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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.
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Citosol/metabolismo , Proteínas de Ligação a DNA/genética , DNA/genética , Interferon Tipo I/genética , Animais , Linhagem Celular , RNA Helicases DEAD-box/genética , Regulação da Expressão Gênica/genética , Humanos , Camundongos , Proteínas Nucleares/genética , Fosfoproteínas/genética , Plasmídeos/genética , RNA Mensageiro/genética , Proteínas de Ligação a RNA/genética , Transdução de Sinais/genética , TransfecçãoRESUMO
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.
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Células Progenitoras Endoteliais/metabolismo , Fragmentos de Imunoglobulinas/biossíntese , Fragmentos de Imunoglobulinas/genética , Biossíntese de Proteínas , Proteínas/genética , Transfecção , Peptídeos beta-Amiloides/metabolismo , Barreira Hematoencefálica/metabolismo , Linhagem Celular , Eletroporação , Células Endoteliais/metabolismo , Expressão Gênica , Genes Reporter , Humanos , Plasmídeos/genética , Agregados ProteicosRESUMO
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.
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Cobalto , Complexos de Coordenação , Cobre , Vírus da Dengue/metabolismo , Queratinócitos/virologia , Carga Viral/efeitos dos fármacos , Zika virus/metabolismo , Animais , Chlorocebus aethiops , Cobalto/química , Cobalto/farmacologia , Complexos de Coordenação/química , Complexos de Coordenação/farmacologia , Cobre/química , Cobre/farmacologia , Culicidae , Células HeLa , Humanos , Queratinócitos/metabolismo , Queratinócitos/patologia , Células Vero , Proteínas do Envelope ViralRESUMO
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.
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Ar , DNA/metabolismo , Gases em Plasma , Plasmídeos/metabolismo , Transfecção/instrumentação , Transfecção/métodos , Animais , Linhagem Celular , Sobrevivência Celular , Genes Reporter , Humanos , Queratinócitos/fisiologia , Luciferases/análise , Melanócitos/fisiologia , CamundongosRESUMO
Activation of human platelets produces a gel-like substance referred to as platelet rich plasma or platelet gel. Platelet gel is used clinically to promote wound healing; it also exhibits antimicrobial properties that may aid in the healing of infected wounds. The purpose of this study was to quantify the efficacy of human platelet gel against the opportunistic bacterial wound pathogens Acinetobacter baumannii, Pseudomonas aeruginosa, and Staphylococcus aureus on skin. These opportunistic pathogens may exhibit extensive antibiotic resistance, necessitating the development of alternative treatment options. The antimicrobial efficacy of platelet gel supernatants was quantified using an in vitro broth dilution assay, an ex vivo inoculated skin assay, and in an in vivo skin decontamination assay. Human platelet gel supernatants were highly bactericidal against A. baumannii and moderately but significantly bactericidal against S. aureus in vitro and in the ex vivo skin model. P. aeruginosa was not inactivated in vitro; a low but significant inactivation level was observed ex vivo. These supernatants were quite effective at inactivating a model organism on skin in vivo. These results suggest application of platelet gel has potential clinical applicability, not only in the acceleration of wound healing, but also against relevant bacteria causing wound infections.
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Infecções Bacterianas/tratamento farmacológico , Infecções Bacterianas/microbiologia , Plaquetas/metabolismo , Infecções Oportunistas/tratamento farmacológico , Infecções Oportunistas/microbiologia , Plasma Rico em Plaquetas/metabolismo , Ferimentos e Lesões/tratamento farmacológico , Ferimentos e Lesões/microbiologia , Acinetobacter baumannii , Animais , Modelos Animais de Doenças , Humanos , Masculino , Testes de Sensibilidade Microbiana , Pseudomonas aeruginosa , Ratos , Pele/lesões , Staphylococcus aureus , CicatrizaçãoRESUMO
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.
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Interleukin-12 (IL-12) gene electrotransfer (GET) delivery is highly effective in inducing long-term, complete regression in mouse and human melanoma and other solid tumors. Therapeutic efficacy is enhanced by immune checkpoint inhibitors, and the combination of IL-12 plasmid GET (pIL-12 GET) and anti-programmed cell death protein 1 (PD-1) monoclonal antibodies has reached clinical trials. In this study, we designed peptides and plasmids encoding the mouse homologs of the pembrolizumab and nivolumab programmed cell death 1 ligand 1 (PD-L1) binding regions. We hypothesized that intratumor autocrine/paracrine peptide expression would block PD-1/PD-L1 binding and provide cancer patients with an effective and cost-efficient treatment alternative. We demonstrated that the mouse homolog to pembrolizumab was effective at blocking PD-1/PD-L1 in vitro. After intratumor plasmid delivery, both peptides bound PD-L1 on tumor cells. We established that plasmid DNA delivery to tumors in vivo or to tumor cells in vitro upregulated several immune modulators and PD-L1 mRNA and protein, potentiating this therapy. Finally, we tested the combination of pIL-12 GET therapy and peptide plasmids. We determined that pIL-12 GET therapeutic efficacy could be enhanced by combination with the plasmid encoding the pembrolizumab mouse homolog.
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Background: Tissues, such as skeletal muscle, have been targeted for the delivery of plasmid DNA (pDNA) encoding vaccines and therapeutics. The application of electric pulses (electroporation or electrotransfer) increases cell membrane permeability to enhance plasmid delivery and expression. However, the molecular effects of DNA electrotransfer on the muscle tissue are poorly characterized. Materials and Methods: Four hours after intramuscular plasmid electrotransfer, we evaluated gene expression changes by RNA sequencing. Differentially expressed genes were analyzed by gene ontology (GO) pathway enrichment analysis. Results: GO analysis highlighted many enriched molecular functions. The terms regulated by pulse application were related to muscle stress, the cytoskeleton and inflammation. The terms regulated by pDNA injection were related to a DNA-directed response and its control. Several terms regulated by pDNA electrotransfer were similar to those regulated by pulse application. However, the terms related to pDNA injection differed, focusing on entry of the plasmid into the cells and intracellular trafficking. Conclusion: Each muscle stimulus resulted in specific regulated molecular functions. Identifying the unique intrinsic molecular changes driven by intramuscular DNA electrotransfer will aid in the design of preventative and therapeutic gene therapies.
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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.
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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.
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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.
Assuntos
DNA/genética , DNA/metabolismo , Sistemas de Liberação de Medicamentos/métodos , Plasmídeos/genética , Animais , Expressão Gênica , Melanoma Experimental/patologia , CamundongosRESUMO
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.
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DNA/genética , DNA/metabolismo , Técnicas de Transferência de Genes , Plasmídeos/genética , Pele/metabolismo , Animais , Eletrodos , Eletroporação , Técnicas de Transferência de Genes/instrumentação , CobaiasRESUMO
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.
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OBJECTIVES: To evaluate the efficacy of antibiotic-impregnated polymethylmethacrylate (PMMA) beads in eradication of an arterial prosthetic graft methicillin-resistant Staphylococcus aureus (MRSA) biofilm in an experimental animal model. METHODS: Forty rats underwent subcutaneous implantation of a MRSA-colonized arterial polytetrafluoroethylene (PTFE) 1 x 1 cm wafer on the back. The effect of regional antibiosis produced by antibiotic PMMA bead placement adjacent to the infected PTFE wafer was determined using four 10-animal study groups: control (no antibiotic), PMMA bead with no antibiotic, PMMA bead with 10% vancomycin, and PMMA bead with 10% daptomycin. After 3 d, the PTFE wafers were explanted and quantitative biofilm cultures, expressed as colony-forming units (CFU) per graft wafer, performed using real-time polymerase chain reaction to assess MRSA eradication. No systemic antibiotic was administered. Bioassays of antibiotic bead bacteriocidal were performed by measuring zone of inhibition diameters on MRSA colonized agar culture plates prior to and following graft explantation. RESULTS: All animal tolerated implantation of the MRSA-infected PTFE wafer and survived the 3 d until graft explantation. Quantitative biofilm cultures demonstrated a significant decrease (P < 0.01) in MRSA CFUs present on the PTFE wafer surfaces in the presence of both the vancomycin- and daptomycin-impregnated beads compared to controls and plain PMMA beads. Both vancomycin and daptomycin PMMA beads retained antibacterial activity after 3 d of implantation with decrease in zones of inhibition of 15% and 45%, respectively. CONCLUSIONS: Regional antibiotic delivery using an antibiotic-impregnated PMMA bead reduced the bacterial biofilm concentration in experimental subcutaneous pocket model of vascular surgical site infection. The delivery of antibiotics via a PMMA bead may be a useful adjunct in the treatment of vascular surgical site infection.
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Antibacterianos/administração & dosagem , Antibacterianos/uso terapêutico , Sistemas de Liberação de Medicamentos/métodos , Staphylococcus aureus Resistente à Meticilina , Infecções Relacionadas à Prótese/tratamento farmacológico , Infecções Estafilocócicas/tratamento farmacológico , Animais , Biofilmes , Daptomicina/administração & dosagem , Daptomicina/uso terapêutico , Modelos Animais de Doenças , Masculino , Microesferas , Polimetil Metacrilato , Infecções Relacionadas à Prótese/microbiologia , Ratos , Ratos Sprague-Dawley , Infecções Estafilocócicas/microbiologia , Resultado do Tratamento , Vancomicina/administração & dosagem , Vancomicina/uso terapêuticoRESUMO
Identification of mycobacterial clinical isolates by pyrosequencing within the hypervariable A region of the 16S rRNA gene was compared to other identification methods. For >90% of isolates, these identifications correlated to the level of complex or species. For identification of many mycobacteria, pyrosequencing offers an inexpensive alternative to traditional sequencing.
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Técnicas de Tipagem Bacteriana/métodos , Infecções por Mycobacterium/microbiologia , Mycobacterium/classificação , RNA Ribossômico 16S/genética , Análise de Sequência de DNA/métodos , Cromatografia Líquida de Alta Pressão , DNA Bacteriano/análise , DNA Ribossômico/análise , Humanos , Mycobacterium/genética , Mycobacterium/isolamento & purificação , Especificidade da EspécieRESUMO
BACKGROUND: Electroporation is an established technique for enhancing plasmid delivery to many tissues in vivo, including the skin. We have previously demonstrated efficient delivery of plasmid DNA to the skin utilizing a custom-built four-plate electrode. The experiments described here further evaluate cutaneous plasmid delivery using in vivo electroporation. Plasmid expression levels are compared to those after liposome mediated delivery. METHODS: Enhanced electrically-mediated delivery, and less extensively, liposome complexed delivery, of a plasmid encoding the reporter luciferase was tested in rodent skin. Expression kinetics and tissue damage were explored as well as testing in a second rodent model. RESULTS: Experiments confirm that electroporation alone is more effective in enhancing reporter gene expression than plasmid injection alone, plasmid conjugation with liposomes followed by injection, or than the combination of liposomes and electroporation. However, with two time courses of multiple electrically-mediated plasmid deliveries, neither the levels nor duration of transgene expression are significantly increased. Tissue damage may increase following a second treatment, no further damage is observed after a third treatment. When electroporation conditions utilized in a mouse model are tested in thicker rat skin, only higher field strengths or longer pulses were as effective in plasmid delivery. CONCLUSION: Electroporation enhances reporter plasmid delivery to the skin to a greater extent than the liposome conjugation method tested. Multiple deliveries do not necessarily result in higher or longer term expression. In addition, some impact on tissue integrity with respect to surface damage is observed. Pulsing conditions should be optimized for the model and for the expression profile desired.
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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.
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Contaminação de Equipamentos , Bactérias Gram-Positivas/genética , Bactérias Gram-Positivas/fisiologia , Plásticos , DNA Bacteriano , Genoma Bacteriano , Propriedades de Superfície , Fatores de TempoRESUMO
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.
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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.
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Eletroporação/métodos , Técnicas de Transferência de Genes , Melanoma Experimental , Plasmídeos , Esferoides Celulares , Animais , DNA , Camundongos , Receptores de Reconhecimento de Padrão/genética , Transfecção/métodos , Células Tumorais Cultivadas , Regulação para CimaRESUMO
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.