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A Rolland

Publications and source records attributed to A Rolland.

At least 19 recordsLinked to original sources
PubMed2001

Immune and clinical responses in patients with metastatic melanoma to CD34(+) progenitor-derived dendritic cell vaccine.

Immunization to multiple defined tumor antigens for specific immune therapy of human cancer has thus far proven difficult. Eighteen HLA A*0201(+) patients with metastatic melanoma received injections s.c. of CD34(+)progenitor-derived autologous dendritic cells (DCs), which included Langerhans cells. DCs were pulsed with peptides derived from four melanoma antigens [(MelAgs) MelanA/MART-1, tyrosinase, MAGE-3, and gp100], as well as influenza matrix peptide (Flu-MP) and keyhole limpet hemocyanin (KLH) as control antigens. Overall immunological effects were assessed by comparing response profiles using marginal likelihood scores. DC injections were well tolerated except for progressive vitiligo in two patients. DCs induced an immune response to control antigens (KLH, Flu-MP) in 16 of 18 patients. An enhanced immune response to one or more MelAgs was seen in these same 16 patients, including 10 patients who responded to >2 MelAgs. The two patients failing to respond to both control and tumor antigens experienced rapid tumor progression. Of 17 patients with evaluable disease, 6 of 7 patients with immunity to two or less MelAgs had progressive disease 10 weeks after study entry, in contrast to tumor progression in only 1 of 10 patients with immunity to >2 MelAgs. Regression of >1 tumor metastases were observed in seven of these patients. The overall immunity to MelAgs after DC vaccination is associated with clinical outcome (P = 0.015).

Adult↗
PubMed2001

Neurophysiological effects of an extract of Eschscholzia californica Cham. (Papaveraceae).

An aqueous alcohol extract of Eschscholzia californica (Ec) has been evaluated for benzodiazepine, neuroleptic, antidepressant, antihistaminic and analgesic properties, in order to complete the study of the sedative and anxiolytic effects previously demonstrated. The plant extract did not protect mice against the convulsant effects of pentylenetetrazol, and did not cause muscle relaxant effects but appeared to possess an affinity for the benzodiazepine receptor: thus, flumazenil, an antagonist of these receptors, suppressed the sedative and anxiolytic effects of the extract. The Ec extract induced peripheral analgesic effects in mice but did not possess antidepressant, neuroleptic or antihistaminic effects.

Acetic Acid↗
PubMed2001

Fusogenic activity of vesicular stomatitis virus glycoprotein plasmid in tumors as an enhancer of IL-12 gene therapy.

We have characterized the fusogenic activity of a plasmid expression system encoding vesicular stomatitis virus G protein (VSVG) in vitro and in vivo. Over 70% of murine colon and renal carcinoma cells (MC38 and Renca, respectively) transfected with VSVG plasmid in vitro fused and formed polykaryons upon incubation with pH 5.5 media. Using a plasmid expression system encoding VSVG and bacterial green fluorescent protein (GFP) formulated in a polyvinyl pyrrolidone (PVP) delivery system, diffusion of GFP throughout the VSVG-induced syncytia was shown in vivo in MC38 and Renca tumors. Moreover, tumor-bearing mice showed tumor growth inhibition following in vivo transfection with VSVG plasmid at an optimal dose of 48 microg. We have previously shown that direct injection of interleukin -12 (IL -12) plasmid complexed with PVP into tumors induces a strong immune response. In the current study, we assessed the ability of VSVG to elicit an antitumor response by enhancing cytokine gene delivery within the tumor mass. Tumor-bearing mice treated intratumorally with both VSVG/PVP and IL-12/PVP (48 and 24 microg, respectively) showed increase in tumor rejection when compared to IL- 12 plasmid alone (75% vs. 50%, respectively). These data suggest that VSVG gene therapy can be used in combination with other therapeutic genes to induce an antitumor response in vivo by enhancing the expression of the gene of interest.

Animals↗
PubMed2000

Topoisomerase I-mediated antiproliferative activity of enantiomerically pure fluorinated homocamptothecins.

Homocamptothecin (hCPT) is an E-ring modified camptothecin (CPT) analogue bearing a methylene spacer between the alcohol and carboxyl functions of the CPT lactone. Combining pronounced inhibitory activity of topoisomerase I (Topo I) with enhanced plasma stability, hCPT constitutes an attractive template for the elaboration of new anticancer agents. Fluorinated hCPT analogues, prepared in enantiomerically pure form, were assayed by their stimulation of Topo I-mediated DNA cleavage. Translation into cytotoxicity against tumor cells was evaluated on HT29 human colon adenocarcinoma and on the multidrug resistant lung and bladder tumor cell lines, A549 and T24r. Good correlation is observed between the ability of the drugs to stimulate Topo I-mediated DNA cleavage and the respective 50% inhibitory concentrations (IC(50) values) of the HT29, A549, and T24r cell growth. Fluorine substitution in the A-ring of hCPT was found to have a pronounced influence on biological activity, providing several compounds which are up to 100-fold more potent than CPT in terms of IC(50). Among these, 10,11-difluoro-hCPT has been selected for further development.

Antineoplastic Agents↗
PubMed2000

Synthetic glycopeptide-based delivery systems for systemic gene targeting to hepatocytes.

PURPOSE: To design, synthesize, and test synthetic glycopeptide-based delivery systems for gene targeting to hepatocytes by systemic administration. METHODS: All peptides were synthesized by the solid phase method developed using Fmoc chemistry on a peptide synthesizer. The binding of galactosylated peptides to HepG2 cells and accessibility of the galactose residues on particle surface was demonstrated by a competition assay using 125I-labeled asialoorosomucoid and RCA lectin agglutination assay, respectively. DNA plasmid encoding chloramphenicol acetyl transferase (CAT) gene was complexed with a tri-galactosylated peptide (GM245.3) or tri-galactosylated lipopeptide (GM246.3) in the presence of an endosomolytic peptide (GM225.1) or endosomolytic lipopeptide (GM227.3) to obtain DNA particles of 100-150 nm in size. The plasmid/peptide complexes were added to HepG2 cell cultures or intravenously administered by tail vein injection into normal mice or rats. Plasmid uptake and expression was quantified by qPCR and ELISA, respectively. RESULTS: Multiple antennary glycopeptides that have the ability to condense and deliver DNA plasmid to hepatocytes were synthesized and complexed with DNA plasmid to obtain colloidally stable DNA/peptide complexes. Addition of DNA/GM245.3/GM225.1 peptide complexes (1:3:1 (-/+/-)) to HepG2 cell cultures yielded CAT expression in transfected cells. The transfection efficiency was significantly reduced in the absence of galactose ligand or removal of endosomolytic peptide. Intravenous administration of DNA/GM245.3 peptide complexes (1:0.5 (-/+)) into the tail vein of normal rats yielded DNA uptake in the liver. Substitution of GM245.3 by galactosylated lipopeptide GM246.3 resulted in more stable DNA particles, and a 10-fold enhancement in liver plasmid uptake. CAT expression was detectable in liver following intravenous administration of DNA/GM246.3 complexes. Addition of endosomolytic lipopeptide GM227.3 into the complexes (DNA/ GM246.3/GM227.3 (1:0.5:1 (-/+/-))) yielded a 5-fold increase in CAT expression. Liver expression was 8-fold and 40-fold higher than lung and spleen, respectively, and localized in the hepatocytes only. The transfection efficiency in liver was enhanced by increasing DNA dose and injection volume. The plasmid uptake and expression in liver using DNA/GM246.3/GM227.3 complexes was 100-200-fold higher than DNA formulated in glucose. Tissue examination and serum biochemistry did not show any adverse effect of the DNA/GM246.3/ GM227.3 (1:0.5:1 (-/+/-)) complexes after intravenous delivery. CONCLUSIONS: Gene targeting to hepatocytes was achieved by systemic administration of a well-tolerated synthetic glycopeptide-based delivery system. The transfection efficiency of this glycopeptide delivery system was dependent on peptide structure, endosomolytic activity, colloidal particle stability, and injection volume.

Amino Acid Sequence↗
PubMed2000

Ultrasound enhancement of cationic lipid-mediated gene transfer to primary tumors following systemic administration.

The impact of a localized application of ultrasound on gene transfer to primary tumors following systemic administration of cationic lipid based transfection complexes was investigated. We have previously shown that systemic administration of DOTMA (N-[(1-(2-3-dioleyloxy) propyl)]-N-N-N-trimethylammonium chloride):cholesterol-based transfection complexes to tumor-bearing mice resulted in expression in the tumor and other tissues, primarily the lungs. Application of ultrasound to the tumor before or after the injection resulted in a significant increase in gene transfer to the tumor with no increase observed in other tissues. The magnitude of increased expression ranged from three- to 270-fold depending upon the DNA dose. The following parameters were optimized for maximal increase: duration of ultrasound application, the time interval between plasmid injection and sonoporation, and plasmid dose. A combination of plasmid quantitation and fluorescence microscopy showed that ultrasound increased tumor uptake of the plasmid and that uptake was limited to the tumor vasculature. Using an IL- 12 expression plasmid, the combination of a single plasmid dose (10 microg) and ultrasound treatment produced significantly higher levels of IL-12 in tumor. This increased expression was sufficient to inhibit tumor growth compared with the control conditions. These data demonstrate the potential application of sonoporation as an effective method for enhancing the expression of systemically administered genes in tumor endothelium for cancer gene therapy.

Animals↗
PubMed2000

Optimization of cationic lipid/DNA complexes for systemic gene transfer to tumor lesions.

Intravenous (i.v.) administration of cationic lipid N-[( 1-(2-3-dioleyloxy)propyl)]-N-N-N-trimethylammonium chloride (DOTMA)-based transfection complexes in mice with subcutaneous squamous cell tumors yielded plasmid delivery and expression in tumor lesions. The efficiency of gene transfer in tumors was significantly lower than in the lung. This was consistent with low plasmid levels associated with the tumor, suggesting that plasmid delivery to the tumor site was a limiting factor. Lowering the lipid/DNA charge ratio from 5:1 to 0.8:1 (+/-) did not change DNA levels in tumor but significantly reduced DNA levels in lung. However, expression levels were significantly reduced in both tissues at lower lipid/DNA charge ratios. Complexes prepared from small unilamellar liposomes gave significantly lower expression levels in the lungs but similar expression levels in tumors when compared to complexes prepared from larger unilamellar liposomes. The small liposome complexes were better tolerated than large liposome complexes. Varying the cationic lipid to colipid (cholesterol or DOPE) molar ratio from 4: 1 to 1: 1 significantly reduced expression levels in both tumor and lung. Cationic lipid substitution, using a cholesterol cationic lipid, diethyldiamino-carbamyl-cholesterol instead of DOTMA, produced reduced expression in all other tissues except tumor. Incorporation of PEG into preformed transfection complexes reduced DNA delivery to lung, increased circulation half-life, and enhanced DNA delivery to tumor. In a lung metastatic mouse tumor model, where the accessibility of the i.v. administered transfection complexes to tumor lesions should be less challenging, DOTMA: CHOL complexes (4: 1 lipid to colipid molar ratio, 3: 1 +/- lipid to plasmid charge ratio) were preferentially localized in tumor lesions. These data demonstrate that systemic gene transfer to distal tumor sites by lipid/ DNA complexes may be limited by low plasmid delivery. Modifying the chemical surface properties of transfection complexes enhanced both DNA delivery and expression in tumor and is one approach that may overcome limitations.

Animals↗
PubMed1999

Crystal structure of Pseudomonas fluorescens 4-hydroxyphenylpyruvate dioxygenase: an enzyme involved in the tyrosine degradation pathway.

BACKGROUND: In plants and photosynthetic bacteria, the tyrosine degradation pathway is crucial because homogentisate, a tyrosine degradation product, is a precursor for the biosynthesis of photosynthetic pigments, such as quinones or tocophenols. Homogentisate biosynthesis includes a decarboxylation step, a dioxygenation and a rearrangement of the pyruvate sidechain. This complex reaction is carried out by a single enzyme, the 4-hydroxyphenylpyruvate dioxygenase (HPPD), a non-heme iron dependent enzyme that is active as a homotetramer in bacteria and as a homodimer in plants. Moreover, in humans, a HPPD deficiency is found to be related to tyrosinemia, a rare hereditary disorder of tyrosine catabolism. RESULTS: We report here the crystal structure of Pseudomonas fluorescens HPPD refined to 2.4 A resolution (Rfree 27.6%; R factor 21.9%). The general topology of the protein comprises two barrel-shaped domains and is similar to the structures of Pseudomonas 2,3-dihydroxybiphenyl dioxygenase (DHBD) and Pseudomonas putida catechol 2,3-dioxygenase (MPC). Each structural domain contains two repeated betaalpha betabeta betaalpha modules. There is one non-heme iron atom per monomer liganded to the sidechains of His161, His240, Glu322 and one acetate molecule. CONCLUSIONS: The analysis of the HPPD structure and its superposition with the structures of DHBD and MPC highlight some important differences in the active sites of these enzymes. These comparisons also suggest that the pyruvate part of the HPPD substrate (4-hydroxyphenylpyruvate) and the O2 molecule would occupy the three free coordination sites of the catalytic iron atom. This substrate-enzyme model will aid the design of new inhibitors of the homogentisate biosynthesis reaction.

4-Hydroxyphenylpyruvate Dioxygenase↗
PubMed1999

Pharmaceutical perspectives of nonviral gene therapy.

The use of nonviral plasmid-based gene medicines represents an attractive in vivo gene transfer strategy that is simple and lacks many risks that are inherent to viral systems. Commercialization of gene medicines requires a thorough analysis of business opportunities, unmet clinical needs, competitive products under development, and issues related to intellectual property. Synthetic gene delivery systems are designed to control the location of a gene within the body by affecting distribution and access of a gene expression system to the target cell, and/or recognition by a cell surface receptor and uptake followed by intracellular and nuclear translocation. Plasmid-based gene expression systems are designed to control the level, fidelity, and duration of in vivo production of a therapeutic gene product. This review will provide insights into the potentials of plasmid-based gene therapy and critical evaluation of gene delivery sciences and clinical applications of gene medicines.

Animals↗
PubMed1999

Systemic inhibition of tumor growth and tumor metastases by intramuscular administration of the endostatin gene.

Tumors require ongoing angiogenesis to support their growth. Inhibition of angiogenesis by production of angiostatic factors should be a viable approach for cancer gene therapy. Endostatin, a potent angiostatic factor, was expressed in mouse muscle and secreted into the bloodstream for up to 2 weeks after a single intramuscular administration of the endostatin gene. The biological activity of the expressed endostatin was demonstrated by its ability to inhibit systemic angiogenesis. Moreover, the sustained production of endostatin by intramuscular gene therapy inhibited both the growth of primary tumors and the development of metastatic lesions. These results demonstrate the potential utility of intramuscular delivery of an antiangiogenic gene for treatment of disseminated cancers.

Animals↗
PubMed1999

Peptide-based gene delivery.

To achieve effective plasmid-based gene therapy, the control of cellular access and uptake, intracellular trafficking and nuclear retention of plasmids must be achieved. Inefficient endosomal release, cytoplasmic transport and nuclear entry of plasmids are amongst some of the key limiting factors in the use of plasmids for effective gene therapy. A number of non-viral gene delivery systems have been designed to overcome these limiting factors. The most common approach to protect and control plasmid distribution is to complex plasmids with cationic lipids or polymers through electrostatic interactions. Endosomal release of plasmids can be achieved, for instance, by using pH-sensitive lipids, inactivated viral particles, endosomolytic peptides and polymers. Among the least explored gene delivery systems are those that consist mainly of synthetic, short peptides. Peptides can be incorporated into multicomponent gene delivery complexes for specific purposes, such as for DNA condensation, cell-specific targeting, endosomolysis or nuclear transport. The aims of this review are to: (i) explore the conceptual and experimental aspects of peptide-DNA interactions; (ii) critically assess the possible use of peptides for efficient gene transfer; and (iii) present an overview on the use of peptides to enhance the effectiveness of other gene delivery systems. On balance, peptide-based gene delivery systems appear to have a significant potential as commercially viable gene delivery products.

Amino Acid Sequence↗
PubMed1998

Homocamptothecins: synthesis and antitumor activity of novel E-ring-modified camptothecin analogues.

Homocamptothecin (hCPT), a camptothecin (CPT) analogue with a seven membered beta-hydroxylactone which combines enhanced plasma stability and potent topoisomerase I (Topo I)-mediated activity, is an attractive template for the elaboration of new anticancer agents. Like CPT, hCPT carries an asymmetric tertiary alcohol and displays stereoselective inhibition of Topo I. The preparation and biological screening of racemic hCPT analogues are described. The 10 hCPTs tested were better Topo I inhibitors than CPT. Fluorinated hCPTs 23c, d,f,g were found to have potent cytotoxic activity on A427 and PC-3 tumor cell lines. Their cytotoxicity remained high on the K562adr and MCF7mdr cell lines, which overexpress a functionally active P-glycoprotein. Fluorinated hCPTs were more efficacious in vivo than CPT on HT-29 xenografts. In this model, a tumor growth delay of 25 days was reached with hCPT 23g at a daily dose of 0.32 mg/kg, compared to 4 days with CPT at 0.625 mg/kg. Thus difluorinated hCPT 23g warrants further investigation as a novel Topo I inhibitor with high cytotoxicity toward tumor cells and promising in vivo efficacy.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1998

Biodistribution and gene expression of lipid/plasmid complexes after systemic administration.

The objectives of this study were to investigate the influence of physicochemical properties of lipid/plasmid complexes on in vivo gene transfer and biodistribution characteristics. Formulations based on 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA) and novel biodegradable cationic lipids, such as ethyl dioleoyl phosphatidylcholine (EDOPC), ethyl palmitoyl myristyl phosphatidylcholine (EPMPC), myristyl myristoyl carnitine ester (MMCE), and oleyl oleoyl L-carnitine ester (DOLCE), were assessed for gene expression after tail vein injection of lipid/plasmid complexes in mice. Gene expression was influenced by cationic lipid structure, cationic lipid-to-colipid molar ratios, plasmid-to-lipid charge ratios, and precondensation liposome size. Detectable levels of human growth hormone (hGH) in serum, human factor IX (hFIX) in plasma, and chloramphenicol acetyltransferase (CAT) in the lung and liver were observed with positively charged lipid/plasmid complexes prepared from 400-nm extruded liposomes with a cationic lipid-to-colipid ratio of 4:1 (mol/mol). Intravenous administration of lipid/CAT plasmid complexes resulted in distribution of plasmid DNA mainly to the lung at 15 min after injection. Plasmid DNA accumulation in the liver increased with time up to 24 hr postinjection. There was a 10-fold decrease in the amount of plasmid DNA in the lung at 15 min after injection, when the lipid/plasmid complex charge ratio was decreased from 3:1 to 0.5:1 (+/-). Bright fluorescent aggregates were evident in in vivo-transfected lung with the positively charged pCMV-CAT/DOLCE:dioleyl phosphatidylethanolamine (DOPE) (1:1, mol/mol) complexes, while more discrete punctate fluorescence was observed with a 4:1 molar ratio of cationic lipid:colipid formulations. Preinjection of polyanions such as plasmid, dextran sulfate, polycytidic acid, and polyinosinic acid decreased hGH expression, whereas the preinjection of both positively charged and neutral liposomes had no effect on hGH serum levels. Of the cationic lipids tested, DOLCE was found to be the most effective potentially biodegradable cationic lipid. A correlation between gene expression and cationic lipid:colipid ratios and lipid-to-plasmid charge ratio was also observed for DOTMA- and DOLCE-based formulations.

Animals↗
PubMed1998

Preface.

Explore the source record for details and available documents.

Journal Article↗
PubMed1998

A physicochemical approach for predicting the effectiveness of peptide-based gene delivery systems for use in plasmid-based gene therapy.

Novel synthetic peptides, based on carrier peptide analogs (YKAKnWK) and an amphipathic peptide (GLFEALLELLESLWELLLEA), have been formulated with DNA plasmids to create peptide-based gene delivery systems. The carrier peptides are used to condense plasmids into nanoparticles with a hydrodynamic diameter (DH) ranging from 40 to 200 nm, which are sterically stable for over 100 h. Size and morphology of the carrier peptide/plasmid complex have been determined by photon correlation spectroscopy (PCS) and transmission electron microscopy (TEM), respectively. The amphipathic peptide is used as a pH-sensitive lytic agent to facilitate release of the plasmid from endosomes after endocytosis of the peptide/plasmid complex. Hemolysis assays have shown that the amphipathic peptide destabilizes lipid bilayers at low pH, mimicking the properties of viral fusogenic peptides. However, circular dichroism studies show that unlike the viral fusion peptides, this amphipathic peptide loses some of its alpha-helical structure at low pH in the presence of liposomes. The peptide-based gene delivery systems were tested for transfection efficiency in a variety of cell lines, including 14-day C2C12 mouse myotubes, using gene expression systems containing the beta-galactosidase reporter gene. Transfection data demonstrate a correlation between in vitro transfection efficiency and the combination of several physical properties of the peptide/plasmid complexes, including 1) DNA dose, 2) the zeta potential of the particle, 3) the requirement of both lytic and carrier peptides, and 4) the number of lysine residues associated with the carrier peptide. Transfection data on 14-day C2C12 myotubes utilizing the therapeutic human growth hormone gene formulated in an optimal peptide gene delivery system show an increase in gene expression over time, with a maximum in protein levels at 96 h (approximately 18 ng/ml).

Amino Acid Sequence↗
PubMed1998

Target specific optimization of cationic lipid-based systems for pulmonary gene therapy.

PURPOSE: Cationic lipids are capable of transferring foreign genes to the pulmonary epithelium in vivo. It is becoming increasingly clear that factors other than lipid molecular structure also influence efficiency of delivery using cationic lipid systems. This study is aimed at evaluating the effect of formulation variables such as cationic lipid structure, cationic lipid/DNA ratio, particle size, co-lipid content and plasmid topology on transgene expression in the lung. METHODS: The effect of varying the surface and colloidal properties of cationic lipid-based gene delivery systems was assessed by intratracheal instillation into rats. An expression plasmid encoding chloramphenicol acetyl transferase (CAT) was used to measure transgene expression. RESULTS: Cationic lipid structure, cationic lipid/DNA ratio, particle size, co-lipid content and topology of the plasmid, were found to significantly affect transgene expression. Complexation with lipids was found to have a protective effect on DNA integrity in bronchoalveolar lavage fluid (BALF). DNA complexed with lipid showed enhanced persistence in rat lungs as measured by quantitative polymerase chain reaction. CONCLUSIONS: Fluorescence microscopy analysis indicated that the instilled formulation reaches the lower airways and alveolar region. Data also suggests cationic lipid-mediated gene expression is primarily localized in the lung parenchyma and not infiltrating cells isolated from the BALF.

Animals↗