Medicine and Health Sciences Commons^™

Gene Expression Under Combined Hypoxia And Acidosis In Chondrosarcoma, Michael Stacey, Kostika Vangjeli, Christopher Osgood

Bioelectrics Publications

Chondrosarcomas are the second most common cause of bone cancer and are removed surgically with wide margins. On recurrence, they are resistant to chemo and radiation therapy and new treatment options are critically required. This tumor type produces hyaline cartilage, a cartilage normally formed under hypoxic and acidic environment due to lack of vasculature in cartilage. Paradoxically, chondrosarcomas arise in the well vascularized, oxygen rich environment of the bone. Hypoxia and acidosis are two stressors where the cellular effects are typically reported separately even though cells experience combined effects of hypoxia and acidosis. Given the mechanistic links between hypoxia and …

Il-12 Gene Electrotransfer Triggers A Change In Immune Response Within Mouse Tumors, Guilan Shi, Chelsea Edelblute, Sezgi Arpag, Cathryn Lundberg, Richard Heller

Bioelectrics Publications

Metastatic melanoma is an aggressive skin cancer with a relatively low survival rate. Immune-based therapies have shown promise in the treatment of melanoma, but overall complete response rates are still low. Previous studies have demonstrated the potential of plasmid IL-12 (pIL-12) delivered by gene electrotransfer (GET) to be an effective immunotherapy for melanoma. However, events occurring in the tumor microenvironment following delivery have not been delineated. Therefore, utilizing a B16F10 mouse melanoma model, we evaluated changes in the tumor microenvironment following delivery of pIL-12 using different GET parameters or injection of plasmid alone. The results revealed a unique immune cell …

Tumor Cell Death After Electrotransfer Of Plasmid Dna Is Associated With Cytosolic Dna Sensor Upregulation, Katarina Znidar, Masa Bosnjak, Nina Semenova, Olga N. Pakhomova, Loree Heller, Maja Cemazar

Bioelectrics Publications

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 …

Upregulation Of Dna Sensors In B16.F10 Melanoma Spheroid Cells After Electrotransfer Of Pdna, Katarina Znidar, Masa Bosnjak, Tanja Jesenko, Loree C. Heller, Maja Cemazar

Bioelectrics Publications

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 …

Electrotransfer Of Different Control Plasmids Elicits Different Antitumor Effectiveness In B16.F10 Melanoma, Masa Bosnjak, Tanjo Jesenko, Urska Kamensek, Gregor Sersa, Jaka Lavrencak, Loree Heller, Maja Cemazar

Bioelectrics Publications

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 …

Wild-Type P53 Enhances Endothelial Barrier Function By Mediating Rac1 Signalling And Rhoa Inhibition, Nektarios Barabutis, Christiana Dimitropoulou, Betsy Gregory, John D. Catravas

Bioelectrics Publications

Inflammation is the major cause of endothelial barrier hyper-permeability, associated with acute lung injury and acute respiratory distress syndrome. This study reports that p53 "orchestrates" the defence of vascular endothelium against LPS, by mediating the opposing actions of Rac1 and RhoA in pulmonary tissues. Human lung microvascular endothelial cells treated with HSP90 inhibitors activated both Rac1- and P21-activated kinase, which is an essential element of vascular barrier function. 17AAG increased the phosphorylation of both LIMK and cofilin, in contrast to LPS which counteracted those effects. Mouse lung microvascular endothelial cells exposed to LPS exhibited decreased expression of phospho-cofilin. 17AAG treatment …

Membrane Channel Gene Expression In Human Costal And Articular Chondrocytes, A. Asmar, R. Barrett-Jolley, A. Werner, R. Kelly Jr., M. Stacey

Bioelectrics Publications

Chondrocytes are the uniquely resident cells found in all types of cartilage and key to their function is the ability to respond to mechanical loads with changes of metabolic activity. This mechanotransduction property is, in part, mediated through the activity of a range of expressed transmembrane channels; ion channels, gap junction proteins, and porins. Appropriate expression of ion channels has been shown essential for production of extracellular matrix and differential expression of transmembrane channels is correlated to musculoskeletal diseases such as osteoarthritis and Albers-Sch€onberg. In this study we analyzed the consistency of gene expression between channelomes of chondrocytes from human …

Gene Electro Transfer Of Plasmid Encoding Vascular Endothelial Growth Factor For Enhanced Expression And Perfusion In The Ischemic Swine Heart, Barbara Y. Hargrave, Robert Strange Jr., Sagar Navare, Michael Stratton, Niculina Burcus, Len Murray, Cathryn Lundberg, Anna A. Bulysheva, Fanying Li, Richard Heller

Bioelectrics Publications

Myocardial ischemia can damage heart muscle and reduce the heart's pumping efficiency. This study used an ischemic swine heart model to investigate the potential for gene electro transfer of a plasmid encoding vascular endothelial growth factor for improving perfusion and, thus, for reducing cardiomyopathy following acute coronary syndrome. Plasmid expression was significantly greater in gene electro transfer treated tissue compared to injection of plasmid encoding vascular endothelial growth factor alone. Higher gene expression was also seen in ischemic versus non-ischemic groups with parameters 20 Volts (p

Electrotransfer Of Single-Stranded Or Double-Stranded Dna Induces Complete Regression Of Palpable B16.F10 Mouse Melanomas, Loree Heller, Vesba Todorovic, Maja Cemazar

Bioelectrics Publications

Enhanced tumor delivery of plasmid DNA with electric pulses in vivo has been confirmed in many preclinical models. Intratumor electrotransfer of plasmids encoding therapeutic molecules has reached Phase II clinical trials. In multiple preclinical studies, a reduction in tumor growth, increased survival or complete tumor regression have been observed in control groups in which vector or backbone plasmid DNA electrotransfer was performed. This study explores factors that could produce this antitumor effect. The specific electrotransfer pulse protocol employed significantly potentiated the regression. Tumor regression was observed after delivery of single-stranded or double-stranded DNA with or without CpG motifs in both …

Electroporation-Mediated Gene Transfer Directly To The Swine Heart, Barbara Hargrave, Harre Downey, Cathryn Lundberg, Annelise Israel, Yeong-Jer Chen, Richard Heller

Bioelectrics Publications

In vivo gene transfer to the ischemic heart via electroporation holds promise as a potential therapeutic approach for the treatment of heart disease. In the current study, we investigated the use of in vivo electroporation for gene transfer using three different penetrating electrodes and one non-penetrating electrode. The hearts of adult male swine were exposed through a sternotomy. Eight electric pulses synchronized to the rising phase of the R wave of the electrocardiogram were administered at varying pulse widths and field strengths following an injection of either a plasmid encoding luciferase or one encoding green fluorescent protein. Four sites on …

Advancing Our Understanding Of The Inheritance And Transmission Of Pectus Excavatum, Lisa Horth, Michael W. Stacey, Virginia K. Proud, Kara Segna, Chelsea Rutherford, Donald Nuss, Robert E. Kelly

Bioelectrics Publications

Pectus excavatum is the most common congenital chest wall abnormality expressed in children, yet its inheritance is poorly understood. Here we present the first comprehensive assessment of the inheritance of this disorder. After evaluating 48 pedigrees and 56 clinical traits of probands and family members, we find strong evidence of autosomal recessive, genetic control for this disorder. Additionally there is likely more than one pectus disease-associated allele, as well as a relatively large number of disease allele carriers in the human population. Some clinical traits appear important and may serve as reliable indicators for predicting the likelihood of pectus excavatum …

Plasmid Injection And Application Of Electric Pulses Alter Endogenous Mrna And Protein Expression In B16.F10 Mouse Melanomas, L. C. Heller, Y. L. Cruz, B. Ferraro, H. Yang, R. Heller

Bioelectrics Publications

The application of electric pulses to tissues causes cell membrane destabilization, allowing exogenous molecules to enter the cells. This delivery technique can be used for plasmid gene therapy. Reporter gene expression after plasmid delivery with eight representative published protocols was compared in B16.F10 mouse melanoma tumors. This expression varied significantly based on the pulse parameters utilized for delivery. To observe the possible influence of plasmid injection and/or pulse application on endogenous gene expression, levels of stress-related mRNAs 4 and 24 h after delivery were determined by PCR array. Increases in mRNA levels for several inflammatory chemokines and cytokines were observed …

Evaluation Of Toxicity Following Electrically Mediated Interleukin-12 Gene Delivery In A B16 Mouse Melanoma Model, Loree Heller, Kathleen Merkler, Jeffrey Westover, Yolmari Cruz, Domenico Coppola, Kaaron Benson, Adil Daud, Richard Heller

Bioelectrics Publications

PURPOSE: Interleukin-12 (IL-12) has potential as an immunotherapeutic agent for the treatment of cancer but is unfortunately associated with toxicity. Delivery of a plasmid encoding IL-12 with electroporation induces an antitumor effect in the B16 mouse melanoma model without serious side effects. To translate this observation to the clinic, an evaluation of toxicity was done in the mouse model.

EXPERIMENTAL DESIGN: Weight change, tumor response, blood chemistry and hematology values, and serum IL-12 levels were evaluated. Multiple tissues were analyzed histopathologically.

RESULTS: A pronounced reduction in tumor volume, including a large percentage of complete regressions, was observed after electrically mediated …

Comparison Of Methods For Dna Isolation From Food Samples For Detection Of Shiga Toxin-Producing Escherichia Coli By Real-Time Pcr, Loree C. Heller, Carisa R. Davis, K. Kealy Peak, David Wingfield, Andrew C. Cannons, Philip T. Amuso, Jacqueline Cattani

Bioelectrics Publications

In this study, food samples were intentionally contaminated with Escherichia coli O157:H7, and then DNA was isolated by using four commercial kits. The isolated DNA samples were compared by using real-time PCR detection of the Shiga toxin genes. The four kits tested worked similarly.

Electrically Mediated Delivery Of Vector Plasmid Dna Elicits An Antitumor Effect, L. Heller, D. Coppola

Bioelectrics Publications

In vivo electroporation is an efficient means of increasing plasmid DNA delivery to normal tissues, such as skin and muscle, as well as directly to tumors. In the experiments described here, plasmid DNA was delivered by in vivo electroporation to B16 mouse melanomas using two very different pulsing protocols. Reporter expression increased 21- or 42-fold, respectively with electroporation over injection alone. The growth of experimental melanomas with an approximate diameter of 4 mm on the day of treatment was monitored after electroporation delivery of reporter plasmid DNA. Remarkably, short-term complete regressions using one of these pulsing protocols occurred in up …

Effect Of Electrically Mediated Intratumor And Intramuscular Delivery Of A Plasmid Encoding Ifn Α On Visible B16 Mouse Melanomas, Loree C. Heller, Stephanie F. Ingram, M. Lee Lucas, Richard A. Gilbert, Richard Heller

Bioelectrics Publications

Interferon α may be used as a single agent therapy for metastatic malignant melanoma or as an adjuvant to chemotherapy. Delivery of interferon α by gene therapy offers an alternative to recombinant protein therapy. Electrically mediated delivery enhances plasmid expression in a number of tissues, for instance skin, liver, muscle and tumors including melanomas. Here we compare the effect of delivery of a plasmid encoding mouse interferon α on growth of visible B16 mouse melanomas following electrically mediated delivery to muscle or directly to the tumor. Intratumoral delivery of interferon α plasmid not only slows melanoma growth, but induces complete, …

Il-12 Plasmid Delivery By In Vivo Electroporation For The Successful Treatment Of Established Subcutaneous B16.F10 Melanoma, M. Lee Lucus, Loree Heller, Domenico Coppola, Richard Heller

Bioelectrics Publications

Interleukin-12 (IL-12) has been used in numerous immunotherapy protocols against melanoma. However, delivery of IL-12 in the form of recombinant protein can result in severe toxicity, and gene therapy has had limited success against B16.F10 murine melanoma. The purpose of this study was to examine the effectiveness of in vivo electroporation for the delivery of plasmid DNA encoding IL-12 as an antitumor agent against B16.F10 melanoma. We treated mice bearing established B16.F10 melanoma tumors with intratumoral (i.t.) or intramuscular (i.m.) injections of a plasmid encoding IL-12, followed by in vivo electroporation. For i.t. treatments, we used an applicator containing six …

Electrically Mediated Plasmid Dna Delivery To Hepatocellular Carcinomas In Vivo, L. Heller, M. J. Jaroszeski, D. Coppola, C. Pottinger, R. Gilbert, Richard Heller

Bioelectrics Publications

Gene therapy by direct delivery of plasmid DNA has several advantages over viral gene transfer, but plasmid delivery is less efficient. In vivo electroporation has been used to enhance delivery of chemotherapeutic agents to tumors in both animal and human studies. Recently, this delivery technique has been extended to large molecules such as plasmid DNA. Here, the successful delivery of plasmids encoding reporter genes to rat hepatocellular carcinomas by in vivo electroporation is demonstrated.

Variant Forms Of Ataxia Telangiectasia, A. M. R. Taylor, E. Flude, B. Laher, Michael W. Stacey, E. Mckay, J. Watt, S. H. Greens, A. E. Harding

Bioelectrics Publications

Two ataxia telangiectasia patients with unusual clinical and cellular features are described. Cultured fibroblasts and PHA stimulated lymphocytes from these two patients showed a smaller increase of radiosensitivity than cells from other A-T patients, as measured by colony forming ability or induced chromosome damage respectively, after exposure to ionising radiation. The response of DNA synthesis to irradiation of these cells was, however, the same as for other A-T patients. Cells from a third patient with some clinical features of A-T but with a very protracted course also showed low levels of radiation induced chromosome damage, but colony forming ability and …