Supramembrane potential-induced electroconformational changes in sodium channel proteins: a potential mechanism involved in electric injury

Effects of imposed large supraphysiological transmembrane potential (TP) pulses on channel proteins, particularly on the voltage-gated Na channels, were investigated. Voltage clamp techniques were used to deliver both shock and stimulation pulses, and to monitor changes in the channel functions. Our experimental results indicated that more than one 4 ms duration TP shock of -450 mV resulted in electroconformational denature of voltage-gated Na channels. This resulted in functional reductions in muscle cells' excitability. We quantified the TP shock-induced decrease in the Na channel currents, compared the pre- and post-shocked Na channel currents' voltage dependency, and studied the reversibility of the electroconformationally denatured ion channel proteins. These observations are particularly relevant to the problem of explaining the neuromuscular damage following high voltage electrical shock injuries despite no evidence of a thermal injury component.     

Enhanced magnitude and breadth of neutralizing humoral response to a DNA vaccine targeting the DHBV envelope protein delivered by in vivo electroporation

We explored in the duck hepatitis B virus (DHBV) model the impact of electroporation (EP)-mediated DNA vaccine delivery on the neutralizing humoral response to viral preS/S large envelope protein. EP enhanced the kinetics and magnitude of anti-preS response compared to the standard needle DNA injection (SI). Importantly, EP dramatically enhanced the neutralizing potency of the humoral response, since antibodies induced by low DNA dose (10 μg) were able to highly neutralize DHBV and to recognize ten antigenic regions, including four neutralization epitopes. Whereas, SI-induced antibodies by the same low DNA dose were not neutralizing and the epitope pattern was extremely narrow, since it was limited to only one epitope. Thus, EP-based delivery was able to improve the dose efficiency of DNA vaccine and to maintain a highly neutralizing, multi-specific B-cell response, suggesting that it may be an effective approach for chronic hepatitis B therapy at clinically feasible DNA dose.     

The use of an in vitro 3D melanoma model to predict in vivo plasmid transfection using electroporation

A large-scale in vitro 3D tumor model was generated to evaluate gene delivery procedures in vivo. This 3D tumor model consists of a “tissue-like” spheroid that provides a micro-environment supportive of melanoma proliferation, allowing cells to behave similarly to cells in vivo. This functional spheroid measures approximately 1 cm in diameter and can be used to effectively evaluate plasmid transfection when testing various electroporation (EP) electrode applicators. In this study, we identified EP conditions that efficiently transfect green fluorescent protein (GFP) and interleukin 15 (IL-15) plasmids into tumor cells residing in the 3D construct. We found that plasmids delivered using a 6-plate electrode applying 6 pulses with nominal electric field strength of 500 V/cm and pulse-length of 20 ms produced significant increase of GFP (7.3-fold) and IL-15 (3.0-fold) expression compared to controls. This in vitro 3D model demonstrates the predictability of cellular response toward delivery techniques, limits the numbers of animals employed for transfection studies, and may facilitate future developments of clinical trials for cancer therapies in vivo.     

Membrane electroporation theories: a review

Electroporation, the transient increase in the permeability of cell membranes when exposed to a high electric field, is an established in vitro technique and is used to introduce DNA or other molecules into cells. When the trans-membrane voltage induced by an external electric field exceeds a certain threshold (normally 0.2–1 V), a rearrangement of the molecular structure of the membrane occurs, leading to pore formation in the membrane and a considerable increase in the cell membrane permeability to ions, molecules and even macromolecules. This phenomenon is, potentially, the basis for many in vivo applications such as electrochemotherapy and gene therapy, but still lacks a comprehensive theoretical basis. This article reviews the state of current electroporation theories and briefly considers current and potential applications in biology and medicine.     

Comparative analysis of the methods of drug and protein delivery for the treatment of cancer,genetic diseases and diagnostics

The methods of protein and drug delivery for the treatment of cancer, genetic diseases and diagnostics were summarized. The potential of protein transduction is discussed and the recent developments in the field are reviewed. An overview is provided of the non-viral delivery methods such as liposomes, polymer-based delivery, cell-penetrating peptides, bacterial secretion, cells, virosomes, physical methods including electroporation, microinjection, osmotic lysis, nanoparticles, sonoporation to locally inject therapeutic molecules. The characteristic properties of non-viral vectors and their use for the delivery of therapeutic molecules for the diagnosis and treatment of disorders and to target tumors are also discussed. The potential of the transduced peptides and proteins was used as new therapeutic compounds against infectious diseases, to complement deficiencies in specific genes, to specifically kill tumour cells, for gene therapy. The protein delivery vectors can enhance the transfection at low concentrations and help to develop future gene delivery systems with reduced toxicity. Vitamin B12, folic acid, biotin, and riboflavin are essential in the treatment of cancer. Ultrasound has a potential in the delivery of therapeutic agents. The new developing technologies of drug delivery and targeting offer the possibility to improve the therapeutic possibilities of the existing drugs and to develop novel therapeutics.     

Screening monoclonal antibodies for cross-reactivity in the ferret model of influenza infection

Influenza virus infections carry a high public health cost, and pandemics are potentially catastrophic. Though the ferret is generally regarded as the best model for human influenza, few reagents are available for the analysis of cellular immunity. We thus screened monoclonal antibodies (mAbs) made for identifying immune cells in other species to see if any were cross-reactive. Flow cytometric analysis of lymphocytes isolated from blood, spleen, and lung of normal and virus-infected ferrets indicated that several mouse mAbs bound to the corresponding antigens in ferrets. Typing bronchoalveolar lavage populations from pneumonic ferrets with mAb to human CD8 showed the massive CD8+ T cell enrichment characteristic of this infection in mice. The availability of this, and several other mAbs that showed cross-reactivity, should allow us to begin the dissection of cell-mediated immunity in the ferret, which, at least from these early results, looks similar to the situation in mice.     

Semi-loose seal Neurobiotin electroporation for combined structural and functional analysis of neurons

Intracellular sharp-electrode, whole-cell patch clamp and juxtacellular labeling methods have previously been developed for combined analysis of neuronal structure and function. We describe a novel electroporation technique for labeling neurons with Neurobiotin, using patch electrodes in a semi-loose seal configuration (R = 100–300 MΩ) with very small amplitude pulses (50 mV). The addition of 2% Neurobiotin to the intracellular solution in the patch electrode reduces the dielectric membrane breakdown voltage threshold by about threefold. The resulting pore formation allows for (1) the stable recording of spontaneous and light-evoked postsynaptic potentials without significant cytoplasmic washout and (2) the passage of dye without spillover. The efficiency and reliability of the method makes it particularly suitable for the serial recording and labeling of multiple neurons in a small area of tissue.     

ETM study of electroporation influence on cell morphology in human malignant melanoma and human primary gingival fibroblast cells

Objective

To estimate electroporation (EP) influence on malignant and normal cells.

Methods

Two cell lines including human malignant melanoma (Me-45) and normal human gingival fibroblast (HGFs) were used. EP parameters were the following: 250, 1 000, 1 750, 2 500 V/cm; 50 µs by 5 impulses for every case. The viability of cells after EP was estimated by MTT assay. The ultrastructural analysis was observed by transmission electron microscope (Zeiss EM 900).

Results

In the current study we observed the intracellular effect following EP on Me-45 and HGF cells. At the conditions applied, we did not observe any significant damage of mitochondrial activity in both cell lines treated by EP. Conversely, we showed that EP in some conditions can stimulate cells to proliferation. Some changes induced by EP were only visible in electron microscopy. In fibroblast cells we observed significant changes in lower parameters of EP (250 and 1 000 V/cm). After applying higher electric field intensities (2 500 V/cm) we detected many vacuoles, myelin-like bodies and swallowed endoplasmic reticulum. In melanoma cells such strong pathological modifications after EP were not observed, in comparison with control cells. The ultrastructure of both treated cell lines was changed according to the applied parameters of EP.

Conclusions

We can claim that EP conditions are cell line dependent. In terms of the intracellular morphology, human fibroblasts are more sensitive to electric field as compared with melanoma cells. Optimal conditions should be determined for each cell line. Summarizing our study, we can conclude that EP is not an invasive method for human normal and malignant cells. This technique can be safely applied in chemotherapy for delivering drugs into tumor cells.     

Chicken HSP70 DNA vaccine inhibits tumor growth in a canine cancer model

Immunization with xenogeneic DNA is a promising cancer treatment to overcome tolerance to self-antigens. Heat shock protein 70 (HSP70) is over-expressed in various kinds of tumors and is believed to be involved in tumor progression. This study tested a xenogeneic chicken HSP70 (chHSP70) DNA vaccine in an experimental canine transmissible venereal tumor (CTVT) model. Three vaccination strategies were compared: the first (PE) was designed to evaluate the prophylactic efficacy of chHSP70 DNA vaccination by delivering the vaccine before tumor inoculation in a prime boost setting, the second (T) was designed to evaluate the therapeutic efficacy of the same prime boost vaccine by vaccinating the dogs after tumor inoculation; the third (PT) was similar to the first strategy (PE), with the exception that the electroporation booster injection was replaced with a transdermal needle-free injection. Tumor growth was notably inhibited only in the PE dogs, in which the vaccination program triggered tumor regression significantly sooner than in control dogs (NT). The CD4⁺ subpopulation of tumor-infiltrating lymphocytes and canine HSP70 (caHSP70)-specific IFN-γ-secreting lymphocytes were significantly increased during tumor regression in the PE dogs as compared to control dogs, demonstrating that specific tolerance to caHSP70 has been overcome. In contrast, no benefit of the therapeutic strategy (T) could be noticed and the (PT) strategy only led to partial control of tumor growth. In summary, antitumor prophylactic activity was demonstrated using the chHSP70 DNA vaccine including a boost via electroporation. Our data stressed the importance of DNA electroporation as a booster to get the full benefit of DNA vaccination but also of cancer immunotherapy initiation as early as possible. Xenogeneic chHSP70 DNA vaccination including an electroporation boost is a potential vaccine to HSP70-expressing tumors, although further research is still required to better understand true clinical potential.     

弓形虫转基因学研究进展

转基因技术在弓形虫的运用加速了对弓形虫功能基因组学的研究。本文对转基因弓形虫载体的构建、转基因方法和转基因弓形虫技术在弓形虫研究中的应用进展加以综述。