电穿孔对原代大鼠肝细胞生物学性状影响

目的 探讨不同电穿孔条件对原代大鼠肝细胞生物学性状的影响,优化外源基因转染原代肝细胞的条件。方法 以低电压（220－400V）、高电容（500－950μF）单脉冲电击原代大鼠肝细胞后接种培养,分别于36h-9d不同时点采用台盼蓝拒染法与MTT法观测肝细胞活力,并检测肝细胞培养上清液中白蛋白（ALB）、ALT与乳酸脱氢酶（LDH）含量。以未电穿孔肝细胞为对照组。结果 培养36h-9d各组肝细胞存活率均高于90％,贴壁率高于95％。培养36h肝细胞上清液中ALB、ALT与LDH值,电穿孔B组（220V,950μF）,C组（400V,950μF）明显高于对照组;但MTT法检测各电穿孔组肝细胞活力与对照组比较差异无显著性。结论 该电穿孔条件不影响肝细胞的存活能力,还能促进肝细胞ALB等蛋白的合成与释放,可作为外源基因转染原代肝细胞的优选方法之一。     

Electrochemotherapy as treatment option for hepatocellular carcinoma,a prospective pilot study

Background and objectives

Electrochemotherapy provides non-thermal ablation of cutaneous as well as deep seated tumors. Based on positive results of the treatment of colorectal liver metastases, we conducted a prospective pilot study on hepatocellular carcinomas with the aim of testing the feasibility, safety and effectiveness of electrochemotherapy.

Processing of tetanus and botulinum A neurotoxins in isolated chromaffin cells

Tetanus and botulinum A neurotoxins were introduced into the cytosol of chromaffin cells by means of an electric field in which the plasma membrane is forced to form pores of approximately 1 m at the sites facing the electrodes. As demonstrated by electron microscopy, both [¹²⁵I] and gold-labelled tetanus toxin (TeTx) diffuse through these transient openings. Dichain TeTx, with its light chain linked to the heavy chain by means of a disulfide bond, causes the block of exocytosis to develop more slowly than does the purified light chain. The disulfide bonds, which in both toxins hold the subunits together, were cleaved by the intrinsic thioredoxin-reductase system. Single chain TeTx, in which the heavy and light chains are interconnected by an additional peptide bond, was far less effective than dichain TeTx at blocking exocytosis, which indicates that proteolysis is the rate-limiting step. The toxins were degraded further to low-molecular weight fragments which, together with intact toxins and subunits, were released by the cells. The intracellular half-life of [¹²⁵ I] dichain TeTx was approximately three days. The number of light-chain molecules required to maintain exocytosis block in a single cell, as calculated by two different methods, was less than 10. The long duration of tetanus poisoning may result from the persistence of intracellular toxin due to a scarcity of free cytosolic proteases. This may also hold for the slow recovery from botulism.     

Characterization of guinea pig T cell responses elicited after EP-assisted delivery of DNA vaccines to the skin

The skin is an ideal target tissue for vaccine delivery for a number of reasons. It is highly accessible, and most importantly, enriched in professional antigen presenting cells. Possessing strong similarities to human skin physiology and displaying a defined epidermis, the guinea pig is an appropriate model to study epidermal delivery of vaccine. However, whilst we have characterized the humoral responses in the guinea pig associated with skin vaccine protocols we have yet to investigate the T cell responses. In response to this inadequacy, we developed an IFN-γ ELISpot assay to characterize the cellular immune response in the peripheral blood of guinea pigs. Using a nucleoprotein (NP) influenza pDNA vaccination regimen, we characterized host T cell responses. After delivery of the DNA vaccine to the guinea pig epidermis we detected robust and rapid T cell responses. The levels of IFN-γ spot-forming units averaged approximately 5000 per million cells after two immunizations. These responses were broad in that multiple regions across the NP antigen elicited a T cell response. Interestingly, we identified a number of NP immunodominant T cell epitopes to be conserved across an outbred guinea pig population, a phenomenon which was also observed after immunization with a RSV DNA vaccine. We believe this data enhances our understanding of the cellular immune response elicited to a vaccine in guinea pigs, and globally, will advance the use of this model for vaccine development, especially those targeting skin as a delivery site.     

Construction of a triple modified p53 containing DNA vaccine to enhance processing and presentation of the p53 antigen

More effective and less toxic treatments are urgently needed in the treatment of patients with cancer. The tumour suppressor protein p53 is a tumour-associated antigen that could serve that purpose when applied in an immunologic approval to cancer. It is mutated in ∼50% of the tumours resulting in p53 overexpression, which can serve as target for therapy. To improve the immunisation results in patients with p53 overexpression tumours we constructed a DNA vaccine that could lead to improved processing and presentation of p53 peptides in the MHC-class I. We constructed a triple modified p53 fusion protein containing DNA vaccine by (1) addition of a xeno-antigen (mouse or rat p53 fragment), (2) potentiation of intra-cytoplasmatic accumulation of p53 by deleting the nuclear signalling part, (3) improving the processing to peptides of p53 by addition of ubiquitin. In-vitro experiments confirmed correct construction of the DNA vaccine. Preliminary testing in normal and HLA-A2 mice of this triple modified p53 containing DNA construct meant for human application showed a trend towards a superior immunogenicity.     

Ostial dimensional changes after pulmonary vein isolation: Pulsed field ablation vs radiofrequency ablation

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The site of administration influences both the type and the magnitude of the immune response induced by DNA vaccine electroporation

We investigated the influence of the site of administration of DNA vaccine on the induced immune response. DNA vaccines were administered by electroporation at three different sites: tibial cranial muscle, abdominal skin and ear pinna. Aiming to draw general conclusions about DNA vaccine delivery, we successively used several plasmids encoding either luciferase and ovalbumin as models or gp160 and P1A as vaccines against HIV and P815 mastocytoma, respectively. Low levels and duration of luciferase transgene expression were observed after electroporation of the abdominal skin, partly explaining its lower immunogenic performance as compared to the other sites of administration. Analyses of OT-I CD8+ and OT-II CD4+ T cell responses highlighted the differential impact of the delivery site on the elicited immune response. Muscle electroporation induced the strongest humoral immune response and both muscle and ear pinna sites induced cellular immunity against gp160. Ear pinna delivery generated the highest level of CTL responses against P1A but electroporation of muscle and ear pinna were equally efficient in delaying P815 growth and improving mice survival. The present study demonstrated that the site of administration is a key factor to be tested in the development of DNA vaccine.     

Efficient gene delivery to articular cartilage using electroporation

Abstract

Effective in vivo gene transfer into articular cartilage has not yet been established. Since chondrocytes are embedded within a rich extracellular matrix, various gene transfer methods have failed to introduce genes into deeper layers of the articular cartilage. In this study, we developed new superfine pointed needle electrodes for in situ electroporation (EP), and investigated the efficiency of gene transfer into articular cartilage with different degrees of degeneration. Full-thickness articular cartilage slices were obtained from the knee joint of a 3–4-month-old rabbit. The cartilage tissues were treated briefly with trypsin to partly remove matrix proteoglycan. Human articular cartilage with different grades of degeneration was also used. For EP, the articular cartilage surface was soaked in a solution containing green fluorescent protein (GFP) plasmid. Then, the superfine pointed 7-needle electrodes were gently stabbed into the surface layer of the articular cartilage and the gene was transfected by an electroporator. GFP expression was examined by immunohistochemical analysis. Cartilage tissue was successfully transfected with the GFP gene by the electrodes and EP. Transfection efficiency was enhanced by depleting the matrix proteoglycan in rabbit articular cartilage. Chondrocytes in the deeper layer of the articular cartilage were also transfected and expressed GFP. In human osteoarthritic cartilage, ca. 30% of the cells in the deeper layer were transfected by selecting optimal EP conditions. No adverse effects of EP on damaged articular cartilage were obvious from histological analysis or TUNEL staining. The results indicated that EP-mediated in vivo gene transfer into articular cartilage may provide a useful therapeutic strategy to treat cartilage degeneration.     

Optimal induction of HPV DNA vaccine-induced CD8 T cell responses and therapeutic antitumor effect by antigen engineering and electroporation

Since human papillomavirus (HPV) E6 and E7 are promising tumor antigens, we engineered E6 and E7 antigens to generate an optimal HPV DNA vaccine by codon optimization (Co), fusion of E6 and E7, addition of a tissue plasminogen activator (tpa) signal sequence, addition of CD40 ligand (CD40L) or Fms-like tyrosine kinase-3 ligand (Flt3L). The resulting constructs were investigated in terms of their antitumor activity as well as induction of HPV-specific CD8⁺ T cell responses. When E6^Co and E7^Co were fused (E67^Co), CD8⁺ T cell responses specific for E6 or E7 antigen decreased, but the preventive antitumor effect rather improved, demonstrating the importance of broad immunity. Interestingly, Flt3L-fused HPV DNA vaccine exhibited stronger E6- and E7-specific CD8⁺ T cell responses as well as therapeutic antitumor effect than that of CD40L linked HPV DNA vaccine. Finally, the optimal construct, tFE67^Co, was generated by including tpa signal sequence, Flt3L, fusion of E6 and E7, and codon optimization, which induces 23 and 25 times stronger E6- and E7-specific CD8⁺ T cell responses than those of initial E67 fusion construct. In particular, inclusion of electroporation in intramuscular immunization of tFE67^Co further enhances HPV-specific CD8⁺ T cell responses, leading to complete tumor regression in a therapeutic setting. Thus, our results provide valuable insight on effective HPV DNA vaccine design and suggest that tFE67^Co delivered with electroporation may be a promising therapeutic HPV DNA vaccine against cervical cancer.