Induction of tumor-specific CTL responses using the C-terminal fragment of Viral protein R as cell penetrating peptide.

The discovery of tumor-associated antigens recognized by T lymphocytes opens the possibility of vaccinating cancer patients with defined antigens. However, one of the major limitation of peptide-based vaccines is the low immunogenicity of antigenic peptides. Interestingly, if these epitopes are directly delivered into the cytoplasm of antigen presenting cells, they can be efficiently presented via the direct MHC class I presentation pathway. To improve antigen entry, one promising approach is the use of cell penetrating peptides (CPPs). However, most studies use a covalent binding of the CPP with the antigen. In the present study, we focused on the C-terminal domain of Vpr which was previously demonstrated to efficiently deliver plasmid DNA into cells. We provide evidence that the peptides Vpr55-91 and Vpr55-82 possess the capacity of delivering proteins and epitopes into cell lines as well as into human primary dendritic cells, without the necessicity for a chemical linkage. Moreover, immunization of HLA-A2 transgenic mice with Vpr55-91 as the sole adjuvant is able to induce antigen-specific cytotoxic T lymphocytes against multiple tumor epitopes.

Polydiacetylenic nanofibers as new siRNA vehicles for in vitro and in vivo delivery.

Polydiacetylenic nanofibers (PDA-Nfs) obtained by photopolymerization of surfactant 1 were optimized for intracellular delivery of small interfering RNAs (siRNAs). PDA-Nfs/siRNA complexes efficiently silenced the oncogene Lim-1 in the renal cancer cells 786-O in vitro. Intraperitoneal injection of PDA-Nfs/siLim1 downregulated Lim-1 in subcutaneous tumor xenografts obtained with 786-O cells in nude mice. Thus, PDA-Nfs represent an innovative system for in vivo delivery of siRNAs.

Severity of ineffective esophageal motility is associated with utilization of skeletal muscle relaxant medications.

BACKGROUND: Ineffective esophageal motility (IEM) is the most common finding on high-resolution esophageal manometry (HREM). The underlying mechanisms for IEM remain to be fully elucidated. The aim of this study was to determine if utilization of skeletal muscle relaxants is associated with IEM, and with more severe subtypes of the disorder. METHODS: Patients with diagnosis of IEM were gender and age matched to patients with normal HREM. Demographic information, symptoms, endoscopic findings, medication usage and medical comorbidities were recorded. Patients with a diagnosis of IEM were divided into subgroups based on mean distal contractile integral (DCI) and percentage of ineffective swallows, and assessed for clinically significant differences among patients with varying severity of underlying IEM. KEY RESULTS: A total of 118 patients were included in each group. There were no significant clinical differences between the group of patients with IEM and the group of patients with normal manometry. Within the group of IEM patients, those with mean DCI < 250 mm Hg/s/cm were more likely to be prescribed skeletal muscle relaxants (27.8% vs 11.0%, P = .044), and those using skeletal muscle relaxants had a larger mean percentage of ineffective swallows (81.1% vs 71.5%, P = .029). There were no significant differences across mean DCI subgroups in usage of any other medication, or in any of the demographic variables or disease comorbidities examined in this study. CONCLUSIONS & INFERENCES: Use of skeletal muscle relaxants is associated with more severe IEM, which may suggest a causal association between this class of medications and weaker esophageal peristalsis.

The role of forensic odontology in the quantification of dental and facial aesthetic impairment. A case report from the civil jurisprudence / El papel de la odontología forense en la valoración del daño estético dental y facial. Un relato de caso de la jurisprudencia civil

Despite uncommon in the scientific literature, the quantification of aesthetic impairment (AI) figures among the procedures performed by forensic dentists in Court. During this procedure, the dentist (as expert witness) must be able to provide a scientific report with forensic evidences to support legal decisions, such as towards indemnifications or not. The present study aims to report a case adjudicated in the civil jurisprudence involving a plaintiff with dental and facial AI that underwent forensic examination with a forensic dentist. A lawsuit founded on financial indemnification was demanded by an adult male against a county in South Brazil. The plaintiff claimed AI after falling with his bicycle into a hole in the asphalt. The expert witness’ report provided to the civil Court indicated dental loss and several maxillofacial lesions observed through the physical exam. The quantification of these lesions suggested substantial severity of AI. After considering all the aspects involved in the case, the civil Court ruled against the county, establishing indemnification of nearly $5,000.00. The present study highlights the important role of dentists as expert witnesses in the civil scenario. Forensic training must be encouraged to enable optimal performances in Court

A pesar de ser poco frecuente en la literatura científica, la valoración del daño estético (DE) figura entre los procedimientos realizados por los odontólogos forenses en la Corte. Durante este procedimiento, el dentista (como perito) debe ser capaz de proporcionar un informe científico con pruebas forenses para apoyar las decisiones legales, como aquellas que implican indemnizaciones. El presente estudio tiene como objetivo informar de un caso juzgado en la jurisprudencia civil que involucra a un demandante con DE odontológico y facial que se sometió a un examen forense. Una demanda fundada en la indemnización financiera fue asegurada por un hombre adulto contra un condado en el sur de Brasil. El demandante reclamó DE después de caer con su bicicleta en un agujero en el asfalto. El informe del perito ante el tribunal civil indicó una pérdida dental y varias lesiones maxilofaciales observadas en el examen físico. La valoración de estas lesiones sugirió gravedad sustancial de la DE. Después de considerar todos los aspectos involucrados en el caso, la Corte Civil decidió contra el condado, estableciendo una indemnización de casi 5.000 dólares. El presente estudio pone de relieve el importante papel de los dentistas como expertos en el escenario civil. El entrenamiento forense debe ser estimulado para permitir un desempeño óptimo en la Corte

NF-kB related transgene expression in mouse tibial cranial muscle after pDNA injection followed or not by electrotransfer.

BACKGROUND: When activated, NF-κB can promote the nuclear import and transcription of DNA possessing NF-κB consensus sequences. Here, we investigated whether NF-κB is involved in the plasmid electrotransfer process. METHODS: Mouse tibial cranial muscles were transfected with plasmids encoding luciferase bearing or not NF-κB consensus sequences. Luciferase transgene expression was evaluated noninvasively by luminescence imaging and the number of pDNA copies in the same muscles by qPCR. RT-PCR of heat shock protein HsP70 mRNA evidenced cell stress. Western blots of phosphorylated IkBα were studied as a marker of NF-κB activation. RESULTS: Intra-muscular injection of a plasmid bearing a weak TATA-like promoter results in a very low muscle transfection level. Electrotransfer significantly increased both the number of pDNA copy and the transgene expression of this plasmid per DNA copy. Insertion of NF-κB consensus sequences into pDNA significantly increased the level of gene expression both with and without electrotransfer. Electrotransfer-induced cellular stress was evidenced by increased HsP70 mRNA. Phosphorylated IκBα was slightly increased by simple pDNA injection and a little more by electrotransfer. We also observed a basal level of phosphorylated IκBα and thus of free NF-κB in the absence of any stimulation. GENERAL SIGNIFICANCE: pDNA electrotransfer can increase transgene expression independently of NF-κB. The insertion of NF-κB consensus sequences into pDNA bearing a weak TATA-like promoter leads to enhanced transgene expression in muscle with or without gene electrotransfer. Finally, our results suggest that the basal amount of free NF-κB in muscle might be sufficient to enhance the activity of pDNA bearing NF-κB consensus sequences.

Pre-treatment of cells with pluronic L64 increases DNA transfection mediated by electrotransfer.

Gene transfer into muscle cells is a key issue in biomedical research. Indeed, it is important for the development of new therapy for many genetic disorders affecting this tissue and for the use of muscle tissue as a secretion platform of therapeutic proteins. Electrotransfer is a promising method to achieve gene expression in muscles. However, this method can lead to some tissue damage especially on pathologic muscles. Therefore there is a need for the development of new and less deleterious methods. Triblock copolymers as pluronic L64 are starting to be used to improve gene transfer mediated by several agents into muscle tissue. Their mechanism of action is still under investigation. The combination of electrotransfer and triblock copolymers, in allowing softening electric field conditions leading to efficient DNA transfection, could potentially represent a milder and more secure transfection method. In the present study, we addressed the possible synergy that could be obtained by combining the copolymer triblock L64 and electroporation. We have found that a pre-treatment of cells with L64 could improve the transfection efficiency. This pre-treatment was shown to increase cell viability and this is partly responsible for the improvement of transfection efficiency. We have then labelled the plasmid DNA and the pluronic L64 in order to gain some insights into the mechanism of transfection of the combined physical and chemical methods. These experiences allowed us to exclude an action of L64 either on membrane permeabilization or on DNA/membrane interaction. Using plasmids containing or not binding sequences for NF-κB and an inhibitor of NF-κB pathway activation we have shown that this beneficial effect was rather related to the NF-κB signalling pathway, as it is described for other pluronics. Finally we address here some mechanistic issues on electrically mediated transfection, L64 mediated membrane permeabilization and the combination of both for gene transfer.

Soluble TNF-α receptor secretion from healthy or dystrophic mice after AAV6-mediated muscle gene transfer.

Muscle is an attractive target because it is easily accessible; it also offers a permissive environment for adeno-associated virus (AAV)-mediated gene transfer and has an abundant blood vascular supply providing an efficient transport system for the secretion of proteins. However, gene therapy of dystrophic muscle may be more difficult than that of healthy tissue because of degenerative-regenerative processes, and also because of the inflammatory context. In this study we followed the expression levels of secreted inhibitors of the proinflammatory tumor necrosis factor (TNF) cytokine after intramuscular (i.m.) injection of AAV6 into dystrophic mdx and healthy C57BL/10 mice. We used two chimeric proteins, namely, the human or murine TNF-soluble receptor I fused with the murine heavy immunoglobulin chain. We conducted an AAV6 dose-response study and determined the kinetics of transgene expression. In addition, we followed the antibody response against the transgenes and studied their expression pattern in the muscle. Our results show that transduction efficiency is reduced in dystrophic muscles as compared with healthy ones. Furthermore, we found that the immune response against the secreted protein is stronger in mdx mice. Together, our results underscore that the pathological state of the muscle has to be taken into consideration when designing gene therapy approaches.

Real-time monitoring of cell transplantation in mouse dystrophic muscles by a secreted alkaline phosphatase reporter gene.

Transplantation of muscle precursor cells (MPCs) is a promising approach for the treatment of muscular dystrophies. However, preclinical and clinical results have shown that the technology is not yet efficient enough for most therapeutic applications. Among the problems that remain unsolved are low cellular survival, poor proliferation and lack of migration of the transplanted cells. One major technical hurdle for the optimization of transplantation protocols is how to follow precisely the fate of the cells after transplantation. In this study, we examined the use of a secreted form of the mouse alkaline phosphatase (mSeAP) enzyme as the reporter system transduced into MPCs using a retroviral vector. We show that circulating mSeAP could be detected in the serum of the transplanted mice at different time points after MPC transplantation. We also found that the level of circulating mSeAP is highly correlated with the number of transplanted cells and that mSeAP is an excellent histological marker. Further, studying the levels of circulating mSeAP compared with the number of muscle fibers positive to mSeAP and to dystrophin, enabled detailed analyses of bottleneck steps for successful transplantation. Taken together, our results show that mSeAP is an excellent quantitative 'real-time' reporter gene for cell therapy preclinical studies.

Rational design of vector and antibiotic peptides using solid-state NMR.

The application of 2H solid-state NMR in determining structure activity relationships and mechanism of action of membrane active peptides is discussed. The enhancement of the disruption of anionic lipids in the membrane by new lead compounds is shown to be a key determinant of both DNA vector and antimicrobial activity.

Synthesis, characterization, and gene transfer application of poly(ethylene glycol-b-ethylenimine) with high molar mass polyamine block.

The study of ethyloxazoline/methyloxazoline (EtOXZ/MeOXZ) copolymerization, initiated by methyl tosylate (MeOTs), showed that (i) incorporation of MeOXZ units into random copolymer becomes effective over DP = 100 and (ii) propagation process proceeds with negligible transfer to monomer up to a DP of 400 despite the presence of MeOXZ in the polymerization medium. These results produced random poly(EtOXZ-co-MeOXZ) copolymers with various molar composition ratios in alkyloxazoline units. The close values found for the comonomer reactivity ratios in acetonitrile (r(1MeOXZ) = 1.18; r(2EtOXZ) = 0.34) implied a random chain organization in short sequences of each repeating unit, which was an important parameter in view of the optimization of their subsequent modification: the alkaline hydrolysis was successfully achieved when the MeOXZ unit content of the polyoxazoline chains reached 75%. Using these results, the diblock copolymer poly(ethylene glycol-b-(ethyloxazoline-co-methyloxazoline)) (poly(EG-b-(EtOXZ-co-MeOXZ))) with high DP was synthesized by cationic copolymerization of EtOXZ/MeOXZ comonomers using CH(3)-PEG(2kDa)-Ts as macroinitiator. The comonomer composition of this new compound was adjusted in order to optimize the hydrolysis step and obtain finally the diblock copolymer poly(ethylene glycol-b-ethylenimine) (poly(EG-b-EI)). The high molar mass of this copolymer was confirmed both by (1)H NMR and SANS measurements. Gene delivery experiments showed that the copolymer has significant DNA transfection capacities.

Linear topology confers in vivo gene transfer activity to polyethylenimines.

Although polyethylenimines (PEIs) are frequently used transfection agents, it is still unclear which of their properties are required for efficient gene delivery. This is even more striking when working in vivo since some PEIs are able to generate significant gene expression, whereas others are not. To facilitate a rational development of compounds with improved transfection activities, studies aimed at identifying the properties involved in the transfection process seem indispensable. In the present work, we investigated how transfection with linear PEI of 22 kDa allows for high reporter gene expression in lungs after intravenous injection, whereas the branched PEI of 25 kDa does not. To this end, we synthesized L-PEI derivatives that are intermediates between linear and branched PEIs. Our results show that the topology plays a crucial role in obtaining in vivo reporter gene expression, whereas the content of primary, secondary, and tertiary amines is only of minor importance.

DNA compaction into new DNA vectors based on cyclodextrin polymer: surface enhanced Raman spectroscopy characterization.

The ability of DNA to bind polycation yielding polyplexes is widely used in nonviral gene delivery. The aim of the present study was to evaluate the DNA compaction with a new DNA vector using Raman spectroscopy. The polyplexes result from an association of a beta-cyclodextrin polymer (polybeta-CD), an amphiphilic cationic connector (DC-Chol or adamantane derivative Ada2), and DNA. The charge of the polymeric vector is effectively controlled by simple addition of cationic connector in the medium. We used surface enhanced Raman spectroscopy (SERS) to characterize this ternary complex, monitoring the accessibility of adenyl residues to silver colloids. The first experiments were performed using model systems based on polyA (polyadenosine monophosphate) well characterized by SERS. This model was then extended to plasmid DNA to study polybeta-CD/Ada2/DNA and polybeta-CD/DC-Chol/DNA polyplexes. The SERS spectra show a decrease of signal intensity when the vector/DNA charge ratio (Z+/-) increases. At the highest ratio (Z+/- = 10) the signal is 6-fold and 3-fold less intense than the DNA reference signal for Ada2 and DC-Chol polyplexes, respectively. Thus adenyl residues have a reduced accessibility as DNA is bound to the vector. Moreover, the SERS intensity variations are in agreement with gel electrophoresis and zeta potential experiments on the same systems. The overall study clearly demonstrates that the cationic charges neutralizing the negative charges of DNA result in the formation of stable polyplexes. In vitro transfection efficiency of those DNA vectors are also presented and compared to the classical DC-Chol lipoplexes (DC-Chol/DNA). The results show an increase of the transfection efficiency 2-fold higher with our vector based on polybeta-CD.

Polyethylenimine-mediated gene delivery: a mechanistic study.

BACKGROUND: Ethylenimine polymers (PEIs) belong to one of the most efficient family of cationic compounds for delivery of plasmid DNA into mammalian cells. The high transfection efficiencies are obtained even in the absence of endosomolytic agents such as fusogenic peptides or chloroquine, which is in contrast to most of the other cationic polymers. It has been hypothesized that the efficiency of PEI is due to its capacity to buffer the endosomes. METHODS: To investigate the importance of the acidification of endosomes during PEI-mediated DNA transfer we used proton pump inhibitors such as bafilomycin A1 and concanamycin A. Moreover, we tested whether PEI is able to destabilize natural membranes per se at neutral or acidic pH by performing erythrocyte lysis assays. RESULTS: PEI-mediated transfection in the presence of bafilomycin A1 resulted in a 7-74-fold decrease in reporter gene expression depending on the cell line used. In contrast, the efficiency of the monocationic lipid, DOTAP, was not importantly altered in the presence of the drug. Furthermore, the present data show that PEI cannot destabilize erythrocyte membranes, even at acidic pH, and that PEI, complexed or not to DNA, can increase the transfection efficiency of the cationic polymer, polylysine, when added at the same time to the cells. CONCLUSIONS: The transfection efficiency of PEIs partially relies on their ability to capture the protons which are transferred into the endosomes during their acidification. In addition, PEI is able to deliver significant amounts of DNA into cells and the DNA complexes involved in the expression of the transgene escape within 4 h from the endosomes.

Efficient DNA transfection mediated by the C-terminal domain of human immunodeficiency virus type 1 viral protein R.

Viral protein R (Vpr) of human immunodeficiency virus type 1 is produced late in the virus life cycle and is assembled into the virion through binding to the Gag protein. It is known to play a significant role early in the viral life cycle by facilitating the nuclear import of the preintegration complex in nondividing cells. Vpr is also able to interact with nucleic acids, and we show here that it induces condensation of plasmid DNA. We have explored the possibility of using these properties in DNA transfection experiments. We report that the C-terminal half of the protein (Vpr(52-96)) mediates DNA transfection in a variety of human and nonhuman cell lines with efficiencies comparable to those of the best-known transfection agents. Compared with polylysine, a standard polycationic transfection reagent, Vpr(52-96) was 10- to 1,000-fold more active. Vpr(52-96)-DNA complexes were able to reach the cell nucleus through a pH-independent mechanism. These observations possibly identify an alternate pathway for DNA transfection.

Glycofection in the presence of anionic fusogenic peptides: a study of the parameters affecting the peptide-mediated enhancement of the transfection efficiency.

Gene delivery mediated by polyplexes such as DNA complexed with polylysine conjugates is limited by the low efficiency of escape of DNA from the endosomes. One of the strategies which favors the transmembrane passage of polyplexes consists of adding anionic amphipathic peptides capable of destabilizing membranes in an acidic medium. Although less efficient than replication-defective adenoviruses, fusogenic peptides increase the expression of the reporter gene by a factor between 100 and 1000 depending on the cell line. However, the activity of a given peptide depends on the composition of the lipid bilayer. We were interested in developing a polyplex (glycoplex) formulation comprising a glycosylated polylysine, a fusogenic peptide and a plasmid which would be useful for efficient transfection (glycofection) of a large panel of cells, even in the presence of serum. We synthesized several peptides and tested their efficiency in combination with different glycoplex formulations. We found that glycofection with a quaternary complex (called one pot formulation) made of lactosylated-polylysine, polylysine, DNA, and the dimeric peptide (E5-WYGG)2-KA was less cell-type dependent than other peptide-based formulations. In addition, its efficiency was not affected by the presence of serum (up to 20%).

Influence of the DNA complexation medium on the transfection efficiency of lipospermine/DNA particles.

Dioctadecylamidoglycylspermine (DOGS, Transfectam) is a cationic lipid able to interact with DNA to form complexes that mediate efficient gene transfer into various eukaryotic cells. The state of condensation of the plasmid changes with the medium composition. We therefore investigated to what extent the DNA condensation buffer influences the transfection efficiency of Transfectam/DNA particles. Our results show that in a variety of cell lines, a greater than 100-fold difference in luciferase gene expression is observed with Transfectam/DNA complexes at a +/- charge ratio of 0.75 depending on the conditions of complex formation. The best transfection conditions consisted of particles formed in RPMI medium, NaHCO3/Na2HPO4 or sodium citrate solutions. Mixing in a 150 mM sodium chloride solution (as recommended) resulted in lower gene expression. When the helper lipid 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) was present in the DNA/cationic lipid formulation, the increase in reporter activity was also observed, although to a lower extent. Thus, choosing the optimal conditions for formulating DNA/lipid complexes considerably reduces the amount of lipid and DNA needed to obtain maximum gene transfer.

Membrane permeabilization and efficient gene transfer by a peptide containing several histidines.

We designed a peptide, H5WYG (GLFHAIAHFIHGGWHGLIHGWYG), that permeabilizes cell membrane at a slightly acidic pH but not at neutral pH. Absorbance, fluorescence, and circular dichroism spectra showed that H5WYG undergoes a dramatic conformational change between pH 7.0 and 6.0 that correlates with the protonation of the histidyl residues. Cell permeabilization studies monitored by flow cytometry on living cells showed that H5WYG permeabilizes the cell membrane with a great efficiency at pH 6.4 but was not active at neutral pH; at pH 6.8, the peptide permeabilized 50% of the cells at 20 degrees C within 10 min. H5WYG increased the expression of genes transferred to cells as glycosylated polylysine-DNA complexes, and the transfection efficiency was not impaired in the presence of serum. Therefore, this peptide containing several histidines that become positively charged when the pH decreased to less than 7.0 is a suitable helper for delivering molecules into the cytosol upon either permeabilization of the plasma membrane induced by lowering the extracellular medium to pH 6.4 or permeabilization of the endosomal membrane induced by acidification of endosomes.

Comparative affinity of synthetic multi-antennary galactosyl derivatives for the Gal/GalNAc receptor of rat hepatocytes and peritoneal macrophages.

The binding affinity of synthetic bi- and triantennary galactose ligands (Kichler, A. and Schuber, F. (1995) Glycoconj. Chem., 12, 275 281) has been determined for the Gal/GalNAc receptors of rat hepatocytes and macrophages. The highest affinities were observed with the triantennary structures, in agreement with the clustering effect known to occur with more complex oligosaccharide structures. However, these ligands present very similar affinities for the receptors of both cell types and thus lack the necessary selectivity for specific hepatocyte targeting.

Coupling of cell-binding ligands to polyethylenimine for targeted gene delivery.

Recently the high transfection potential of the cationic polymer polyethylenimine (PEI) was described (Boussif O et al. Proc Natl Acad Sci USA 1995; 92: 7297-7301). To combine the promising DNA delivering activity of PEI with the concept of receptor-mediated gene delivery, cell-binding ligands (transferrin or antiCD3 antibody) were incorporated by covalent linkage to PEI. DNA complexes of PEI or ligand-PEI conjugates were tested for transfection of cultured neuroblastoma Neuro 2A cells, melanoma B16 or H225 cells, erythroid leukemic K562 cells and T cell leukemia Jurkat E6.1 cells. Depending on the cell line, incorporation of the cell-binding ligand resulted in an up to 1000-fold increased transfection efficiency. This activity depends on ligand-receptor interaction and was observed also at low PEI cation:DNA anion ratios where ligand-free PEI lacks efficiency. Depending on the cell-binding ligand, specific targeting (CD3 antibody, Jurkat cells) can be achieved. Gene transfer can be augmented by the addition of an endosome-destabilizing influenza peptide, but is not dependent on the presence of additional endosomolytic agents. Application of transferrin-PEI for the production of murine interleukin-2 in B16 cells resulted in exceptionally high secretion rates of 19 micrograms IL-2 protein per 10(6) cells per 24 h.