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Find video protocols related to scientific articles indexed in Pubmed.
Secure and effective gene delivery system of plasmid DNA coated by polynucleotide.
J Drug Target
PUBLISHED: 08-22-2014
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Abstract Polynucleotides are anionic macromolecules which are expected to transfer into the targeted cells through specific uptake mechanisms. So, we developed polynucleotides coating complexes of plasmid DNA (pDNA) and polyethylenimine (PEI) for a secure and efficient gene delivery system and evaluated their usefulness. Polyadenylic acid (polyA), polyuridylic acid (polyU), polycytidylic acid (polyC), and polyguanylic acid (polyG) were examined as the coating materials. pDNA/PEI/polyA, pDNA/PEI/polyU, and pDNA/PEI/polyC complexes formed nanoparticles with a negative surface charge although pDNA/PEI/polyG was aggregated. The pDNA/PEI/polyC complex showed high transgene efficiency in B16-F10 cells although there was little efficiency in pDNA/PEI/polyA and pDNA/PEI/polyU complexes. An inhibition study strongly indicated the specific uptake mechanism of pDNA/PEI/polyC complex. Polynucleotide coating complexes had lower cytotoxicity than pDNA/PEI complex. The pDNA/PEI/polyC complex showed high gene expression selectively in the spleen after intravenous injection into mice. The pDNA/PEI/polyC complex showed no agglutination with erythrocytes and no acute toxicity although these were observed in pDNA/PEI complex. Thus, we developed polynucleotide coating complexes as novel vectors for clinical gene therapy, and the pDNA/PEI/polyC complex as a useful candidate for a gene delivery system.
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Novel siRNA delivery system using a ternary polymer complex with strong silencing effect and no cytotoxicity.
Biol. Pharm. Bull.
PUBLISHED: 08-05-2014
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We developed a novel small interfering RNA (siRNA) delivery system using a ternary complex with polyethyleneimine (PEI) and ?-polyglutamic acid (?-PGA), which showed silencing effect and no cytotoxicity. The binary complexes of siRNA with PEI were approximately 73-102 nm in particle size and 45-52 mV in ?-potential. The silencing effect of siRNA/PEI complexes increased with an increase of PEI, and siRNA/PEI complexes with a charge ratio greater than 16 showed significant luciferase knockdown in a mouse colon carcinoma cell line regularly expressing luciferase (Colon26/Luc cells). However, strong cytotoxicity and blood agglutination were observed in the siRNA/Lipofectamine complex and siRNA/PEI16 complex. Recharging cationic complexes with an anionic compound was reported to be a promising method for overcoming these toxicities. We therefore prepared ternary complexes of siRNA with PEI (charge ratio 16) by the addition of ?-PGA to reduce cytotoxicity and deliver siRNA. As expected, the cytotoxicity of the ternary complexes decreased with an increase of ?-PGA content, which decreased the ?-potential of the complexes. A strong silencing effect comparable to siRNA/Lipofectamine complex was discovered in ternary complexes including ?-PGA with an anionic surface charge. The high incorporation of ternary complexes into Colon26/Luc cells was confirmed with fluorescence microcopy. Having achieved knockdown of an exogenously transfected gene, the ability of the complex to mediate knockdown of an endogenous housekeeping gene, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), was assessed in B16-F10 cells. The ternary complex (siRNA/PEI16/?-PGA12 complex) exhibited a significant GAPDH knockdown effect. Thus, we developed a useful siRNA delivery system.
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Radiolabeled ?-polyglutamic acid complex as a nano-platform for sentinel lymph node imaging.
J Control Release
PUBLISHED: 07-15-2014
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We established a ternary anionic complex constructed with polyamidoamine dendrimer (4th generation; G4) modified with chelating agents (diethylenetriamine pentaacetic acid (DTPA) derivative), polyethyleneimine (PEI), and ?-polyglutamic acid (PGA) as a safe nano-platform for molecular imaging. We prepared indium-111-labeled DTPA-G4/PEI/?-PGA, and evaluated the effectiveness as a nuclear imaging probe for sentinel lymph node (LN), the first LN that drains the primary tumor. (111)In-DTPA-G4/PEI with strong cationic charge agglutinated with erythrocytes and showed extremely high cytotoxicity. By contrast, the anionic (111)In-DTPA-G4/PEI/?-PGA had little agglutination activity with erythrocytes and no cytotoxicity, indicating their high biocompatibility. (111)In-DTPA-G4/PEI/?-PGA was highly taken up by macrophage cells (high populations in LNs) comparable to (111)In-DTPA-G4/PEI. The uptake mechanisms of (111)In-DTPA-G4/PEI/?-PGA were suggested to be both phagocytosis and ?-PGA-specific pathway. Upon administration of each (111)In-labeled nano-platform into rat footpads intradermally, significantly higher radioactivity of (111)In-DTPA-G4/PEI/?-PGA was observed in the first draining popliteal LN when compared with that of (111)In-DTPA-G4/PEI. Moreover, (111)In-DTPA-G4/PEI/?-PGA clearly visualized the sentinel LN with single photon emission computed tomography (SPECT) compared with (111)In-DTPA-G4/PEI. Thus, (111)In-DTPA-G4/PEI/?-PGA can be useful as a nano-platform for molecular imaging including sentinel LN imaging.
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A Randomized Prospective Study of Bowel Preparation for Colonoscopy with Low-Dose Sodium Phosphate Tablets versus Polyethylene Glycol Electrolyte Solution.
Gastroenterol Res Pract
PUBLISHED: 06-26-2014
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Optimal bowel preparation is essential for the safety and outcome of colonoscopy. A solution containing polyethylene glycol (PEG) is often used as a bowel cleansing agent, but some patients are intolerant of PEG, and this may lead to discontinuation of colonoscopy. Sodium phosphates (NaP) tablets are designed to improve patient acceptance and compliance. The objective of this study was to compare bowel preparation efficiency and patient acceptance of a 30?NaP tablet preparation (L-NaP) and a 2?L?PEG preparation. Patients were randomized into either the L-NaP or PEG group. The primary endpoint was the efficiency of colon cleansing as assessed by a validated four-point scale according to the Aronchick scale by endoscopists and was verified by blinded investigators. The secondary endpoints were patients' tolerability and acceptance. Colon-cleansing efficiency was not significantly different between the two preparations. However, patients' overall judgment was significantly in favor of L-NaP, reflecting better acceptance of L-NaP than PEG. Additionally, more patients favored L-NaP over PEG in a hypothetical future occasion requiring colonoscopy.
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The Relationship between H. pylori Infection and Osteoporosis in Japan.
Gastroenterol Res Pract
PUBLISHED: 04-30-2014
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Background and Objective. H. pylori infection causes a chronic inflammation in the gastric mucosa. However, this local inflammation may result in extra-digestive conditions. Our aim is to investigate the relationship between H. pylori infection and osteoporosis in Japan. Methods. This cross-sectional study was conducted among outpatients at the Juntendo University Hospital between 2008 and 2014. Participants for patient profile, H. pylori infection status, comorbidity, internal medical therapies, lumbar dual-energy X-ray absorptiometry (DXA), and bone turnover marker were collected and upper gastrointestinal endoscopy for reflux esophagitis, hiatal hernia, peptic ulcer disease (PUD), and endoscopic gastric mucosal atrophy (EGA) was performed. The diagnosis of osteoporosis was performed in accordance with the Japanese criteria. We investigated risk factors of osteoporosis. Results. Of the eligible 200 study subjects, 41 cases were of osteoporosis. Bivariate analysis showed that age, being female, BMI, alcohol, smoking, H. pylori, bone-specific ALP, PUD, and EGA were related to osteoporosis. Multivariate analysis showed that age (OR 1.13; 95%CI 1.07-1.20), being female (OR 4.77; 95%CI 1.78-12.77), BMI (OR 0.79; 95%CI 0.68-0.92), H. pylori (OR 5.33; 95%CI 1.73-16.42), and PUD (OR 4.98; 95%CI 1.51-16.45) were related to osteoporosis. Conclusions. H. pylori infection may be a risk factor of osteoporosis in Japan.
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Altered brain response to others? pain in major depressive disorder.
J Affect Disord
PUBLISHED: 04-24-2014
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Empathy has a central role in successful interpersonal engagement. Several studies have reported altered empathy in major depressive disorder (MDD), which could lead to interpersonal difficulties. However, the neural basis of altered empathy in the disorder is still largely unknown. To address this, we performed functional magnetic resonance imaging that tested empathy for others? pain in MDD patients.
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Biodegradable nanoparticles composed of dendrigraft poly-L-lysine for gene delivery.
Eur J Pharm Biopharm
PUBLISHED: 04-23-2014
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We developed novel gene vectors composed of dendrigraft poly-L-lysine (DGL). The transgene expression efficiency of the pDNA/DGL complexes (DGL complexes) was markedly higher than that of the control pDNA/poly-L-lysine complex. However, the DGL complexes caused cytotoxicity and erythrocyte agglutination at high doses. Therefore, ?-polyglutamic acid (?-PGA), which is a biodegradable anionic polymer, was added to the DGL complexes to decrease their toxicity. The resultant ternary complexes (DGL/?-PGA complexes) were shown to be stable nanoparticles, and those with ?-PGA to pDNA charge ratios of >8 had anionic surface charges. The transgene expression efficiency of the DGL/?-PGA complexes was similar to that of the DGL complexes; however, they exhibited lower cytotoxicity and did not induce erythrocyte agglutination at high doses. After being intravenously administered to mice, the DGL6 complex demonstrated high transfection efficiency in the liver, lungs, and spleen, whereas the DGL6/?-PGA8 complex only displayed high transfection efficiency in the spleen. Future studies should examine the utility of DGL and DGL/?-PGA complexes for clinical gene therapy.
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Hepatic gene delivery system electrostatically assembled with glycyrrhizin.
Mol. Pharm.
PUBLISHED: 04-21-2014
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In this study, a novel liver-targeted gene delivery vector was developed by electrostatically coating the cationic complex of pDNA and polyethylenimine (PEI) with glycyrrhizin (GL). The ternary complex, pDNA/PEI/GL, had approximately 100 nm stable particles with a negative charge surface. pDNA/PEI/GL showed high gene expression comparable to that of the complex of pDNA and PEI (pDNA/PEI) in human hepatoma cell line HepG2 without cytotoxicity and agglutination. After intravenous injection of pDNA/PEI/GL into mice, the highest gene expression was observed in the liver. pDNA/PEI/GL showed significantly higher gene expression in parenchymal cells than in nonparenchymal cells. On the basis of these results, we evaluated the pharmacological activity of the ternary complex including the pDNA encoding insulin (pCMV-Ins). The pCMV-Ins/PEI/GL decreased blood glucose concentrations 24 h after its intravenous administration to mice. The ternary complex of pDNA, PEI, and GL may be a promising liver-targeted gene vector.
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Ternary complex of plasmid DNA electrostatically assembled with polyamidoamine dendrimer and chondroitin sulfate for effective and secure gene delivery.
Biol. Pharm. Bull.
PUBLISHED: 04-04-2014
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The purpose of this study was to develop a ternary complex of plasmid DNA (pDNA) electrostatically assembled with polyamidoamine (PAMAM) dendrimer and chondroitin sulfate (CS) for effective and secure gene delivery. PAMAM dendrimers are new cationic polymers that are expected to be used as gene delivery vectors. However, cationic non-viral gene vectors showed cytotoxicity by binding to negative cellular membranes. We therefore prepared a ternary complex by adding CS, an anionic polymer, and examined its usefulness. The pDNA/PAMAM dendrimer complex (PAMAM dendriplex) and the PAMAM dendriplex coated by CS (CS complex) showed nanoparticles with positive ?-potential and negative ?-potential, respectively. The CS complex had no cytotoxicity against B16-F10 cells and no agglutination activity, although severe cytotoxicity and high agglutination were observed in the PAMAM dendriplex. As a result of an in vitro gene expression study of B16-F10 cells, not only the PAMAM dendriplex but also the CS complex showed high transfection efficiency. The transfection efficiency of the CS complex was significantly inhibited by clathrin-mediated endocytosis inhibitor (chlorpromazine), caveolae-mediated endocytosis inhibitor (genistein), and hypothermia. Tail-vein injection of the CS complex into mice led to significantly higher gene expression in the spleen than the PAMAM dendriplex. Thus, the ternary complex of pDNA electrostatically assembled with PAMAM denriplex and CS showed safe high gene expression in the spleen. This vector is expected to be useful for useful gene delivery.
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Influence of different additives and their concentrations on corneal toxicity and antimicrobial effect of benzalkonium chloride.
Cornea
PUBLISHED: 03-13-2014
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The aim of this study was to examine the ophthalmic additives responsible for modulating acute corneal epithelial toxicity induced by benzalkonium chloride (BAC) and investigate the ability of polyoxyethylene hydrogenated castor oil 40 (HCO-40) and polysorbate 80 (PS-80) to reduce the corneal toxicity and antimicrobial effects of BAC.
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Pentosan polysulfate treatment ameliorates motor function with increased serum soluble vascular cell adhesion molecule-1 in HTLV-1-associated neurologic disease.
J. Neurovirol.
PUBLISHED: 02-20-2014
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The main therapeutic strategy against human T lymphotropic virus type I (HTLV-I)-associated myelopathy/tropical spastic paraparesis (HAM/TSP) characterized by lower extremity motor dysfunction is immunomodulatory treatment, with drugs such as corticosteroid hormone and interferon-?, at present. However, there are many issues in long-term treatment with these drugs, such as insufficient effects and various side effects. We now urgently need to develop other therapeutic strategies. The heparinoid, pentosan polysulfate sodium (PPS), has been safely used in Europe for the past 50 years as a thrombosis prophylaxis and for the treatment of phlebitis. We conducted a clinical trial to test the effect of subcutaneous administration of PPS in 12 patients with HAM/TSP in an open-labeled design. There was a marked improvement in lower extremity motor function, based on reduced spasticity, such as a reduced time required for walking 10 m and descending a flight of stairs. There were no significant changes in HTLV-I proviral copy numbers in peripheral blood contrary to the inhibitory effect of PPS in vitro for intercellular spread of HTLV-I. However, serum soluble vascular cell adhesion molecule (sVCAM)-1 was significantly increased without significant changes of serum level of chemokines (CXCL10 and CCL2). There was a positive correlation between increased sVCAM-1and reduced time required for walking 10 m. PPS might induce neurological improvement by inhibition of chronic inflammation in the spinal cord, through blocking the adhesion cascade by increasing serum sVCAM-1, in addition to rheological improvement of the microcirculation. PPS has the potential to be a new therapeutic tool for HAM/TSP.
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Enantioselective formal ?-allylation of nitroalkanes through a chiral iminophosphorane-catalyzed Michael reaction-Julia-Kocienski olefination sequence.
Chem. Commun. (Camb.)
PUBLISHED: 02-20-2014
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A two-step sequence for the asymmetric formal ?-allylation of nitroalkanes is disclosed. This new methodology relies on the development of a highly diastereo- and enantioselective conjugate addition of nitroalkanes to vinylic 2-phenyl-1H-tetrazol-5-ylsulfones using chiral triaminoiminophosphorane as a requisite base catalyst and subsequent Julia-Kocienski olefination under kinetic conditions.
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Kidney-selective gene transfection using anionic bubble lipopolyplexes with renal ultrasound irradiation in mice.
Nanomedicine
PUBLISHED: 02-14-2014
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This study assessed the ability of a new ultrasound (US) responsive gene delivery carrier, bubble lipopolyplexes, to deliver genes to the kidneys. The bubble lipopolyplexes showed highly selective gene expression in kidney tubules, but only after renal irradiation with US. These bubble lipopolyplexes, however, did not increase the expression of biomarkers of kidney injury, including blood urea nitrogen, serum creatinine, kidney injury molecule-1 mRNA, and clusterin mRNA, or induce any histopathological abnormalities in the kidney. Furthermore, pDNA containing CMV early enhancer/chicken beta-actin promoter prolonged gene expression by the bubble lipopolyplexes in the kidney for 42days. This novel renal gene delivery method, in which transfection of bubble lipopolyplexes was followed by US irradiation of the kidneys, resulting in cell-selective, high, and long-term gene expression without renal injury in mice, may have future applications in patient treatment.
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A randomized prospective study comparing the efficacy of on-demand therapy versus continuous therapy for 6 months for long-term maintenance with omeprazole 20 mg in patients with gastroesophageal reflux disease in Japan.
Scand. J. Gastroenterol.
PUBLISHED: 01-21-2014
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To assess the efficacy of continuous therapy (cont) and on-demand therapy (on-demand) as maintenance therapy for gastroesophageal reflux disease (GERD).
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Nanoparticle formulation enhanced protective immunity provoked by PYGPI8p-transamidase related protein (PyTAM) DNA vaccine in Plasmodium yoelii malaria model.
Vaccine
PUBLISHED: 01-02-2014
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We have previously reported the new formulation of polyethylimine (PEI) with gamma polyglutamic acid (?-PGA) nanoparticle (NP) to have provided Plasmodium yoelii merozoite surface protein-1 (PyMSP-1) plasmid DNA vaccine with enhanced protective cellular and humoral immunity in the lethal mouse malaria model. PyGPI8p-transamidase-related protein (PyTAM) was selected as a possible candidate vaccine antigen by using DNA vaccination screening from 29 GPI anchor and signal sequence motif positive genes picked up using web-based bioinformatics tools; though the observed protection was not complete. Here, we observed augmented protective effect of PyTAM DNA vaccine by using PEI and ?-PGA complex as delivery system. NP-coated PyTAM plasmid DNA immunized mice showed a significant survival rate from lethal P. yoelii challenge infection compared with naked PyTAM plasmid or with NP-coated empty plasmid DNA group. Antigen-specific IgG1 and IgG2b subclass antibody levels, proportion of CD4 and CD8T cells producing IFN-? in the splenocytes and IL-4, IFN-?, IL-12 and TNF-? levels in the sera and in the supernatants from ex vivo splenocytes culture were all enhanced by the NP-coated PyTAM DNA vaccine. These data indicates that NP augments PyTAM protective immune response, and this enhancement was associated with increased DC activation and concomitant IL-12 production.
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Secure splenic delivery of plasmid DNA and its application to DNA vaccine.
Biol. Pharm. Bull.
PUBLISHED: 11-06-2013
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In this experiment, we developed a novel safe and effective gene delivery vector coated with ?-polyglutamic acid (?-PGA-coated complexes). The ?-PGA-coated complex was composed of chiseled spherical nano-particles with anionic charges. The plasmid DNA/polyethyleneimine complex (non-coated complex) showed high transgene efficiency in the spleen and lung after intravenous administration in mice, with high liver toxicity and lethality. On the other hand, ?-PGA-coated complex selectively showed high transgene efficiency in the spleen without such toxicity. Furthermore, the ?-PGA-coated complex highly accumulated and showed high gene expression in the marginal zone of the spleen. Those results strongly indicated that ?-PGA-coated complex was suitable as a DNA vaccine vector. We therefore applied ?-PGA-coated complex to melanoma DNA vaccine, pUb-M. The ?-PGA-coated complex containing pUb-M significantly inhibited the growth and metastasis of a melanoma cell line, B16-F10 cells. In conclusion, we developed a splenic gene vector, ?-PGA-coated complex, as a novel technology for clinical vaccination.
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Ternary complex of plasmid DNA with protamine and ?-polyglutamic acid for biocompatible gene delivery system.
Biol. Pharm. Bull.
PUBLISHED: 11-06-2013
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The purpose of the present study was to investigate the usefulness of the ternary complex with protamine and ?-polyglutamic acid (?-PGA), which are biodegradable materials for foods and medical products, as a safe gene delivery vector. We formed cationic binary complexes (plasmid DNA (pDNA)/protamine complexes) with high transfection efficiency. The binary complex showed slight toxicity probably related to its total cationic charge. Then, we formed ternary complexes (pDNA/protamine/?-PGA complexes) by addition of anionic polymer, ?-PGA, and they showed no cytotoxicity. The transfection efficiency of the pDNA/protamine/?-PGA complexes was as high as that of the pDNA/protamine complexes, although their zeta potentials were different. Inhibition study of the gene expressions in B16-F10 cells suggested that pDNA/protamine complexes were taken up by caveolae-mediated endocytosis and macropinocytosis. On the other hand, pDNA/protamine/?-PGA complexes were taken up by clathrin-mediated endocytosis and macropinocytosis. Thus, we succeeded in developing the ternary complex as a safe gene delivery vector with biocompatible materials.
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A case of neuroendocrine tumor G1 with unique histopathological growth progress.
World J Gastrointest Endosc
PUBLISHED: 10-24-2013
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A gastric neuroendocrine tumor (NET) is generated from deep within the tissue mucosal layers. In many cases, NETs are discovered as submucosal tumor (SMT)-like structures by forming a tumor mass. This case has a clear mucosal demarcation line and developed like a polyp. A dilated blood vessel was found on the surface. The mass lacked the yellow color characteristic of NETs, and a SMT-like form was evident. Therefore, a nonspecific epithelial lesion was suspected and we performed endoscopy with magnifying narrow-band imaging (M-NBI). However, this approach did not lead to the diagnosis, as we diagnosed the lesion as a NET by biopsy examination. The lesion was excised by endoscopic submucosal dissection. The histopathological examination proved that the lesion was a polypoid lesion although it was also a NET because the tumor cells extended upward through the normal gland ducts scatteredly. To our knowledge, there is no previous report of NET G1 with such unique histopathological growth progress and macroscopic appearance shown by detailed examination using endoscopy with M-NBI.
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Development of anionic bubble lipopolyplexes for efficient and safe gene transfection with ultrasound exposure in mice.
J Control Release
PUBLISHED: 09-29-2013
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Anionic bubble lipopolyplexes have been developed as anionic ultrasound (US)-responsive gene delivery carriers with biocompatible compounds for efficient and safe transfection in mice. The particles of the anionic bubble lipopolyplexes were approximately 450-600nm with an anionic surface charge. In the absence of US exposure, the bubble lipopolyplexes showed extremely low gene expression in the human vascular endothelial cell line EAhy926. The anionic bubble lipopolyplexes, however, delivered pDNA into cells without endocytosis and showed markedly high gene expression following US exposure. The anionic bubble lipopolyplexes showed little cytotoxicity in EAhy926 cells and little aggregation with erythrocytes. Following intravenous administration into mice, the anionic bubble lipopolyplexes showed high levels of gene expression in the liver, kidney, and spleen only after US exposure to the abdominal area. The level of gene expression in liver non-parenchymal cells was significantly higher than that in parenchymal cells. In addition, the anionic bubble lipopolyplexes did not show any severe hepatic toxicity and did not enhance the production of proinflammatory cytokines. Overall, we have succeeded in preparing anionic bubble lipopolyplexes for efficient and safe transfection with US exposure in mice.
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[Second-line therapy for Helicobacter pylori].
Nippon Rinsho
PUBLISHED: 08-24-2013
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Currently, the recommended second-line eradication regimen for Helicobacter pylori is a quadruple therapy consisting of PPI, bismuth, tetracycline and metronidazole. Recently, levofloxacin-based (PPI, levofloxacin and amoxicillin) and moxifloxacin-based (PPI, moxifloxacin and amoxicillin) triple therapy represent promising alternative. In Japan, PPI-based triple therapy (PPI, amoxicillin and metronidazole) is used for second-line regimen after failure of first-line eradication. The rate of second-line eradication is still high(approximately 90% (ITT)) and triple therapy with PPI, AMPC, and MNZ may still be considered useful for second-line eradication therapy of Helicobacter pylori in Japan. High-dose dual therapy with PPI and amoxicillin for 2 weeks may be useful treatment strategy after failure of first-line eradication.
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Trends of second-line eradication therapy for Helicobacter pylori in Japan: a multicenter study in the Tokyo metropolitan area.
Helicobacter
PUBLISHED: 06-18-2013
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In Japan, the eradication rate of first-line therapy for Helicobacter pylori (H. pylori) with a proton pump inhibitor (PPI), amoxicillin (AMPC) and clarithromycin (CAM) has been decreasing because of a high prevalence of CAM resistance. A possible decrease of the eradication rate for second-line therapy with a PPI, AMPC and metronidazole (MNZ) is of concern. The aim of this study is to assess the trends in second-line eradication therapy for H. pylori in Japan.
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Safe and effective delivery of small interfering RNA with polymer- and liposomes-based complexes.
Biol. Pharm. Bull.
PUBLISHED: 06-04-2013
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We developed binary and ternary complexes based on polymers and liposomes for safe and effective delivery of small interfering RNA (siRNA). Anti-luciferase siRNA was used as a model of nucleic acid medicine. The binary complexes of siRNA were prepared with cationic polymers and cationic liposomes such as polyethylenimine (PEI), polyamidoamine (PAMAM) dendrimer, poly-L-arginine (PLA), trimethyl[2,3-(dioleoxy)-propyl]ammonium chloride (DOTMA), and cholesteryl 3?-N-(dimetylaminnoethyl)carbamate hydrochloride (DC-Chol). The ternary complexes were constructed by the addition of ?-polyglutamic acid (?-PGA) to the binary complexes. The complexes were approximately 54-153 nm in particle size. The binary complexes showed a cationic surface charge although an anionic surface charge was observed in the ternary complexes. The polymer-based complexes did not show a silencing effect in the mouse colon carcinoma cell line expressing luciferase regularly (Colon26/Luc cells). The binary complexes based on liposomes and their ternary complexes coated by ?-PGA showed a significant silencing effect. The binary complexes showed significant cytotoxicity although the ternary complexes coated by ?-PGA did not show significant cytotoxicity. The ternary complexes coated by ?-PGA suppressed luciferase activity in the tumor after their direct injection into the tumors of mice bearing Colon26/Luc cells. Thus, we have newly identified safe and efficient ternary complexes of siRNA for clinical use.
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?-1,6-Fucosyltransferase (FUT8) inhibits hemoglobin production during differentiation of murine and K562 human erythroleukemia cells.
J. Biol. Chem.
PUBLISHED: 04-22-2013
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Erythropoiesis results from a complex combination of the expression of several transcription factor genes and cytokine signaling. However, the overall view of erythroid differentiation remains unclear. First, we screened for erythroid differentiation-related genes by comparing the expression profiles of high differentiation-inducible and low differentiation-inducible murine erythroleukemia cells. We identified that overexpression of ?-1,6-fucosyltransferase (Fut8) inhibits hemoglobin production. FUT8 catalyzes the transfer of a fucose residue to N-linked oligosaccharides on glycoproteins via an ?-1,6 linkage, leading to core fucosylation in mammals. Expression of Fut8 was down-regulated during chemically induced differentiation of murine erythroleukemia cells. Additionally, expression of Fut8 was positively regulated by c-Myc and c-Myb, which are known as suppressors of erythroid differentiation. Second, we found that FUT8 is the only fucosyltransferase family member that inhibits hemoglobin production. Functional analysis of FUT8 revealed that the donor substrate-binding domain and a flexible loop play essential roles in inhibition of hemoglobin production. This result clearly demonstrates that core fucosylation inhibits hemoglobin production. Third, FUT8 also inhibited hemoglobin production of human erythroleukemia K562 cells. Finally, a short hairpin RNA study showed that FUT8 down-regulation induced hemoglobin production and increase of transferrin receptor/glycophorin A-positive cells in human erythroleukemia K562 cells. Our findings define FUT8 as a novel factor for hemoglobin production and demonstrate that core fucosylation plays an important role in erythroid differentiation.
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Ten-Year Trend of the Cumulative Helicobacter pylori Eradication Rate for the Japanese Eradication Strategy
Digestion
PUBLISHED: 03-05-2013
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Background/Aims: In Japan, a systematic eradication strategy for Helicobacter pylori has been constructed, and consists of a proton pump inhibitor/amoxicillin and clarithromycin (PPI/AC) therapy as the first-line regimen and proton pump inhibitor/amoxicillin and metronidazole (PPI/AM) therapy as the second-line regimen. The cumulative rate of H. pylori eradication has not been reported. Therefore, we investigated the annual and cumulative eradication rate to verify the efficacy of the Japanese eradication strategy. Methods: Patients who received first-line PPI/AC therapy and, if necessary, second-line PPI/AM therapy between 2000 and 2009 were retrospectively analyzed. The annual cumulative eradication rate was calculated. Data were subjected to intention-to-treat analysis. Results: PPI/AC was administered to 1973 patients (male n = 1,162, female n = 811; mean age: 55.8 years, range: 15-87), and 250 patients received PPI/AM. The eradication rate for the PPI/AC regimen was 65.3%, and it gradually but significantly decreased over 10 years (p < 0.05). For the PPI/AM regimen, the eradication rate was 84.0%, with no change in the annual eradication rate. The cumulative eradication rates were 76.0% in intention-to-treat analysis and 98.4% in per-protocol analysis, respectively, which provided a consistent annual eradication rate without decreases in effectiveness. Conclusion: Although the eradication rate for the first-line PPI/AC regimen decreased over time, the Japanese eradication strategy provided a sufficient eradication rate. © 2013 S. Karger AG, Basel.
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Efficacy of prosultiamine treatment in patients with human T lymphotropic virus type I-associated myelopathy/tropical spastic paraparesis: results from an open-label clinical trial.
BMC Med
PUBLISHED: 02-26-2013
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Human T lymphotropic virus type I (HTLV-I)-associated myelopathy/tropical spastic paraparesis (HAM/TSP) is a chronic myelopathy characterized by motor dysfunction of the lower extremities and urinary disturbance. Immunomodulatory treatments are the main strategy for HAM/TSP, but several issues are associated with long-term treatment. We conducted a clinical trial with prosultiamine (which has apoptotic activity against HTLV-I-infected cells) as a novel therapy in HAM/TSP patients.
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Development and clinical usability of a new traction device "medical ring" for endoscopic submucosal dissection of early gastric cancer.
Surg Endosc
PUBLISHED: 02-15-2013
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Although various traction devices exist for endoscopic submucosal dissection (ESD), the effects of the material used in the devices on the human body has not been considered. Moreover, there has been no report on a device that facilitates dissection both on the oral and anal side of the lesion. We made a traction device that has no deleterious effects on the body and is noninvasive, easy to use, and enables a bilateral approach in ESD. We report the process of its creation and a prospective evaluation of its usage in actual ESD procedures.
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Ocular gene delivery systems using ternary complexes of plasmid DNA, polyethylenimine, and anionic polymers.
Biol. Pharm. Bull.
PUBLISHED: 01-11-2013
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In this experiment, we developed anionic ternary complexes for effective and safe ocular gene delivery. Ternary complexes were constructed by coating plasmid DNA (pDNA)/polyethylenimine (PEI) complex with anionic polymers such as ?-polyglutamic acid (?-PGA) and chondroitin sulfate (CS). The cationic pDNA/PEI complex showed high gene expression on the human retinal pigment epithelial cell line, ARPE-19 cells. The pDNA/PEI complexes, however, also showed high cytotoxicity on the cells and aggregated strongly in the vitreous body. On the other hand, the anionic ternary complexes showed high gene expression on ARPE-19 cells without such cytotoxicity and aggregation. After intravitreous administration of the complexes, the anionic ternary complexes showed high gene expression in the retina. These results strongly indicate that anionic ternary complexes are suitable for effective and safe ocular gene therapy.
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Food-drug interaction of tacrolimus with pomelo, ginger, and turmeric juice in rats.
Drug Metab. Pharmacokinet.
PUBLISHED: 11-29-2011
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Tacrolimus is a well-known potent immunosuppressant agent, which has various drug-drug or food-drug interactions. Previously, we found a renal transplant recipient who increased tacrolimus blood concentrations after ingestion of pomelo as a rare case. So, we investigated the effect of pomelo after its administration for one day or 3 consecutive days on the pharmacokinetics of tacrolimus in rats. We also confirmed the effects of grapefruit, turmeric, and ginger. The tacrolimus blood concentrations of the rats pre-treated with 100% pomelo juice were significantly higher than those pre-treated with water. On the other hand, the tacrolimus blood concentrations of the rats pre-treated with 50% pomelo juice were not significantly different from those pre-treated with water. The pomelo-tacrolimus interaction showed concentration dependency. Even low concentration of pomelo juice could enhance the blood concentrations of tacrolimus by repeated administration. The inhibitory effect of 100% pomelo juice disappeared 3 days after intake. The AUC values of tacrolimus in the rats pre-treated with grapefruit juice, ginger juice, and turmeric juice were significantly larger than those pre-treated with water. We could confirm the pomelo-tacrolimus interaction, which we discovered in a case study, quantitatively. We newly found the influence of turmeric and ginger on tacrolimus pharmacokinetics, comparable to pomelo.
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Population pharmacokinetic investigation for optimization of amiodarone therapy in Japanese patients.
Ther Drug Monit
PUBLISHED: 11-23-2011
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Optimal use of amiodarone (AMD) requires information regarding the drugs pharmacokinetics and the influence of various factors on the drugs disposition. This study was conducted to establish the role of patient characteristic in estimating doses of AMD using nonlinear mixed effects modeling in Japanese patients treated with oral therapy.
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Multiple components in serum contribute to hepatic transgene expression by lipoplex in mice.
J Gene Med
PUBLISHED: 10-18-2011
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Interaction of cationic liposome/plasmid DNA complex (lipoplex) with serum was not a limiting factor for in vivo transfection. After intraportal injection of lipoplex, hepatic transgene expression was enhanced by interaction with serum in mice. In the present study, we analyzed the mechanism of enhanced hepatic transgene expression of lipoplex by interaction with serum components.
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Immunogenicity of novel nanoparticle-coated MSP-1 C-terminus malaria DNA vaccine using different routes of administration.
Vaccine
PUBLISHED: 08-05-2011
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An important aspect in optimizing DNA vaccination is antigen delivery to the site of action. In this way, any alternative delivery system having higher transfection efficiency and eventual superior antibody production needs to be further explored. The novel nanoparticle, pDNA/PEI/?-PGA complex, is one of a promising delivery system, which is taken up by cells and is shown to have high transfection efficiency. The immunostimulatory effect of this novel nanoparticle (NP) coated plasmid encoding Plasmodium yoelii MSP1-C-terminus was examined. Groups of C57BL/6 mice were immunized either with NP-coated MSP-1 plasmid, naked plasmid or NP-coated blank plasmid, by three different routes of administration; intravenous (i.v.), intraperitoneal (i.p.) and subcutaneous (s.c). Mice were primed and boosted twice at 3-week intervals, then challenged 2 weeks after; and 100%, 100% and 50% mean of survival was observed in immunized mice with coated DNA vaccine by i.p., i.v. and s.c., respectively. Coated DNA vaccine showed significant immunogenicity and elicited protective levels of antigen specific IgG and its subclass antibody, an increased proportion of CD4(+) and CD8(+) T cells and INF-? and IL-12 levels in the serum and cultured splenocyte supernatant, as well as INF-? producing cells in the spleen. We demonstrate that, NP-coated MSP-1 DNA-based vaccine confers protection against lethal P. yoelii challenge in murine model across the various route of administration and may therefore, be considered a promising delivery system for vaccination.
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Polyoxyethylene hydrogenated castor oil modulates benzalkonium chloride toxicity: comparison of acute corneal barrier dysfunction induced by travoprost Z and travoprost.
J Ocul Pharmacol Ther
PUBLISHED: 06-29-2011
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To determine the element that modulates benzalkonium chloride (BAC) toxicity by using a new electrophysiological method to evaluate acute corneal barrier dysfunction induced by travoprost Z with sofZia (Travatan Z(®)), travoprost with 0.015% BAC (Travatan(®)), and its additives.
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Development of effective cancer vaccine using targeting system of antigen protein to APCs.
Pharm. Res.
PUBLISHED: 06-18-2011
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To develop a novel cancer vaccine using the targeting system of antigen protein to antigen-presenting cells (APCs) for efficient and safe cancer therapy.
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Fluorescence investigation of the retinal delivery of hydrophilic compounds via liposomal eyedrops.
Biol. Pharm. Bull.
PUBLISHED: 06-02-2011
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We examined the feasibility of using submicron-sized liposomes (ssLips) for retinal delivery of hydrophilic compounds, which would also have a wide range of applications. To evaluate the uptake into conjunctival cell line and the intraocular behavior of hydrophilic compound-containing ssLips after eyedrop application, fluorometric investigation was carried out by using a hydrophilic fluorescence probe, 5(6)-carboxyfluorescein (CF). A relatively high amount of CF (>50%) could be incorporated into an internal phase of ssLips by a calcium acetate gradient method. CF being entrapped within the liposomes markedly enhanced both the uptake of CF into conjunctival cells and CF-oriented emission in the retina in mice after eyedrop application, while the free CF did not clear delivery efficiency in both in vitro and in vivo study. In addition, the cellular uptake and luminescence intensity in the retina were higher when a ssLip formulation composed of L-?-distearoyl phosphatidylcholine was applied than when a ssLip formulation composed of egg phosphatidylcholine was applied. Consequently, ssLips of appropriate composition were considered to have good potential to carry hydrophilic compounds into the retina.
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Self-assemble gene delivery system for molecular targeting using nucleic acid aptamer.
Gene
PUBLISHED: 05-26-2011
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We have developed a novel vector constructed with pDNA, polyethylenimine (PEI), and mucin 1 (MUC1) aptamer for tumor-targeted gene delivery. The MUC1 aptamer and non-specific aptamer were employed to coat the pDNA/PEI complexes electrostatically and stable nanoparticles were formed. The addition of a non-specific aptamer to the pDNA/PEI complex decreased gene expression in the human lung cancer cell line, A549 cells expressing MUC1 regularly. At the same time, the pDNA/PEI/MUC1 aptamer complex showed higher gene expression than pDNA/PEI/non-specific aptamer complex. Furthermore, the pDNA/PEI/MUC1 aptamer complex showed markedly high gene expression in tumor-bearing mice; thus, pDNA/PEI/MUC1 aptamer complexes are useful as a tumor-targeted gene delivery system with high transfection efficiency.
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Nanoparticles electrostatically coated with folic acid for effective gene therapy.
Mol. Pharm.
PUBLISHED: 05-18-2011
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We developed a novel vector, electrostatically coated poly(ethylenimine) (PEI)/pDNA complexes with folic acid (FA). Without covalent binding, the FA molecules could coat the PEI/pDNA complexes, and stable anionic nanoparticles were formed at a charge ratio greater than 60. The addition of FA markedly decreased the cytotoxicity of the cationic PEI/pDNA complexes to the melanoma cell line, B16-F10 cells, which regularly expressed FA-specific receptor (FR). Furthermore, the anionic FA60/PEI/pDNA complexes showed high transgene efficiency via the FR-mediated pathway in B16-F10 cells. The FA60/PEI/pDNA complexes did not show agglutination with erythrocytes. After the intravenous injection of FA60/PEI/pDNA complexes into mice, a higher transgene efficiency than PEI/pDNA complexes was observed in the liver, kidney, spleen, and lung with FR. The gene expressions of FA60/PEI/pDNA complexes were significantly inhibited by preadministration of FA. Thus, the FA60/PEI/pDNA complexes were useful for effective gene therapy.
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Donepezil significantly improves abilities in daily lives of female Down syndrome patients with severe cognitive impairment: a 24-week randomized, double-blind, placebo-controlled trial.
Int J Psychiatry Med
PUBLISHED: 04-19-2011
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Down syndrome (DS) patients share certain neuropathological features with Alzheimer disease patients. A randomized, double-blind, placebo-controlled study was performed to investigate the efficacy and safety of donepezil, an Alzheimer disease drug, for DS patients.
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Methotrexate chronotherapy is effective against rheumatoid arthritis.
Chronobiol. Int.
PUBLISHED: 04-02-2011
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Methotrexate (MTX) is the most important drug for treating rheumatoid arthritis (RA). It has been stated that cytokines play an important role in the pathogenesis of RA, and that cytokine levels increase and show 24-h rhythms in RA patients. Previously, we found that arthritis was relieved after the administration of MTX at specific times in synchronization with the 24-h rhythm of tumor necrosis factor (TNF)-? in collagen-induced arthritis (CIA) animals. Based on our findings in an earlier study of the dosing time-dependent effects of MTX in MRL/lpr mice, which develop autoimmune disorders that share similarities with human RA, we examined here the utility of MTX chronotherapy in Japanese RA patients. In an initial animal modeling study, we collected blood from MRL/lpr mice at different times (2, 6, 10, 14, 18, or 22 hours after the light was turned on [HALO]), and we measured TNF-? mRNA expression in leukocytes. MTX was administered to the mice at two different dosing times (6 or 18 HALO), and various blood parameters were measured to estimate arthritis activity. TNF-? mRNA levels showed a clear 24-h rhythm with a peak at 22 HALO and a trough at 18 HALO after RA had developed. In these MRL/lpr mice, inflammation and TNF-? were markedly reduced when the MTX dosing time was matched to the time (18 HALO) when the TNF-? level began to increase. We then applied these findings to Japanese RA patients by switching them from the standard MTX three times/wk (day 1: after breakfast and supper; day 2: after breakfast schedule), to chronotherapy, in which the dose and number of doses/wk were not changed but MTX was administered once-a-day at bedtime. Disease Activity Score (DAS)28, modified health assessment questionnaire (MHAQ), and adverse effects were assessed. With MTX chronotherapy, DAS28, which is commonly used to quantitatively assess RA symptoms, was significantly improved at all follow-up clinical visit times compared with the baseline (vs. 1 mo: p?=?.0197, 2 mos: p?=?.0107, 3 mos: p?=?.0087). Significant symptom recovery was observed in 41.2% of patients, and 23.5% of patients achieved clinical remission during the 3 mos of follow-up. Functional capacity of RA patients, as indicated by the MHAQ, was markedly improved by chronotherapy. There were no severe adverse effects. Thus, we demonstrated (i) inflammation and plasma TNF-? concentrations were significantly reduced in MRL/lpr mice treated with MTX at 18 HALO, the time when TNF-? mRNA level began to increase; and (ii) MTX bedtime chronotherapy was safe, markedly reduced disease activity, and improved the functional capacity of RA patients. The findings on RA patients show that bedtime MTX chronotherapy can improve RA symptoms compared to the current standard dosing methods.
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Relationship between lipophilicity and absorption from the liver surface of paraben derivatives and antipyrine in rats.
J. Pharm. Pharmacol.
PUBLISHED: 03-31-2011
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The importance of drug lipophilicity on absorption from the liver surface was examined in rats using paraben derivatives, antipyrine, Sudan III, and Sudan blue.
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Secure and effective gene vector of polyamidoamine dendrimer pharmaceutically modified with anionic polymer.
J Pharm Sci
PUBLISHED: 03-01-2011
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The purpose of this study was to develop a new type of gene vector, polyamidoamine (PAMAM) dendriplex pharmaceutically modified, based on electrostatic interactions, by various anionic polymers. The ?-polyglutamic acid (?-PGA)/PAMAM dendriplex and the ?-PGA/PAMAM dendriplex formed a stable complex, although ?-polyaspartic acid and heparin released pDNA from the complex. The addition of anionic polymer decreased the ?-potential, although it did not greatly affect the size of the complex. As a result of an in vitro gene expression study of mouse melanoma cells, we found that the ?-PGA/PAMAM dendriplex showed high gene expression comparable to the PAMAM dendriplex, although the ?-PGA/PAMAM dendriplex showed lower gene expression. Tail vein injection of the ?-PGA/PAMAM dendriplex into mice also led to high gene expression in the spleen and lung. The ?-PGA/PAMAM dendriplex showed no cytotoxicity and no agglutination, although severe cytotoxicity and agglutination were observed in the PAMAM dendriplex. Thus, we discovered that complexes of pDNA, PAMAM dendrimers, and ?-PGA showed higher gene expression in vitro and in vivo, and markedly lower toxicity. This complex is valuable and is expected to be a safe and effective gene vector.
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Mechanism of the cardioprotective effects of docetaxel pre-administration against adriamycin-induced cardiotoxicity.
J. Pharmacol. Sci.
PUBLISHED: 02-24-2011
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We revealed that pre-treatment with docetaxel (DOC) 12 h before adriamycin (ADR) administration significantly reduced ADR-induced toxic death compared with the simultaneous dosing schedule that was commonly used in previous studies. We considered that pre-treatment with DOC relieves ADR-induced cardiotoxicity. In this study, we investigated the influence of DOC on the pharmacokinetics and pharmacodynamics of ADR in order to clarify the mechanism by which DOC pre-treatment relieves ADR-induced cardiotoxicity. When ADR and/or DOC was intravenously administered, the DOC pre-treatment (DOC-ADR) group showed significantly less toxic death than the ADR-alone group. We examined hepatopathy, nephropathy, leukopenia, and cardiotoxicity, all of which can cause toxic death. Of these toxicities, ADR-induced cardiotoxicity was significantly relieved in the DOC-ADR group. To elucidate the mechanism by which DOC pre-treatment relieved ADR-induced cardiotoxicity, lipid peroxidation as a proxy for the free radical level and the pharmacokinetics of ADR were measured. There was no difference in the pharmacokinetics of ADR between the ADR and DOC-ADR groups. On the other hand, the DOC-ADR group showed significantly inhibited lipid peroxidation in the heart compared with the ADR group. It was considered that DOC pre-administration inhibited ADR-induced free radicals and decreased cardiotoxicity.
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Injection site and pharmacokinetics after intravitreal injection of immunoglobulin G.
J Ocul Pharmacol Ther
PUBLISHED: 12-23-2010
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The aim of this study was to investigate whether the pharmacokinetics differ after immunoglobulin G (IgG) intravitreal injections are done in the different sites of the vitreous cavity of rabbit eyes.
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Chondroitin sulfate capsule system for efficient and secure gene delivery.
J Pharm Pharm Sci
PUBLISHED: 11-25-2010
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In this study, we developed various ternary complexes of encapsulated polyplexes and lipoplexes using chondroitin sulfate (CS) and investigated their universal usefulness for gene delivery.
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Frontal midline theta rhythm: comment on Chan and Hsu (2010).
Percept Mot Skills
PUBLISHED: 09-28-2010
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That the frontal theta rhythm of EEGs increases in magnitude during various mental tasks which require focusing of attention is well-known. The task includes visual-search tasks using a visual display terminal (VDT) and Chinese characters.
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Influence of alkyl chain length of benzalkonium chloride on acute corneal epithelial toxicity.
Cornea
PUBLISHED: 08-31-2010
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To evaluate acute corneal epithelial toxicity induced by benzalkonium chloride (BAC) homologs with different alkyl chain lengths using an in vivo electrophysiological method.
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Sensitive and real-time method for evaluating corneal barrier considering tear flow.
Biol. Pharm. Bull.
PUBLISHED: 01-05-2010
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We developed a new electrophysiological method mimicking tear flow to evaluate the epithelial tight junction of rabbit cornea quantitatively. We investigated the effect of tear flow on the corneal damage induced by ophthalmic preservatives using this method. An Ussing chamber system with Ag/AgCl electrodes was used in the electrophysiological experiment. The excised rabbit cornea was mounted in the Ussing chamber and the precorneal solution in the chamber was perfused with a peristaltic pump at the rate of human tear flow. Corneal transepithelial electrical resistance (TEER) was monitored as corneal barrier ability. In the electrophysiological method mimicking tear flow, we observed stable TEER, which rapidly decreased with benzalkonium chloride (BAC), an eye drop preservative. Using this system, we first found that 0.004% BAC decreased corneal TEER reversibly. A high concentration of BAC showed strong irreversible damage to the tight junction. The influence of BAC on corneal TEER was not only concentration-dependent but also tear flow rate-dependent. The electrophysiological method mimicking tear flow was useful to evaluate the corneal barrier quantitatively. Using this method, we clarified that the tear flow was important to protect the corneal damage induced by preservatives.
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Therapeutic index of methotrexate depends on circadian cycling of tumour necrosis factor-alpha in collagen-induced arthritic rats and mice.
J. Pharm. Pharmacol.
PUBLISHED: 10-10-2009
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Rheumatoid arthritis is an autoimmune disorder of unknown aetiology. Morning stiffness, a characteristic feature of rheumatoid arthritis, shows a 24-h rhythm. Noticing this rhythm, we hypothesized the presence of a similar rhythm for a rheumatoid arthritis indicator, in addition to dosing-time dependency of the anti-rheumatic effect of methotrexate in arthritis induced by collagen in rats and mice, which reflect the symptomatology of rheumatoid arthritis patients.
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Gamma-polyglutamic acid-coated vectors for effective and safe gene therapy.
J Control Release
PUBLISHED: 07-29-2009
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In the present study, we developed some novel gene delivery vectors, coated cationic complexes with gamma-polyglutamic acid (gamma-PGA) for effective and safe gene therapy. Cationic complexes were constructed with pDNA and cationic vectors, such as poly-L-arginine hydrochloride (PLA), poly-L-lysine hydrobromide (PLL), N-[1-(2, 3-dioleyloxy) propyl]-N, N, N-trimethylammonium chloride (DOTMA)-cholesterol (Chol) liposomes, and DOTMA-dioleylphosphatidylethanolamine (DOPE) liposomes. The cationic complexes showed high gene expression with strong cytotoxicity in melanoma B16-F10 cells. The cationic complexes were also strongly toxic to erythrocytes. On the other hand, the gamma-PGA was able to coat all cationic complexes and form stable nano-sized particles with negative charges. These gamma-PGA-coated complexes had high gene expression without cytotoxicity and toxicities to the erythrocytes. In in vivo transfection experiments, polyplexes showed high transfection efficiency over 10(5) RLU/g in the lung tissue after intravenous injection, although gamma-PGA-coated polyplexes showed a high value in the spleen. High transfection efficiency in lipoplexes and gamma-PGA-coated lipoplexes was observed in the spleen and lung. Thus, gamma-PGA-coated vectors are useful for clinical gene therapy.
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Liver- and lobe-specific gene transfer following the continuous microinstillation of Plasmid DNA onto the liver surface in mice: effect of instillation speed.
Biol. Pharm. Bull.
PUBLISHED: 07-03-2009
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Development of technology to deliver foreign gene(s) to a specific organ/tissue is one of the major challenges in gene therapy. Here, we show liver- and lobe-specific gene transfer following the continuous microinstillation of plasmid DNA (pDNA) onto the liver surface in mice. Naked pDNA was continuously instilled onto the right medial liver lobe using syringe pump in male ddY mice. Our previous studies showed liver- and lobe-selective gene expression after instillation of 30 mul of pDNA solution onto the liver surface, but gene expression was also found in the other liver lobe, kidney and spleen. To improve target site selectivity of gene expression, the instillation volume was decreased; however, non-specific gene expression in the other liver lobe and diaphragm was still detected. To prevent immediate diffusion of the pDNA solution, we performed continuous microinstillation of pDNA using a syringe pump; as a result, target site selectivity was greatly improved. As for instillation speed, 5 min infusion was enough to prevent diffusion of pDNA solution. Furthermore, transfection efficiency in the target site was maintained when instillation speed was slowed. Wiping off residual pDNA solution from the applied liver lobe resulted in a further improvement in selectivity, suggesting not only immediate diffusion, but also gradual diffusion, are important factors for successful target site-specific gene transfer. Information in this study will be useful for further development of an effective gene delivery system targeted to a specific organ/tissue by use of other non-viral or viral vectors.
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Cationic liposomes-mediated plasmid DNA delivery in murine hepatitis induced by carbon tetrachloride.
J Liposome Res
PUBLISHED: 02-25-2009
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In order to elucidate the influence of hepatic disease stage on cationic liposomes-mediated gene delivery, we investigated the cationic liposomes-mediated plasmid DNA delivery with time in murine hepatitis induced by subcutaneous injection of CCl(4). Liver injury after injection of CCl(4) was confirmed by the determination of serum aspartate aminotransferase and alanine aminotransferase activities. Two kinds of liposomes constructed with N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethlylammoniumchloride and dioleylphosphatidylethanolamine (DOTMA-DOPE) or DOTMA and cholesterol (DOTMA-CHOL) were used for the gene-delivery vector. We determined luciferase activities in various organs after the intravenous administration of the lipoplexes. The CCl(4)-treated mice administered with DOTMA-DOPE lipoplexes showed the more significant decreases of transgene expression in the liver and spleen at 18 hours after CCl(4) injection. On the other hand, the CCl(4)-treated mice administered with DOTMA-CHOL lipoplexes showed a significant increase in the liver at 48 hours. In conclusion, our findings demonstrate that murine hepatitis induced by CCl(4) can influence cationic liposomes-mediated plasmid DNA delivery. The extent of influences was also affected by lipid contents. These results indicate the necessity of considering the timing and the formulation for gene therapy according to the disease stage.
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Pulmonary gene delivery of hybrid vector, lipopolyplex containing N-lauroylsarcosine, via the systemic route.
J Control Release
PUBLISHED: 02-09-2009
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We newly prepared lipopolyplexes containing N-lauroylsarcosine (LS) as a hybrid vector for pulmonary gene delivery via the systemic route. Lipopolyplexes were composed of polyethylenimine (PEI), N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethlylammonium chloride (DOTMA), LS, and plasmid DNA (pDNA). The particle size of lipopolyplex of PEI, DOTMA, and pDNA (lipopolyplex 2P-2D) was not largely changed by the addition of LS, although the addition of LS decreased the high zeta potential. Lipopolyplexes containing LS with low zeta potential showed little aggregation with erythrocytes and low cytotoxicity. In HepG2 cells, lipopolyplexes containing LS showed high transgene efficiency comparable to lipopolyplex 2P-2D. After intravenous injection of the complexes into mice, lipopolyplexes containing LS showed high transgene efficiency, comparable to lipopolyplex 2P-2D. In particular, lipopolyplexes containing LS showed extremely high transgene efficiency in the lung. As a result of the analysis to identify optimum formulations, we discovered that LS contributed to the high transgene efficiency in the lung as 76.7% of the contribution index. These results suggest that lipopolyplexes containing LS are safe and useful gene delivery vectors with lung directivity.
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The development of a gene vector electrostatically assembled with a polysaccharide capsule.
Biomaterials
PUBLISHED: 02-01-2009
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The purpose of this study was to develop a gene vector electrostatically assembled with a polysaccharide capsule. We used pDNA/polyethylenimine (PEI) complexes as efficient non-viral vectors. The pDNA/PEI complex was electrostatically encapsulated with various polysaccharides such as fucoidan, lambda-carrageenan, xanthan gum, alginic acid, hyaluronic acid, and chondroitin sulfate (CS). The pDNA/PEI complex was shown as nanoparticles with positive zeta-potential, although the ternary complexes encapsulated with polysaccharides were shown as nanoparticles with negative zeta-potential. The pDNA/PEI complex showed high agglutination activity and cytotoxicity, although the ternary complexes encapsulated with polysaccharides had no agglutination activities and lower cytotoxicities. The pDNA/PEI complex showed high uptake and high transgene efficiency in B16-F10 cells. On the other hand, most of the ternary complexes show little uptake and gene expression. The ternary complex encapsulated by CS, however, showed comparable transgene efficiency to the pDNA/PEI complex. The uptake and gene expression of the ternary complex encapsulated by CS were significantly inhibited by hypothermia and the addition of CS, suggesting that the ternary complex was taken by CS-specific receptor-mediated energy-dependent process.
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Ternary complexes of pDNA, polyethylenimine, and gamma-polyglutamic acid for gene delivery systems.
Biomaterials
PUBLISHED: 01-29-2009
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We discovered a vector coated by gamma-polyglutamic acid (gamma-PGA) for effective and safe gene delivery. In order to develop a useful non-viral vector, we prepared several ternary complexes constructed with pDNA, polyethylenimine (PEI), and various polyanions, such as polyadenylic acid, polyinosinic-polycytidylic acid, alpha-polyaspartic acid, alpha-polyglutamic acid, and gamma-PGA. The pDNA/PEI complex had a strong cationic surface charge and showed extremely high transgene efficiency although it agglutinated with erythrocytes and had extremely high cytotoxicity. Those polyanions changed the positive zeta-potential of pDNA/PEI complex to negative although they did not affect the size. They had no agglutination activities and lower cytotoxicities but most of the ternary complexes did not show any uptake and gene expression; however, the pDNA/PEI/gamma-PGA complex showed high uptake and gene expression. Most of the pDNA/PEI/gamma-PGA complexes were located in the cytoplasm without dissociation and a few complexes were observed in the nuclei. Hypothermia and the addition of gamma-PGA significantly inhibited the uptake of pDNA/PEI/gamma-PGA by the cells, although l-glutamic acid had no effect. These results strongly indicate that the pDNA/PEI/gamma-PGA complex was taken up by gamma-PGA-specific receptor-mediated energy-dependent process. Thus, the pDNA/PEI/gamma-PGA complex is useful as a gene delivery system with high transfection efficiency and low toxicity.
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AAPS-FIP summary workshop report: Pharmacogenetics in individualized medicine: methods, regulatory, and clinical applications.
AAPS J
PUBLISHED: 01-23-2009
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The workshop "Pharmacogenetics in Individualized Medicine: Methods, Regulatory, and Clinical Applications" was held November 15-16, 2008 in Atlanta, Georgia, USA. This workshop provided an opportunity for pharmaceutical scientists, clinical practitioners, clinical laboratory scientists, and FDA to discuss methods, regulatory, and the application of pharmacogenetics in clinical practice and drug discovery. Key highlights of the workshop were: (a) the use of genetic information in individualized medicine has significant potential in advancing drug development and human health by optimizing drug response, drug efficacy, and preventing adverse drug reactions; (b) various barriers exist preventing the advance of the individualized medicine in the society, industry, and clinical practice; and (c) the barriers may be overcome by integrated approaches; the education of researchers, clinical practitioners, and patients and fostering interactive communication among stakeholders. By targeting the AAPS audience, this workshop was one step among many steps that AAPS-FIP is intending to take towards removing the barriers to widespread uptake of pharmacogenetics in drug discovery and clinical practice.
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Retinal drug delivery using eyedrop preparations of poly-L-lysine-modified liposomes.
Eur J Pharm Biopharm
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The purpose of this study was to develop surface-modified liposomes that enhance the efficiency of eye drop drug delivery to the retina. Various molecular weights and concentrations of the water-soluble cationic polymer poly-L-lysine (PLL) were used to modify the surface of submicronized (100 nm) liposomes. Physicochemical properties of surface-modified liposomes were determined in vitro, and the efficiency of drug delivery to the retina was investigated in vivo. Using coumarin-6 as a model drug and fluorescent marker, we show that liposome surface modification by PLL dramatically increased delivery to mouse retina segments after eye drop administration. However, when PLL of high molecular weight (>30,000) was used at higher concentrations (>0.05%), aggregation of surface-modified liposomes increased particle size and hampered distribution to inner ocular tissues. As a result, the efficiency of drug delivery of these aggregated surface-modified liposomes was the same as unmodified liposomes. The optimal molecular weight and concentration of PLL in drug-delivering liposomes were 15,000-30,000 and 0.005%, respectively. Under these conditions, PLL-modified liposomes were not cytotoxic in corneal or conjunctival cells. In conclusion, surface-modified liposomes have great potential as effective retinal drug delivery carriers in eye drop formulations.
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Chronopharmacology of mizoribine in collagen-induced arthritis rats.
J. Pharmacol. Sci.
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We previously reported that higher therapeutic effects were obtained in rheumatoid arthritis (RA) patients and RA model animals when the dosing-times of methotrexate and tacrolimus were chosen according to the 24-h rhythms of the inflammatory response. Mizoribine (MZR) is an immunosuppressive agent and is used against RA in the same manner as methotrexate and tacrolimus. In this study, we examined whether a dosing-time dependency of the therapeutic effect of MZR could be detected in collagen-induced arthritis (CIA) rats. To measure C-reactive protein (CRP) and tumor necrosis factor (TNF)-? levels, blood was collected from CIA rats at different times. MZR was administered at two different dosing-times based on these findings and its effects and toxicity were examined. CRP and TNF-? concentrations in blood showed significant 24-h rhythms. The exacerbation of arthritis and excessive increase in leukocytes in CIA rats were markedly lower in the group treated with MZR at the dark phase than those of the group treated with MZR at the light phase. These findings suggest that the therapeutic index of RA therapy may be improved by administering MZR at a time in the day when the inflammatory reaction begins to activate.
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Effect of viscous additives on the absorption and hepatic disposition of 5-fluorouracil (5-FU) after application to liver surface in rats.
J. Pharm. Pharmacol.
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Objectives? The aim was to study the effect of viscous additives on the absorption and hepatic disposition of 5-fluorouracil (5-FU) after application to the liver surface in rats. Methods? 5-FU solution with or without viscous additives was applied to the rat liver surface with a cylindrical diffusion cell. Then, blood and the remaining solution in the diffusion cell were collected at selected times, followed by excision of the liver. The excised liver was divided into three sites and assayed for 5-FU content. Key findings? The absorption rate of 5-FU from the liver surface was decreased in the presence of carboxymethylcellulose sodium (CMC-Na) and polyvinyl alcohol (PVA) as compared with the control. The k(a) values of PVA 15% and CMC-Na 1% were reduced to about 80 and 67% of the control. The maximum plasma concentration of 5-FU was decreased by incorporation of viscous additives. The 5-FU concentration at the diffusion cell attachment site of the liver (site 1) plateaued at 180?min in the absence of viscous additives. On the other hand, the concentration of 5-FU at site 1 increased in a time-dependent manner until 360?min in the presence of viscous additives. Conclusion? Viscous additives might be useful for retaining drugs at their application site and controlling the rate of absorption from the liver surface.
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Efficient in vivo gene transfer by intraperitoneal injection of plasmid DNA and calcium carbonate microflowers in mice.
Mol. Pharm.
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Gene transfer to intraperitoneal organs is thought to be a promising approach to treat such conditions as peritoneal fibrosis and peritoneal dissemination of cancers. We previously discovered that simple instillation of naked plasmid DNA (pDNA) onto intraperitoneal organs such as the liver and stomach could effectively transfer foreign genes in mice. In this study, we developed a novel nonviral method to enhance transfection efficiency of naked pDNA to intraperitoneal organs using a calcium carbonate suspension containing pDNA. Using commercially available calcium carbonate, we successfully transfected pDNA to the stomach. Handling of commercially available calcium carbonate, however, was troublesome owing to rapid precipitation and caking. To obtain slowly settling particles of calcium carbonate, we tried to synthesize novel versions of such particles and succeeded in creating flower-shaped particles, named calcium carbonate microflowers. Sedimentation of calcium carbonate microflowers was sufficiently slow for in vivo experiments. Moreover, the transfection efficiency of the suspension of calcium carbonate microflowers to the stomach was more effective than that of commercially available calcium carbonate, especially at low concentrations. Intraperitoneal injection of the suspension of calcium carbonate microflowers containing pDNA greatly enhanced naked pDNA transfer to whole intraperitoneal organs in mice. Furthermore, lactate dehydrogenase activities in intraperitoneal fluid and plasma were not raised by the suspension of calcium carbonate microflowers.
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Clinical feature of asymptomatic reflux esophagitis in patients who underwent upper gastrointestinal endoscopy.
J. Gastroenterol. Hepatol.
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Prevalence of gastroesophageal reflux disease (GERD) varies in regions, but few reports on clinical features and quality of life (QOL) of asymptomatic GERD exist in Japan.
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Analysis of brain activity during visceral stimulation.
J. Gastroenterol. Hepatol.
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Functional magnetic resonance imaging (fMRI) is a useful technology for investigating regional metabolic activity in the brain. Many experiments using fMRI have been performed, but because of variations in protocols and analytic techniques, the results vary. When a priori information of the task is known, a model-based technique, such as statistical parametric mapping, is often used for analysis. In the case of acid stimulation of the esophagus the task model is unclear, so we analyzed brain activity during an acid or isotonic saline infusion to the esophagus using independent component analysis (ICA), which does not depend on a priori information of the task.
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Investigation of protective effects of sodium hyaluronate eyedrop against corneal epithelial disorders using an electrophysiological method.
J Ocul Pharmacol Ther
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The purpose of this study was to investigate the protective effects of sodium hyaluronate eyedrop against corneal epithelial disorders caused by antiglaucomatous eyedrops using an electrophysiological method.
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What is Visualize?

JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.

How does it work?

We use abstracts found on PubMed and match them to JoVE videos to create a list of 10 to 30 related methods videos.

Video X seems to be unrelated to Abstract Y...

In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.