Calcineurin A and CaMKIV transactivate PGC-1α promoter, but differentially regulate cytochrome c promoter in rat skeletal muscle

In skeletal muscle, slow-twitch fibers are highly dependent on mitochondrial oxidative metabolism suggesting the existence of common regulatory pathways in the control of slow muscle-specific protein expression and mitochondrial biogenesis. In this study, we determined whether peroxisome proliferator-activated receptor γ co-activator-1α (PGC-1α) could transactivate promoters of nuclear-encoded mitochondrial protein (cytochrome c) and muscle-specific proteins (fast troponin I, MyoD). We also investigated if calcineurin A (CnA) and calcium/calmodulin kinase IV (CaMKIV) were involved in the regulation of PGC-1α and cytochrome c promoter. For this purpose, we took advantage of the gene electrotransfer technique, which allows acute expression of a gene of interest. Electrotransfer of a PGC-1α expression vector into rat Tibialis anterior muscle induced a strong transactivation of cytochrome c promoter (P < 0.001) independent of nuclear respiratory factor 1. PGC-1α gene electrotransfer did not transactivate fast troponin I promoter, whereas it did transactivate MyoD promoter (P < 0.05). Finally, whereas electrotransfers of CnA or CaMKIV expression vectors transactivated PGC-1α promoter (P < 0.001), gene electrotransfer of CaMKIV was only able to transactivate cytochrome c promoter. Taken together, these data suggest that CnA triggers PGC-1α promoter transactivation to drive the expression of non-mitochondrial proteins.     

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.     

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.     

Review of current thermal ablation treatment for lung cancer and the potential of electrochemotherapy as a means for treatment of lung tumours

Lung cancer remains the most common cancer diagnosed worldwide and has one of the lowest survival rates of all cancers. Surgery remains the only curative treatment option but because most patients are either diagnosed at advanced stages or are unfit for surgery, less than a third of all lung cancer patients will undergo a surgical resection. Thermal ablation has emerged as an alternative option in patients who are unfit to undergo surgery. Thermal ablative therapies used in clinical practice to date include Radiofrequency Ablation (RFA), Microwave Ablation (MWA) and Cryoablation This article will focus on the advantages and limitations of thermal ablative therapy and investigates the potential of a relatively new treatment modality, Electrochemotherapy (ECT), as a novel treatment for lung cancer.     

Intraoperative electrochemotherapy of colorectal liver metastases: A prospective phase II study

Background and objectivesA previous pilot study proved the feasibility, safety and efficacy of electrochemotherapy in the treatment of colorectal liver metastases. The aim of this study was to evaluate long-term effectiveness and safety of electrochemotherapy in the treatment of unresectable colorectal liver metastases.Patients and methodsIn this prospective phase II study, patients with metachronous colorectal liver metastases were included. In all patients, at least one metastasis was unresectable due to its central location or a too-small future remnant liver volume. Patients were treated by electrochemotherapy using intravenously administered bleomycin during open surgery. Treated were 84 metastases in 39 patients. Local tumor control, progression-free survival and overall survival were evaluated.ResultsThe objective response was 75% (63% CR, 12% PR). The median duration of the response was 20.8 months for metastases in CR and 9.8 months for metastases in PR. The therapy was significantly more effective for metastases smaller than 3 cm in diameter than for larger ones. There was no difference in response according to the metastatic location, i.e., metastases in central vs. peripheral locations. Progression-free survival was better in patients who responded well to electrochemotherapy compared to those metastases that had a partial response or progressive disease. However, there was no difference in overall survival, with a median of 29.0 months.ConclusionsElectrochemotherapy has proven to be safe and effective in the treatment of colorectal liver metastases, with a durable response. It provides local tumor control that enables patients with unresectable metastases to receive further treatments.     

Enhancement of the immunogenicity of an alphavirus replicon-based DNA vaccine against classical swine fever by electroporation and coinjection with a plasmid expressing porcine interleukin 2

Alphavirus replicon-based DNA vaccines have emerged as a promising approach to generation of antigen-specific immune responses. However, due to their low immunogenicity, there is a need for other approaches to enhance the vaccine potency. In this study, electroporation (EP) and a plasmid expressing porcine interleukin 2 (IL-2) were used to improve the immunogenicity of an alphavirus replicon-based DNA vaccine pSFV1CS-E2 against classical swine fever (CSF). Pigs were immunized with pSFV1CS-E2 alone or together with IL-2 by EP or by simple intramuscular injection. The results showed that EP combined with IL-2 resulted in marked enhancement of E2-specific antibody responses. Moreover, CSFV-specific lymphocyte proliferation, IFN-γ and IL-4 responses were increased significantly in the pSFV1CS-E2+IL-2/EP group. Pigs immunized with pSFV1CS-E2 plus IL-2 by EP were completely protected from lethal challenge, which is comparable to the sterilizing immunity and full protection offered by the live attenuated vaccine C-strain and in contrast with the incomplete protection conferred by pSFV1CS-E2 without or with IL-2 or EP alone, as demonstrated by the presence of pathological changes or/and viral loads. We conclude that EP in combination with IL-2 can significantly improve the immunogenicity of the plasmid DNA vaccine.     

Enhancement of protein vaccine potency by in vivo electroporation mediated intramuscular injection

Protein-based vaccines have emerged as a potentially promising approach for the generation of antigen-specific immune responses. However, due to their low immunogenicity, there is a need for innovative approaches to enhance protein-based vaccine potency. One approach to enhance protein-based vaccine potency is the employment of toll-like receptor ligands, such as CpG oligonucleotides, to activate the antigen-specific T cell immune responses. Another approach involves employing a method capable of improving the delivery of protein-based vaccine intramuscularly to lead to the slow release of the protein, resulting in improved vaccine potency. In the current study, we aimed to determine whether intramuscular injection of protein-based vaccines in conjunction with CpG followed by electroporation can lead to increased delivery of the protein-based vaccine into muscle cells, resulting in enhanced protein-based vaccine potency. We found that intramuscular injection followed by electroporation can effectively transduce the protein-based vaccine into the muscle cells. Furthermore, we found that intramuscular vaccination with OVA protein in combination with CpG followed by electroporation generates the best OVA-specific CD8+ T cell immune responses as well as the best protective and therapeutic antitumor effects in vaccinated mice. CD8+ T cells were found to play an important role in the observed protective antitumor effects generated by the vaccination. Similar results were observed using the HPV-16 E7 protein-based vaccination system. Thus, our data indicate that intramuscular administration of protein-based vaccines in conjunction with CpG followed by electroporation can significantly enhance the antigen-specific CD8+ T cell immune responses. The clinical implications of the study are discussed.     

Comparison of immune responses generated by optimized DNA vaccination against SIV antigens in mice and macaques

Optimized DNA vectors were constructed comprising the proteome of SIV including the structural, enzymatic, regulatory, and accessory proteins. In addition to native antigens as produced by the virus, fusion proteins and modified antigens with altered secretion, cellular localization and stability characteristics were generated. The DNA vectors were tested for expression upon transfection in human cells. In addition, the vectors were tested either alone or in combinations in mice and macaques, which provided an opportunity to compare immune responses in two animal models. DNA only immunization using intramuscular injection in the absence or presence of in vivo electroporation did not alter the phenotype of the induced T cell responses in mice. Although several fusion proteins induced immune responses to all the components of a polyprotein, we noted fusion proteins that abrogated immune response to some of the components. Since the expression levels of such fusion proteins were not affected, these data suggest that the immune recognition of certain components was altered by the fusion. Testing different DNA vectors in mice and macaques revealed that a combination of DNAs producing different forms of the same antigen generated more balanced immune responses, a desirable feature for an optimal AIDS vaccine.     

Complete protection against a H5N2 avian influenza virus by a DNA vaccine expressing a fusion protein of H1N1 HA and M2e

Most influenza vaccines target hemagglutinin (HA) in order to protect the host against infection. However, theses vaccines are strain-specific due to major antigenic variations of HA. Since it is difficult to predict epidemic and pandemic strains of influenza virus, the development of effective vaccines against divergent influenza viruses is urgently needed. Although M2e-based vaccines are associated with weaker protection than HA-based vaccines that induce neutralizing antibodies against challenge virus matched-strain, the extracellular domain of Matrix 2 protein (M2e) is one of a potential broad-spectrum immunogen because it contains highly conserved sequences among influenza A viruses. In this study, M2e sequence was fused to H1N1 HA DNA (M2e-HA) and the immunogenicity and antiviral efficacy of this DNA vaccine was evaluated in response to challenge with a heterosubtypic H5N2 avian influenza virus. Compared to vaccination with HA or M2e DNA alone, vaccination with M2e-HA DNA or combination of M2e DNA and HA DNA (M2e DNA + HA DNA) induced a broad immunity without evidence of immune interference. In addition, HA-specific CD8⁺ and M2e-specific T cell responses elicited by M2e-HA DNA vaccination were significantly higher than those of HA or M2e DNA vaccine alone, respectively. Following challenge with a heterosubtypic influenza virus infection, vaccination with M2e-HA DNA conferred complete protection against mortality. In combination, these results suggest that DNA vaccines expressing a fusion protein, M2e-HA, may provide an attractive approach for the development of broad-spectrum influenza vaccines.     

Effects of menstrual cycle on gene transfection through mouse vagina for DNA vaccine

Human immunodeficiency virus (HIV) infections mainly occur through the vaginal and rectal mucosal membranes. In the present study, to develop a DNA vaginal vaccine against viral and bacterial infections, the effects of the menstrual cycle on DNA transfection through the vaginal mucosa in female mice and transfection enhancement by electroporation, a chelating agent, cell-penetrating peptides (CPP) and nuclear localizing signals (NLS) were investigated. The transfection efficiencies of a marker plasmid DNA (pDNA), pCMV-Luc, on the vaginal mucosal membrane in mice at the stages of metestrus and diestrus were significantly higher than those at the stages of proestrus and estrus. The gene expression was markedly enhanced by electroporation and by pretreatment with the chelating agent. The highest level of expression was obtained by 2h pretreatment with 5% citric acid solution combined with electroporation with 15 pulses at 250 V/cm for 5 milliseconds (ms). Furthermore, a synergistic promoting effect on pDNA transfection was obtained by co-administration of CPP, the Tat peptide analog, and NLS, the NF-kappaB analog. These results indicate that effective DNA vaccination administered through the vaginal tract is possible by selecting the menstrual stage and overcoming the mucosal barrier using a combination of methods that promotes uptake.