Clinical evaluation of safety and human tolerance of electrical sensation induced by electric fields with non-invasive electrodes.

This paper reports the first clinical safety study of human tolerance of electrical sensation using non-invasive, flexible surface-type electrodes and exponentially decaying electric pulses. The study evaluated the effect of electric fields in the absence of a drug and an anesthetic, and was performed in light of potential applications in the field of erectile dysfunction (ED). Twenty impotent patients who had previously received injection or intraurethral therapies were enrolled in the study. Voltage escalations from 50 to 80 V (in 10-V increments) with a single pulse of 3-ms duration were performed with meander-type electrodes placed on the shaft and part of the glans of the penis. The electric fields-induced sensation was assessed via a pain scale from 0 to 10. All 20 patients, who were free to withdraw from the study at any point, completed the voltage escalation study. No clinical safety concerns were apparent and no skin irritation was observed after electric treatment. Our initial study indicates that the pulses in the tested voltage range were well tolerated by most patients. In previous animal experiments under analogous experimental conditions, the application of 50 V has been found effective for transdermal drug delivery into the penis.

Topical delivery of lidocaine in healthy volunteers by electroporation, electroincorporation, or iontophoresis: an evaluation of skin anesthesia.

BACKGROUND AND OBJECTIVES: This study was designed to compare the onset, duration, and depth of local anesthesia after the topical delivery of lidocaine using electroporation (EP), electroincorporation (EI), and iontophoresis (IP) in healthy volunteers. EP and EI were performed with prototype devices and IP with a commercial drug/device product. METHODS: A double-blind, crossover study design was used for 10 randomized volunteers selected for EP treatment with either 4%, 10%, or 20% lidocaine or placebo normal saline. Because it was impossible to blind between IP, EP, and EI, an open label study with randomized volunteers was selected for IP and EP treatments. Onset and duration of anesthesia was determined by measuring warm sensation, cool sensation, and hot pain; depth of anesthesia was determined by measurement of pain sensation to a 27-gauge needle passed through the skin. RESULTS: For EP, all concentrations of lidocaine produced significant changes from baseline on 2 or 3 efficacy measures, however, the 4% concentration appeared to be the most efficacious when delivered by the EP method. The EP and EI methods produced a significant elevation in all 3 thermal thresholds, whereas IP produced significant elevations in cool and warm thresholds only. However, IP resulted in a greater depth of anesthesia. Plasma lidocaine levels were undetectable. CONCLUSIONS: The transdermal delivery of lidocaine by IP, EP, and EI results in similar surface skin anesthesia; however, IP results in a greater depth of anesthesia. Reg Anesth Pain Med 2001;26:229-238.

Increased DNA vaccine delivery and immunogenicity by electroporation in vivo.

DNA vaccines have been demonstrated to be potent in small animals but are less effective in primates. One limiting factor may be inefficient uptake of DNA by cells in situ. In this study, we evaluated whether cellular uptake of DNA was a significant barrier to efficient transfection in vivo and subsequent induction of immune responses. For this purpose, we used the technique of electroporation to facilitate DNA delivery in vivo. This technology was shown to substantially increase delivery of DNA to cells, resulting in increased expression and elevated immune responses. The potency of a weakly immunogenic hepatitis B surface Ag DNA vaccine was increased in mice, as seen by a more rapid onset and higher magnitude of anti-hepatitis B Abs. In addition, the immunogenicity of a potent HIV gag DNA vaccine was increased in mice, as seen by higher Ab titers, a substantial reduction in the dose of DNA required to induce an Ab response, and an increase in CD8+ T cell responses. Finally, Ab responses were enhanced by electroporation against both components of a combination HIV gag and env DNA vaccine in guinea pigs and rabbits. Therefore, cellular uptake of DNA is a significant barrier to transfection in vivo, and electroporation appears able to overcome this barrier.

Electroporation therapy of solid tumors.

The curative effects of some chemotherapeutic drugs are impeded by their poor permeation through the cell membrane. This limitation can be overcome by a novel approach called electroporation therapy (EPT), electrochemotherapy (ECT), or electrical impulse chemotherapy (EIC). The method involves application of brief electrical pulses, which destabilize the cell membrane barrier, allowing intracellular access of chemotherapeutic drugs that otherwise would not be able to penetrate the cell membrane effectively. EPT makes it possible to lower the drug dose, thereby relieving the patient of adverse side effects associated with conventional chemotherapy. Even with the lower drug dose, EPT has shown significantly higher efficacy than has conventional chemotherapy. The method is currently being evaluated clinically for treating various cancer indications using the anticancer drugs bleomycin or cisplatin. This article provides a historical perspective and current insights into this new modality of cancer treatment, including basic physical, biological, and medical facts about EPT; computer-assisted development of electrical pulse generators and electrodes necessary to create effective electrical fields in the treatment area; results of cancer cell and tumor treatments in vitro, in animals, and in humans; safety aspects of EPT; potential combined delivery of chemotherapeutic drugs and biological agents to reduce or eliminate metastatic disease; and intracellular delivery of DNA by electroporation for cancer gene therapy.

Improving glove barrier effectiveness.

Perioperative staff members depend on surgical gloves to prevent disease transmission between themselves and patients, but these gloves frequently fail during use. Three approaches can make surgical gloves more effective barriers: preventing glove failures, monitoring glove integrity, and improving glove quality. Failure prevention includes modifying surgical techniques, improving instruments and equipment, streamlining teamwork, selecting the most appropriate gloves, double gloving, and performing preventive glove changes. Glove integrity monitoring can be performed visually or by feel, by wearing glove pairs with color-puncture indicators, or by using electronic monitoring devices. Glove quality improvements must be accompanied by testing methods that reflect in-use conditions. A glove rating system that is based on in-use performance may enhance glove safety substantially.

The risks and challenges of surgical glove failure.

The relationship between infection control standards and surgical gloving practices is an important issue in combating infectious disease. For their own and their patients' protection, health care personnel must rely on the barrier qualities of gloves more than ever before. Both surgical and examination gloves fail frequently, which puts health care providers and patients at risk of acquiring fluid-borne pathogens. This paper discusses current guidelines, the risk of infection by viral and bacterial pathogens, controversial issues involving gloving practices, and recommendations to improve infection control practices.

Surgical glove failures in clinical practice settings.

Health care personnel often pay little attention to the barrier effectiveness of the surgical gloves they use in clinical settings. They may assume that all surgical gloves provide adequate protection against the transfer of bloodborne pathogens, chemicals, or mutagenic substances. Perioperative staff members frequently are unaware that their surgical gloves have failed until they find blood on their hands after operative procedures are completed. In this first article of a three-part series, the authors review current surgical glove testing standards, define surgical glove failure, and describe the reasons that surgical glove failure occurs in clinical practice settings.

Transient and stable expression of foreign DNA introduced into plant protoplasts by electroporation.

Electroporation utilizes high-voltage electric fields for cell permeabilization, This technique has been used for promoting the cellular uptake of exogenous molecules and macromolecules, including nucleotides, dyes, RNA, DNA, and even small proteins (1-7). Electroporation's useful attributes are its simplicity and its general effectiveness with a wide range of cell types. Because of its general efficacy, electroporation is becoming a valuable technique for the introduction of DNA into cell types that are resistant to transformation by other procedures (6-8). In this chapter, we will outline procedures for introducing foreign DNAs into plant protoplasts by electroporation. DNA uptake is verified by the transient expression of chloramphenicol acetyltransferase (CAT), and by the recovery of plants stably transformed to kanamycin resistance through the introduction of the neomycin phosphotransferase gene.

IL-3A virus infection of a Chlorella-like green alga induces a DNA restriction endonuclease with novel sequence specificity.

A type II restriction endonuclease, named CviJI, was isolated from a eukaryotic Chlorella-like green alga infected with the dsDNA containing virus IL-3A. CviJI is the first restriction endonuclease to recognize the sequence PuGCPy; CviJI cleaves DNA between the G and C. Methylation of the cytosine in PuGCPy sequences prevents cleavage by CviJI. CviJI cleaved DNA into smaller but defined fragments in the presence of ATP. This "star" activity was stimulated by dithiothreitol and/or S-adenosylmethionine but did not occur under conditions which favor "star" activity of other restriction endonucleases.

Restriction endonuclease activity induced by PBCV-1 virus infection of a Chlorella-like green alga.

An enzyme was isolated from a eucaryotic, Chlorella-like green alga infected with the virus PBCV-1 which exhibits type II restriction endonuclease activity. The enzyme recognized the sequence GATC and cleaved DNA 5' to the G. Methylation of deoxyadenosine in the GATC sequence inhibited enzyme activity. In vitro the enzyme cleaved host Chlorella nuclear DNA but not viral DNA because host DNA contains GATC and PBCV-1 DNA contains GmATC sequences. PBCV-1 DNA is probably methylated in vivo by the PBCV-1-induced methyltransferase described elsewhere (Y. Xia and J. L. Van Etten, Mol. Cell. Biol. 6:1440-1445). Restriction endonuclease activity was first detected 30 to 60 min after viral infection; the appearance of enzyme activity required de novo protein synthesis, and the enzyme is probably virus encoded. Appearance of enzyme activity coincided with the onset of host DNA degradation after PBCV-1 infection. We propose that the PBCV-1-induced restriction endonuclease participates in host DNA degradation and is part of a virus-induced restriction and modification system in PBCV-1-infected Chlorella cells.

Initiation of transcription at phage T4 late promoters with purified RNA polymerase.

We have previously identified T4 late promoters governing the in vivo expression of T4 late genes 23 and 24 (P23 and P24). T4 late transcription in vivo is known to involve the binding of at least five phage-coded proteins to the bacterial RNA polymerase and normally requires concurrent DNA replication for DNA template activation. We show here that in vitro transcription, primarily of plasmids carrying T4 genes 23 and 24, by RNA polymerase purified from Escherichia coli at late times after T4 infection allows specific initiation at P23 and P24 in the absence of DNA replication. These promoters are not utilized by E. coli RNA polymerase holoenzyme, by RNA polymerase core, or by T4-modified RNA polymerase purified from cells infected with a T4 gene 55 mutant (gene 55 codes for an RNA polymerase binding protein required for late transcription). The utilization of P23 and P24 in vitro is sharply inhibited by NaCl concentrations greater than 100 mM, and this inhibition is partly reversed by the addition of 10% DMSO. Relaxation of plasmid DNA containing P23 (with topoisomerase I) reduces P23 utilization at low salt (50 mM Na+) and nearly abolishes it at high salt (250 mM Na+). P23 utilization is discernible in linear, glucosylated hydroxymethylcytosine-containing T4 virion DNA.

Construction and properties of a cell-free system for bacteriophage T4 late RNA synthesis.

A cell-free system for synthesizing bacteriophage T4 late RNA is described. The system, which is based on the "cellophane disc" technique introduced by Schaller and co-workers (Schaller, H., Otto, B., Nüsslein, V., Huf, J., Hermann, R., and Bonnhoeffer, F. (1972) J. Mol. Biol. 63, 183-200), provides favorable conditions for T4 DNA and RNA synthesis in vitro. Total RNA synthesis can be sustained for more than 1 h at 25 degrees C and initiation of early and late RNA chains occurs in vitro. The capacity to yield cell-free systems which make T4 late RNA in vitro is acquired by virus-infected cells as they make late RNA in vivo. The in vitro synthesized RNA is highly asymmetric. The conditions which optimize the in vitro system with respect to several parameters (total extent of T4 transcription, rate of transcription, asymmetry, fraction of T4 late RNA, and sensitivity to inhibition by rifampicin and streptolydigin) are described.

Phage T4-modified RNA polymerase transcribes T4 late genes in vitro.

Initiation of T4 late RNA synthesis has been achieved in an in vitro system prepared from Escherichia coli cells infected with wild-type or maturation-defective mutant T4 phage. The system uses a cellophane membrane as a mechanical support for concentrated cell lysates and for added streptolydigin-resistant RNA polymerases. Transcriptional activity and selectivity of added RNA polymerases are tested while endogenous RNA polymerase activity is inhibited by streptolydigin. T4-modified RNA polymerase is required for substantial stimulation of T4 late RNA synthesis.