PubMed Health⌕ Search

Biomedical subjects

M Manthorpe

Publications and source records attributed to M Manthorpe.

At least 19 recordsLinked to original sources
PubMed2001

Vaxfectin enhances antigen specific antibody titers and maintains Th1 type immune responses to plasmid DNA immunization.

Antigen specific immune responses were characterized after intramuscular immunization of BALB/c mice with 5 antigen encoding plasmid DNAs (pDNAs) complexed with Vaxfectin, a cationic lipid formulation. Vaxfectin increased IgG titers for all of the antigens with no effect on the CTL responses to the 2 antigens for which CTL assays were performed. Both antigen specific IgG1 and IgG2a were increased, although IgG2a remained greater than IgG1. Furthermore, Vaxfectin had no effect on IFN-gamma or IL-4 production by splenocytes re-stimulated with antigen, suggesting that the Th1 type responses typical of intramuscular pDNA immunization were not altered. Studies with IL-6 -/- mice suggest that the antibody enhancement is IL-6 dependent and results in a correlative increase in antigen specific antibody secreting cells.

Adjuvants, Immunologic↗
PubMed2001

Vaxfectin enhances the humoral immune response to plasmid DNA-encoded antigens.

This report characterizes Vaxfectin, a novel cationic and neutral lipid formulation which enhances antibody responses when complexed with an antigen-encoding plasmid DNA (pDNA). In mice, intramuscular injection of Vaxfectin formulated with pDNA encoding influenza nucleoprotein (NP) increased antibody titers up to 20-fold, to levels that could not be reached with pDNA alone. As little as 1 microg of pDNA formulated with Vaxfectin per muscle resulted in higher anti-NP titers than that obtained with 25 microg naked pDNA. The antibody titers in animals injected with Vaxfectin-pDNA remained higher than in the naked pDNA controls for at least 9 months. The enhancement in antibody titers was dependent on the Vaxfectin dose and was accomplished without diminishing the strong anti-NP cytolytic T cell response typical of pDNA-based vaccines. In rabbits, complexing pDNA with Vaxfectin enhanced antibody titers up to 50-fold with needle and syringe injections and also augmented humoral responses when combined with a needle-free injection device. Vaxfectin did not facilitate transfection and/or increase synthesis of beta-galactosidase reporter protein in muscle tissue. ELISPOT assays performed on bone marrow cells from vaccinated mice showed that Vaxfectin produced a three- to five-fold increase in the number of NP-specific plasma cells. Thus, Vaxfectin should be a useful adjuvant for enhancing pDNA-based vaccinations.

Adjuvants, Immunologic↗
PubMed2000

Sodium phosphate enhances plasmid DNA expression in vivo.

Intramuscular injection of plasmid DNA results in myofiber cell expression of proteins encoded by the DNA. The preferred vehicle for plasmid DNA injections has been saline (154 mM sodium chloride) or PBS (154 mM NaCl plus 10 mM sodium phosphate). Here, it is shown that injection of luciferase or beta-galactosidase encoding plasmid DNA in a 150 mM sodium phosphate vehicle into murine muscle resulted in a two- to seven-fold increase in transgene expression compared with DNA injected in saline or PBS. When the DNA encoded secreted alkaline phosphatase, preproinsulin or interferon, sodium phosphate vehicle increased their serum levels by two- to four-fold. When the DNA encoded mouse erythropoietin, sodium phosphate vehicle increased hematocrits by two-fold compared with DNA injected in saline. When the DNA encoded influenza nucleoprotein, sodium phosphate increased anti-nucleoprotein antibody titers by two-fold. The expression of luciferase from plasmid DNA instilled into lung was increased five-fold compared with that in vehicle without sodium phosphate. Incubation of plasmid DNA with muscle extract or serum showed that sodium phosphate protected the DNA from degradation. Thus, a change from sodium chloride to sodium phosphate vehicle can enhance the expression of plasmid DNA in a tissue, possibly by inhibiting DNA degradation. Gene Therapy (2000) 7, 1171-1182.

Alkaline Phosphatase↗
PubMed1998

Enhancer and promoter chimeras in plasmids designed for intramuscular injection: a comparative in vivo and in vitro study.

Enhancers and promoters from various muscle-specific genes were substituted for or combined with the enhancer/promoter of the human cytomegalovirus (CMV) IE gene in a luciferase reporter gene plasmid in an effort to identify new promoter chimeras with increased expression activity after direct intramuscular injection. The regulatory sequence substitutions or additions varied in content, location, and orientation relative to the CMV regulatory sequences. The expression activities of the derivative and parent plasmids were compared quantitatively in vivo using a standard mouse intramuscular injection assay, and in vitro by transfection of differentiated C2C12 mouse myoblasts and BHK hamster kidney cells, to test whether cultured cell transfection could substitute for at least some animal experimentation. In vivo, 1 of 19 of the enhancer/promoter chimeras increased expression levels. In vitro, some chimeras showed significant expression augmentation in C2C12 cells, but not in BHK cells. We conclude that because of differences in plasmid expression profiles, these cell culture systems cannot readily substitute for in vivo testing of new plasmid constructs.

Animals↗
PubMed1997

Synthetic NGF peptide derivatives prevent neuronal death via a p75 receptor-dependent mechanism.

Cyclized peptides corresponding to beta-loop regions of NGF were purified by HPLC and assayed for neurotrophic activity using DRG neurons. Peptides with the highest activity corresponded to loop region 29-35, a domain likely to interact with the p75 receptor. Unexpectedly, activity was confined to late-eluting HPLC fractions containing peptide multimers and primarily promoted neuronal survival without neurite outgrowth. Directed synthesis of dimer and monomer cyclized peptides demonstrated that dimers acted as partial NGF agonists in that they had both survival-promoting and NGF-inhibiting activity while monomer and linear peptides were inactive. Dimer activity was not affected by the Trk inhibitor K252a but was blocked by p75 receptor antibody and absent using p75 null mutant neurons. These studies suggest that region 29-35 peptide derivatives inhibit neuronal death via a structure- and p75-dependent mechanism.

Animals↗
PubMed1997

Development of improved vectors for DNA-based immunization and other gene therapy applications.

Optimizing gene expression and delivery are necessary steps in the production of vectors for DNA-based immunization as well as for other gene therapy applications. A mouse muscle/reporter gene assay system was used to systematically improve a plasmid DNA vector. The optimized vector VR1255 contained: (1) CMV promoter and enhancer; (2) CMV IE Intron A; (3) kanamycin resistance gene; (4) deleted SV40 origin of replication; (5) optimized lux coding region; and (6) a minimal synthetic terminator from the rabbit beta globin gene, mRBG. The vector VR1255 expressed 137 times greater than an earlier prototype RSV-based vector. For plasmid vector delivery into nonmuscle tissues, a recently synthesized cationic lipid, GAP-DLRIE, was found to greatly enhance the uptake and expression of plasmid DNA by 100-fold when instilled into the mouse lung. The time-course of CAT expression with GAP-DLRIE indicated that peak expression occurs 2-5 days after intranasal administration and expression diminished to about one-third the peak value by day 21. This cationic lipid may be useful for immunization by pulmonary and perhaps other nonmuscle routes.

Animals↗
PubMed1997

Spatial-temporal patterns of gene expression in mouse skeletal muscle after injection of lacZ plasmid DNA.

Gene therapy for muscular diseases requires the efficient transfection of a large proportion of myofiber cells within a given muscle. In the present experiments, patterns of beta-galactosidase expression were examined in mouse rectus femoris muscles at various time-points after a single injection of lacZ encoded plasmid DNA. beta-Galactosidase expression was detected 3 h after injection and rose to peak levels at 3-14 days, and then stabilized at lower levels. beta-Galactosidase staining was detected in an average of about 6% (up to 15%) of the total 4000 myofiber cells, and in about 70% of those myofibers located in the discrete area containing the greatest proportion of transfected cells. Soon after injection of DNA encoding cytoplasmic or nuclear-targeted beta-galactosidase, expression was noted predominantly in the myotendinous junction areas, after which beta-galactosidase activity progressed toward the central parts of the myofibers. This preferential transgene expression at the myotendinous junction may result from some unique, local property of the myofiber cells and/or from a restricted diffusion or binding of the injected plasmid DNA at tendinous surfaces. A better understanding of the reasons for this pattern of reporter gene expression in muscle may suggest procedures for increasing the number of myofiber cells transfected by direct DNA injections.

Animals↗
PubMed1996

A novel cationic lipid greatly enhances plasmid DNA delivery and expression in mouse lung.

Effective gene therapy for lung tissue requires the use of efficient vehicles to deliver the gene of interest into lung cells. When plasmid DNA encoding chloramphenicol acetyltransferase (CAT) was administered intranasally to BALB/c mice without carrier lipids, CAT activity was detected in mouse lung extracts. Plasmid DNA delivered with optimally formulated commercially available transfection reagents expressed up to 10-fold more CAT activity in lung than observed with naked DNA alone. Liposome formulations consisting of (+/-)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis (dodecyloxy)-1-propanaminium bromide (GAP-DLRIE) plus the neutral colipid dioleoylphosphatidylethanolamine (DOPE) enhanced CAT expression by more than 100-fold relative to plasmid DNA alone. A single administration of GAP-DLRIE liposome-CAT DNA complexes to mouse lung elicited peak expression at days 1-4 posttransfection, followed by a gradual return to baseline by day 21 postadministration. Readministration of GAP-DLRIE liposome CAT complexes at day 21 led to another transient peak of reporter gene expression. Histological examination of lungs treated with GAP-DLRIE complexed beta-galactosidase DNA revealed that alveolar epithelial cells were the primary locus of expression and that up to 1% of all alveoli contained epithelial cells expressing the transgene.

Administration, Intranasal↗
PubMed1996

An improved plasmid DNA expression vector for direct injection into skeletal muscle.

In previous work, the direct injection of 50 micrograms of a plasmid DNA vector encoding firefly luciferase (VR1205) into murine quadriceps muscle produced an average of 6.5 ng of luciferase per muscle at 7 days postinjection. In this report, various elements of the VR1205 vector were modified to increase gene expression levels or to eliminate undesired viral sequences. Expression of the modified vectors was then compared to VR1205 using the intramuscular injection assay. In general, modifications to promoter, enhancer, and intronic sequences either decreased luciferase expression levels or had no effect. However, modifications to the polyadenylation and transcriptional termination sequences, plasmid backbone elements, and the luciferase gene itself each increased luciferase expression levels. The best-expressing vector, designated VR1255, contained a combination of these incrementally beneficial changes. A single intramuscular injection of 50 micrograms of VR1255 produced 300 ng of luciferase at 7 days postinjection, an expression level 46-fold higher than the VR1205 vector (or 22-fold higher, excluding modifications to the luciferase gene) and 154-fold higher than a commercially available luciferase expression vector. Thus, VR1255 represents an improved plasmid DNA vector that may be useful for gene therapy applications.

Animals↗
PubMed1996

Converting an alcohol to an amine in a cationic lipid dramatically alters the co-lipid requirement, cellular transfection activity and the ultrastructure of DNA-cytofectin complexes.

Cytofectins are positively charged lipophilic molecules that readily form complexes with DNA and other anionic polynucleotides. Normally, cytofectins are combined with an activity-augmenting phospholipid such as dioleoylphosphatidylethanolamine (DOPE), and a film of dried, mixed lipid is prepared and hydrated to form cationic liposomes. The liposome solution is then mixed with a plasmid DNA solution to afford cytofectin-DNA complexes which, when presented to living cells, are internalized and the transgene is expressed. One of the most potent cytofectins, dimyristoyl Rosenthal inhibitor ether (DMRIE), is presently being used to deliver transcriptionally active DNA into human tumor tissues. Here we report the remarkable consequences of replacing the alcohol moiety of DMRIE with a primary amine. The resulting cytofectin, called beta-aminoethyl-DMRIE (betaAE-DMRIE), promoted high level transfection over a broad range of DNA and cationic lipid concentrations. A comparison of in vitro transfection activity between DMRIE and betaAE-DMRIE in 10 cell types revealed that betaAE-DMRIE was more active than DMRIE, and that betaAE-DMRIE, unlike DMRIE, was maximally effective in the absence of colipid. The consequences of the alcohol-to-amine conversion on the structure of the cytofectin/DNA complex was also examined by Atomic Force Microscopy. Strikingly dissimilar images were found for plasmid DNA alone and for the plasmid complexes of betaAE-DMRIE and DMRIE/DOPE.

Alcohols↗
PubMed1996

Novel, high expressing and antibiotic-controlled plasmid vectors designed for use in gene therapy.

The promise of effective gene therapy can only be accomplished by high-level expression and regulatable delivery of gene products. To achieve this end, a eukaryotic expression plasmid was modified to make transcription dependent on a tetracycline(Tc)-regulated chimeric transactivator. Mouse muscle injected with this two plasmid cis/trans control system expressed reporter proteins at levels five- to 10-fold greater than the cytomegalovirus immediate-early promoter-controlled parental plasmid. Tetracycline could be useful to either repress or activate transactivator-controlled expression based on the position of the tetO control sequences within the reporter plasmid. Finally, a prototype single plasmid construct was made and shown to express a self-regulating bicistronic transcript containing both the reporter and the transactivator. These Tc-controlled plasmids, termed maximum expression and regulated vectors (MERVs), have the potential to target a variety of gene therapy applications.

Animals↗
PubMed1995

Improved cationic lipid formulations for in vivo gene therapy.

The problem of assessing in vivo activity of gene delivery systems is complex. The reporter gene must be carefully chosen depending on the application. Plasmids with strong promoters, enhancers and other elements that optimize transcription and translation should be employed, such as the CMVint and pCIS-CAT constructs. Formulation aspects of cationic lipid-DNA complexes are being studied in several laboratories, and the physical properties and molecular organization of the complexes are being elucidated. Likewise, studies on the mechanism of DNA delivery with cationic lipids are accumulating which support the basic concept that the complexes fuse with biological membranes leading to the entry of intact DNA into the cytoplasm. Naked plasmid DNA administered by various routes is expressed at significant levels in vivo. This observation is not restricted to skeletal and heart muscle, but has been observed in lung, dermis, and in undefined tissues following intravenous administration. Most of the widely available cationic lipids, including Lipofectin, Lipofectamine and DC-cholesterol have a very poor ability to enhance DNA expression above the baseline naked DNA level, at least in lung. In this report we have revealed a novel cationic lipid, DLRIE, which can significantly enhance CAT expression in mouse lung by 25-fold above the naked DNA level. Other compounds are currently being evaluated which can enhance the naked DNA expression even higher. Plasmid vector improvements have led to further increase in in vivo lung expression, so that the net improvement is > 5,000-fold. Results of this nature are advancing the pharmaceutical gene therapy opportunities for synthetic cationic lipid based gene delivery systems.

Animals↗
PubMed1995

Cancer gene therapy using plasmid DNA: safety evaluation in rodents and non-human primates.

To evaluate the safety of a plasmid DNA-lipid complex, a series of good laboratory practice (GLP) safety studies were conducted with VCL-1005, a plasmid DNA expression vector containing both the human class I MHC HLA-B7 heavy-chain and the beta 2-microglobulin (beta 2m) light-chain genes formulated with the cationic lipid, DMRIE/DOPE. In mice, the repeated intravenous injection of VCL-1005 at plasmid DNA doses of 0.1, 1.0, or 10 micrograms for 14 days had only incidental effects on clinical chemistry and hematology, and did not result in any organ pathology. Repeated intrahepatic injections of VCL-1005 in mice did not result in significant liver histopathology or significant alterations in liver enzymes. In cynomolgus monkeys, the repeated intravenous administration of VCL-1005 at a cumulative dose of 720 micrograms of DNA had no effects on clinical chemistry, hematology, or organ pathology. Thus, systemic administration of a plasmid DNA expression vector containing the coding sequence for a foreign MHC class I molecule did not result in significant toxicity or a pathological immune response in animals. These results suggest that the direct transfer of VCL-1005, a plasmid DNA-lipid complex, could be used for the safe in vivo delivery of recombinant DNA for a cancer gene therapy trial.

Animals↗
PubMed1994

Immunization with plasmid DNA using a pneumatic gun.

We characterize a method by which the Med-E-Jet pneumatic vaccination gun can be used to propel intact, supercoiled plasmid DNA through skin and into skeletal muscles of mice. Intramuscular injection of plasmids containing the firefly luciferase gene linked to the human cytomegalovirus promoter resulted in the expression of several hundred picograms of luciferase enzyme in quadriceps muscles. Intramuscular injections of a plasmid containing the influenza A nuclear protein gene regulated by the same promoter resulted in the generation of potent and specific anti-nuclear protein humoral and cellular immune responses. This convenient and rapid injection method would be well-suited for genetic immunization of humans.

Animals↗
PubMed1993

Gene therapy by intramuscular injection of plasmid DNA: studies on firefly luciferase gene expression in mice.

Direct injection of nonviral, covalently closed circular plasmid DNA into muscle results in expression of the DNA in myofiber cells. We have examined the expression of firefly luciferase DNA constructs injected into adult murine skeletal muscle. Considerable variation in luciferase enzyme expression was noted among constructs with different regulatory elements, among different batches of the same DNA construct, and among similar transfection experiments performed at different times. This variation was minimized by using single batches of plasmid DNA and by performing comparable sets of experiments concurrently. A quantitative experimental protocol was defined for comparing various aspects of the transfection process. We report that a luciferase construct containing the human cytomegalovirus immediate-early gene promoter plus intron A (a construct termed "p-CMVint-lux") showed the highest expression among several constructs tested. Dose-response and time course analyses of p-CMVint-lux DNA injections showed that maximal luciferase expression was achieved with 25 micrograms of DNA at 7-14 days post-injection. Selected manipulations of the transfection process were examined for their influence on luciferase expression. Variations in the rate of DNA injection, needle size, injection volume, and vehicle temperature had no significant effect on luciferase expression. The presence of endotoxin, cationic peptide, muscle stimulants or relaxants, vasoconstrictors, metal chelators, or lysosomal lytic reagents had no significant effect on expression. However, linearization of the DNA, injection of the DNA in water rather than saline, or inclusion of a DNA intercalating agent nearly abolished luciferase expression. And finally, increasing the injection dose by giving multiple injections over a 10-day period increased expression proportionally to the number of injections.

Amino Acid Sequence↗
PubMed1992

The localization of nerve growth factor-like immunoreactivity in the adult rat basal forebrain and hippocampal formation.

The role of nerve growth factor (NGF) as a target derived neurotrophic agent for specific cell populations in the peripheral nervous system has been well documented and much evidence suggests that NGF may serve a similar neurotrophic role in the CNS supporting the cholinergic neurons of the basal forebrain. Previous attempts to localize NGF by immunocytochemical methods, however, have not yielded evidence confirming the regional distribution expected based upon reported levels of extractable NGF. In the present study, affinity purified polyclonal antibodies to beta-NGF and a modified immunohistochemical protocol were used to demonstrate specific NGF-like immunoreactivity in the adult rat hippocampal formation and basal forebrain. In the hippocampal formation, NGF-like immunoreactivity was localized primarily within the hilus of the dentate gyrus and within stratum lucidum of the CA3 and CA2 hippocampal subfields. Staining appeared to be associated with cell processes and was similar to the reported distribution of mossy fibers suggesting that granule cells may either serve as a primary source of hippocampal NGF or that mossy fibers selectively accumulate NGF produced by other cell populations. In the basal forebrain, NGF-like immunoreactivity was localized within neuronal cell bodies of the medial septum, diagonal band, and nucleus basalis of Meynert and was further demonstrated to colocalize exclusively with LNGF-R positive neurons. These findings demonstrate the presence of an NGF-like antigen in association with cholinergic neurons of the basal forebrain and strongly support the hypothesis that NGF may serve as an endogenous trophic factor for this adult neuronal population.

Animals↗
PubMed1992

Nerve growth factor receptor immunoreactivity in neurons of the normal adult rat spinal cord and its modulation after peripheral nerve lesions.

Motoneurons of the rat spinal cord express low-affinity nerve growth factor receptor (LNGFR) and corresponding mRNA during development, and re-express it after their axotomy by peripheral nerve injury. The present study establishes the anatomical and quantitative baseline of LNGFR immunoreactive (LNGFR-IR) neurons of the entire normal adult female rat and then investigates the temporal course for the re-expression of LNGFR-IR in lumbar motoneurons after either a crush lesion (which is followed by regeneration and reconnection to the muscle) or a cut lesion with removal of the distal stump (where a neuroma but no reconnection is formed). In the normal adult spinal cord, two types of LNGFR-IR neurons were recognized: (1) small populations of large motoneurons located in the ventral horn mainly in correspondence to the regions of the phrenic, cremasteric and dorsolateral nuclei, and (2) a more numerous and more dorsally located population of small neurons. With a sciatic cut lesion, the number of LNGFR-IR motoneurons at spinal levels L4-L6 rapidly and dramatically increased to a maximum between post-lesion days 1 and 7, apparently involving most axotomized motoneurons of the region, and returned to the baseline level by day 30. With a crush lesion, similar numbers and virtually the same time-course of LNGFR-IR appearance were seen, but the onset of progressive disappearance of LNGFR-IR neurons was delayed by one week, so that at 30 days, the most caudal motoneurons (which are last to reach their target) were still LNGFR-IR. Comparison of these two time courses gives clues to the kind of signals that may be involved in initiating and/or maintaining the LNGFR response.

Animals↗