F10 cells, inducing effects in vitro and potentially in vivo.

Plasma-activated air mediates plasmid DNA delivery in vivo.

Plasma-activated air (PAA) provides a noncontact DNA transfer platform. In the current study, PAA was used for the delivery of plasmid DNA in a 3D human skin model, as well as in vivo. Delivery of plasmid DNA encoding luciferase to recellularized dermal constructs was enhanced, resulting in a fourfold increase in luciferase expression over 120 hours compared to injection only (P < 0.05). Delivery of plasmid DNA encoding green fluorescent protein (GFP) was confirmed in the epidermal layers of the construct. In vivo experiments were performed in BALB/c mice, with skin as the delivery target. PAA exposure significantly enhanced luciferase expression levels 460-fold in exposed sites compared to levels obtained from the injection of plasmid DNA alone (P < 0.001). Expression levels were enhanced when the plasma reactor was positioned more distant from the injection site. Delivery of plasmid DNA encoding GFP to mouse skin was confirmed by immunostaining, where a 3-minute exposure at a 10 mm distance displayed delivery distribution deep within the dermal layers compared to an exposure at 3 mm where GFP expression was localized within the epidermis. Our findings suggest PAA-mediated delivery warrants further exploration as an alternative approach for DNA transfer for skin targets.

Donor platelet plasma components inactivate sensitive and multidrug resistant Acinetobacter baumannii isolates.

Acinetobacter baumannii is an environmentally resilient healthcare-associated opportunistic pathogen responsible for infections at many body sites. In the last 10 years, clinical strains resistant to many or all commonly used antibiotics have emerged globally. With few antimicrobial agents in the pharmaceutical pipeline, new and alternative agents are essential. Platelets secrete a large number of proteins, including proteins with antimicrobial activity. In a previous study, we demonstrated that donor platelet supernatants and plasma significantly inhibited the growth of a reference strain of A. baumannii in broth and on skin. This inhibition appeared to be unrelated to the platelet activation state. In this study, we demonstrate that this growth inhibition extends to clinical multidrug resistant isolates. We also demonstrate that there is no relationship between this activity and selected platelet-derived antimicrobial proteins. Instead, the donor plasma components complement and alpha-2 macroglobulin are implicated.

Activated air produced by shielded sliding discharge plasma mediates plasmid DNA delivery to mammalian cells.

Cold plasma is emerging as a potential method for medical applications. The current study assessed the efficacy of a novel cold plasma reactor based on shielded sliding discharge producing cathode-directed streamers generated in ambient air for the delivery of plasmid DNA. Experiments were performed with mouse melanoma cells (B16F10) and human keratinocyte cells (HaCaT) inoculated with plasmid DNA encoding luciferase. Quantitative results measured over a 72-h period displayed luciferase expression levels as high as 5-fold greater in cells exposed to plasma-activated air (PAA) than levels obtained from the inoculation of plasmid DNA alone (P < 0.05, P < 0.01). No effect on cell viability was observed. Delivery of plasmid encoding GFP to HaCaT cells seeded on polycaprolactone (PCL) scaffolds was confirmed by immunostaining. The use of cold plasma for DNA delivery is attractive as it provides a non-viral, non-invasive method where the electrode or the plasma itself never directly contacts the exposed site. The current device design provides localized DNA transfer using a novel technology. Our report suggests PAA warrants further exploration as an alternative or supplemental approach for DNA transfer.

Gene electrotransfer clinical trials.

Plasmid or non-viral gene therapy offers an alternative to classic viral gene delivery that negates the need for a biological vector. In this case, delivery is enhanced by a variety of approaches including lipid or polymer conjugation, particle-mediated delivery, hydrodynamic delivery, ultrasound or electroporation. Electroporation was originally used as a laboratory tool to deliver DNA to bacterial and mammalian cells in culture. Electrode development allowed this technique to be modified for in vivo use. After preclinical therapeutic studies, clinical delivery of cell impermeant chemotherapeutic agents progressed to clinical delivery of plasmid DNA. One huge benefit of this delivery technique is its malleability. The pulse protocol used for plasmid delivery can be fine-tuned to control the levels and duration of subsequent transgene expression. This fine-tuning allows transgene expression to be tailored to each therapeutic application. Effective and appropriate expression induces the desired clinical response that is a critical component for any gene therapy. This chapter focuses on clinical trials using in vivo electroporation or electrotransfer as a plasmid delivery method. The first clinical trial was initiated in 2004, and now more than fifty trials use electric fields for gene delivery. Safety and tolerability has been demonstrated by several groups, and early clinical efficacy results are promising in both cancer therapeutic and infectious disease vaccine applications.

Surface-dependent inactivation of model microorganisms with shielded sliding plasma discharges and applied air flow.

Cold atmospheric plasma inactivates bacteria through reactive species produced from the applied gas. The use of cold plasma clinically has gained recent interest, as the need for alternative or supplementary strategies are necessary for preventing multi-drug resistant infections. The purpose of this study was to evaluate the antibacterial efficacy of a novel shielded sliding discharge based cold plasma reactor operated by nanosecond voltage pulses in atmospheric air on both biotic and inanimate surfaces. Bacterial inactivation was determined by direct quantification of colony forming units. The plasma activated air (afterglow) was bactericidal against Escherichia coli and Staphylococcus epidermidis seeded on culture media, laminate, and linoleum vinyl. In general, E. coli was more susceptible to plasma exposure. A bacterial reduction was observed with the application of air alone on a laminate surface. Whole-cell real-time PCR revealed a decrease in the presence of E. coli genomic DNA on exposed samples. These findings suggest that plasma-induced bacterial inactivation is surface-dependent.