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A D Miller

Publications and source records attributed to A D Miller.

At least 73 records · Page 4Linked to original sources
PubMed1998

An RGD-oligolysine peptide: a prototype construct for integrin-mediated gene delivery.

We have synthesized a linear, bifunctional peptide that comprises an integrin-targeting domain containing an arginine-glycine-aspartic acid tripeptide motif and a DNA-binding moiety consisting of a short stretch of 16 lysine residues. This peptide can form distinctive, condensed complexes with DNA and is capable of mediating its delivery and expression in a variety of mammalian cells in culture. Internalization is mediated by cell surface integrin receptors via a mechanism that is known to be phagocytic. We have analyzed the relationship between DNA and peptide and have investigated the conditions suitable for optimal gene delivery. The formation of condensed peptide DNA complexes leads to resistance to nuclease degradation. The level of reporter gene expression obtained is dependent on the peptide-to-DNA ratio and is enhanced in the presence of the endosomal buffer chloroquine, polyethyleneimine, and deactivated adenovirus during gene delivery. Under optimal conditions the levels of reporter gene expression obtained approach or even exceed those obtained with DNA delivered with the commercial liposome Lipofectamine. The ability to produce an efficient gene delivery system using small, easily modified, and well-defined constructs that have no constraint of particle size demonstrates the advantages of integrin-targeting peptides for gene transfer.

3T3 Cells↗
PubMed1998

Upper airway motor outputs during vomiting versus swallowing in the decerebrate cat.

Swallowing and vomiting are antagonistic motor acts; nevertheless, vomiting can be immediately followed by swallowing. The purpose of this study was to clarify the interrelationship between these two behaviors, particularly in regard to comparing the upper airway motor patterns at the end of the expulsion phase with those during subsequent swallowing. Experiments were conducted using both paralyzed and non-paralyzed decerebrate cats, in which recordings were obtained either from upper airway muscles, the diaphragm and abdominal muscles or from the nerves that innervate those muscles. The activity patterns of most nerves recorded in paralyzed animals were consistent with the behavior recorded in non-paralyzed animals from the muscles innervated by those nerves, with the exception of the cricothyroid and stylopharyngeus muscles. Vomiting can be divided into a series of retches followed by expulsion, which itself can be further subdivided into three phases. The final stage of expulsion, characterized by burst-like exaggerated activity of the laryngeal elevator thyrohyoid and the pharyngeal constrictors, proved to be different from pharyngeal swallowing, as judged from differences in the spatio-temporal patterns of the upper airway motor outputs. However, post-vomiting swallowing activity was still observed even after total deafferentation of the laryngeal and pharyngeal areas in paralyzed animals. It is therefore likely that the central processes for vomiting and swallowing closely relate in generating these two behaviors.

Afferent Pathways↗
PubMed1998

Recovery of retching after lesions involving the nucleus of the solitary tract.

The nucleus of the solitary tract (NTS) in the caudal brainstem receives various inputs that trigger vomiting. Chemical (kainic acid) NTS lesions in decerebrate, paralyzed cats temporarily suppressed fictive vomiting induced by different emetics. Subsequently, retching but not expulsion, resumed in 2-3.5 h. Since the NTS does not appear essential for retching, antiemetic drugs targeted at the NTS may not completely suppress vomiting.

Animals↗
PubMed1998

beta-Galactosidase staining following intracoronary infusion of cationic liposomes in the in vivo rabbit heart is produced by microinfarction rather than effective gene transfer: a cautionary tale.

The myocardium is a potential target for the expression of exogenous genes to treat inherited and acquired diseases. Although adenovirus-mediated gene transfer has resulted in high-level gene transfer in vivo via direct intramyocardial injection and via a percutaneous intra-arterial route, the time-course of gene expression is limited by host immune responses. It was the aim of this study to test whether cationic liposome-mediated gene transfer, which does not suffer from the aforementioned problems, was feasible in the adult rabbit myocardium via a percutaneous transluminal approach. Doses of plasmid DNA encoding lacZ from 200-800 micrograms complexed to cationic liposomes resulted in X-gal conversion at day 3 with associated myocardial damage. We hypothesised that the damage was associated with macro-aggregates of cationic liposomes-DNA occluding the microcirculation. When such aggregates were excluded no X-gal conversion was seen in vivo. In order to show that X-gal conversion occurs in areas of infarction in the myocardium we caused closed chest infarction by deploying a platinum micro-embolisation coil in the circumflex coronary artery. At day 3 X-gal conversion was observed in the territory supplied by the occluded artery. Thus, microinfarction causes the false positive appearance of gene transfer when using a lacZ reporter gene.

Animals↗
PubMed1998

Endothelial cell transfection with cationic liposomes and herpes simplex-thymidine kinase mediated killing.

Endothelial cells are a promising target for cancer gene therapy because neoangiogenesis is vital for the supply of oxygen and nutrients to solid tumours. However, endothelial cells have been reported to be difficult to transfect. We demonstrate high rates of transfection with the reporter gene pSV40 beta gal using DC-Chol/DOPE cationic liposomes and lower rates with the novel polyamine cationic liposomes ACHx/DC-Chol/DOPE and ACO/DC-Chol/DOPE. Endothelial cells transfected with HSV-thymidine kinase using DC-Chol/DOPE demonstrated 3 log10 increased cytotoxicity compared with controls when exposed to the prodrug ganciclovir, thereby demonstrating significant biological effect.

Cations↗
PubMed1998

Enhanced in vitro and in vivo gene delivery using cationic agent complexed retrovirus vectors.

Retroviruses are, at present, the most efficient integrative vectors available for gene delivery. However, these viruses are still limited by relatively low titres. Although several protocols exist to improve virus titre most of them are time-consuming and unable to provide sufficient virus for in vivo applications. Virus titre can be enhanced by polybrene and other cationic agents. By investigating a broad range of cationic agents for their ability to enhance virus infectivity we found that both ecotropic and amphotropic retrovirus infection could be increased. In particular, the lipopolyamine dioctadecylamidoglycylspermine (DOGS) gave up to one order of magnitude enhancement above polybrene-mediated infection without cytotoxicity. To increase virus infectivity further we combined the enhancing effect of DOGS on virus infectivity with concentration of virus particles by ultrafiltration to reach titres of 1 x 10(9) IU/ml. The in vivo transduction of regenerating rat liver, by an amphotropic retrovirus was increased approximately five-fold by the addition of DOGS compared with virus alone. There was no animal toxicity observed following the administration of DOGS. The improved transduction efficiency seen both in vitro and in vivo following the co-administration of DOGS/virus complexes may be useful for future gene therapy applications.

3T3 Cells↗
PubMed1998

Liposomes enhance delivery and expression of an RGD-oligolysine gene transfer vector in human tracheal cells.

Nonviral gene delivery systems consist predominantly of lipoplexes or receptor-targeting and nontargeting polyplexes. We examined integrin-mediated gene delivery using an Arg-Gly-Asp/oligo-L-lysine ([K]16RGD) cyclic peptide and investigated its gene transfer efficiency when associated with a cationic liposome. We demonstrated that human cystic fibrosis and noncystic fibrosis tracheal epithelial cells in culture express integrins that recognise the RGD integrin-binding motif. We found a 10-fold (P < 0.01) increased expression of a luciferase encoding plasmid in these cells when complexing the plasmid to the [K]16RGD peptide as compared with plasmid alone. This increase was specific to the [K]16RGD peptide since neither a [K]16RGE nor a [K]16 peptide gave a comparable increase. Expression was further enhanced 30-fold (P < 0.01) with lipofectamine and the ratio of DNA/peptide/lipofectamine was critical for specificity and expression. Fluorescence and radioactive labelling of the complex showed that the [K]16RGD peptide increased the endocytic uptake of DNA into cells. The cell association of both DNA and peptide increased even further with lipofectamine. Confocal microscopy showed that the [K]16RGD peptide and the DNA internalised together within 30 min and localised to vesicles in the perinuclear region. These results show that an integrin-binding ligand can deliver genetic material to airway cells and that a cationic liposome can enhance the efficacy of this nonviral vector system.

Cation Exchange Resins↗
PubMed1998

Retrovirus packaging cells expressing the Mus dunni endogenous virus envelope facilitate transduction of CHO and primary hematopoietic cells.

Mus dunni endogenous virus (MDEV) infects a wide variety of cell types from many different species. To take advantage of this broad host range, we have constructed packaging cells (PD223) that produce virions bearing the MDEV envelope. PD223 cells are able to package Moloney murine leukemia virus-based vectors at a titer of 4 x 10(5) infectious units per ml in the absence of contaminating replication-competent retrovirus. Vectors packaged by PD223 cells are able to transduce CHO cells, which are resistant to transduction by many retroviruses, at >/=25-fold higher efficiency than vectors having other pseudotypes. A vector packaged by PD223 was found to be among the most efficient for transducing primary baboon and human CD34(+) cells.

3T3 Cells↗
PubMed1998

Successful readministration of adeno-associated virus vectors to the mouse lung requires transient immunosuppression during the initial exposure.

The airway is an important target for gene transfer to treat cystic fibrosis and other diseases that affect the lung. We previously found that marker gene expression did not persist in the bronchial epithelium following adeno-associated virus (AAV) vector administration to the rabbit lung. In an attempt to promote continued expression, we tested repeat vector administration, but no additional transduction was observed, and the block to transduction correlated with the appearance of neutralizing antibodies to the viral capsid. Here we show that mice exhibit a similar response but that treatment with anti-CD40 ligand antibody (MR1) and a soluble CTLA4-immunoglobulin fusion protein (CTLA4Ig) at the time of primary AAV vector exposure allowed successful repeat transduction and prevented production of neutralizing antibodies. We also tested the possibility that an immune response caused the loss of marker-positive cells in the epithelial population in rabbits by evaluating AAV vector expression in immunocompetent and immunodeficient mice. In contrast to results in rabbits, marker protein expression persisted in the lung in both groups of mice. AAV vector transduction occurred in alveolar cells, airway epithelial cells, and smooth muscle cells, and vector expression persisted for at least 8 months. Although data on persistence of AAV vector expression in the human lung are not available, it is likely that repeat transduction will be necessary either due to loss of expression or to the need for repeat administration to deliver effective amounts of AAV vectors. Results presented here indicate that transient immunosuppression will allow such repeat vector treatment of the lung.

Abatacept↗
PubMed1998

Sequence analysis of Mus dunni endogenous virus reveals a hybrid VL30/gibbon ape leukemia virus-like structure and a distinct envelope.

Mus dunni endogenous virus (MDEV) can be activated from M. dunni cells by exposing the cells to hydrocortisone or 5-iodo-2'-deoxyuridine. Interference analysis has revealed that MDEV uses a receptor for cell entry that is different from those used by other murine retroviruses. The entire genome has now been sequenced, revealing a long terminal repeat (LTR)-gag-pol-env-LTR structure typical of simple retroviruses of the murine leukemia virus genus, with no additional open reading frames between env and the 3' LTR. The LTRs and other noncoding regions of MDEV are most closely related to those of VL30 elements, while the majority of the coding sequences are most closely related to those of gibbon ape leukemia virus. MDEV represents the first example of a naturally occurring, replication-competent virus with sequences closely related to VL30 elements. The U3 region of MDEV contains six nearly perfect 80-bp repeats and the beginning of a seventh, and the region expected to contain the packaging sequence contains approximately four imperfect 33-bp repeats. The receptor specificity domains of the envelope are unique among retroviruses and show no apparent similarity to regions of known proteins.

Animals↗
PubMed1998

Behaviors of hypoglossal hyoid motoneurons in laryngeal and vestibular reflexes and in deglutition and emesis.

Reflex responses of hypoglossal motoneurons innervating the geniohyoid (GH) and thyrohyoid (TH) muscles from the superior laryngeal (SLN) and vestibular nerves and their behaviors during fictive swallowing and vomiting were examined by recording both the extracellular activities of 11 single cells in the hypoglossal nucleus and GH and TH muscle nerve activity in eight decerebrate, paralyzed, and artificially ventilated cats. The majority of TH motoneurons were either active and/or exhibited shortened antidromic latencies during early expiration. In contrast, GH motoneurons did not exhibit any respiratory-related activity. Electrical single-shock stimulation of the SLN never evoked an excitatory reflex response on GH or TH motoneurons but rather evoked inhibitory responses on the THs. Unlike other hypoglossal motoneurons, GH and TH motoneurons do not appear to receive vestibular inputs. However, they can exhibit robust activities during fictive swallowing and vomiting, particularly during expulsion. Thus these motoneurons may play an important role in airway protection during swallowing and vomiting but not in controlling upper airway patency regulated by vestibular afferents.

Animals↗
PubMed1998

The Escherichia coli chaperonin 60 (groEL) is a potent stimulator of osteoclast formation.

Chaperonins (cpns) are intracellular oligomeric protein complexes that fold and refold proteins in a catalytic manner and aid in the transmembrane transport of cellular proteins. We reported previously that the lipopolysaccharide-free recombinant cpn60 of Escherichia coli (groEL) is able to stimulate the breakdown of murine calvarial bone in culture and showed that such resorption is potently inhibited by an inhibitor of the enzyme cyclo-oxygenase and to a lesser extent by inhibitors of 5-lipoxygenase. In this study, we have investigated the effects of groEL on the resorptive activity and formation of osteoclasts in culture. In low density, osteoclast-containing cultures from neonatal rats incubated for 24 or 96 h on dentine discs, groEL (1-1000 ng/ml) stimulated resorption pit formation up to 4-fold, but this effect was essentially dependent on cell number. Using 12-day cultures of mouse bone marrow to assess osteoclast recruitment, groEL (1-1000 ng/ml) caused a dramatic dose-dependent stimulation of the formation of tartrate-resistant acid phosphatase-positive multinucleated cells and the resorption of the dentine on which bone marrow cells were cultured. Osteoclast formation elicited by groEL was almost completely abolished by indomethacin, an inhibitor of cyclo-oxygenase, but was unaffected by inhibitors of 5-lipoxygenase, suggesting that prostaglandins but not leukotrienes may mediate the action of groEL on osteoclastogenesis. It is possible that bacterial cpn60s such as groEL may play a role in the osteolysis associated with bone infections. Whether endogenous ("self") chaperonins have a role in other bone loss disorders, such as osteoporosis, is an intriguing possibility.

Acid Phosphatase↗
PubMed1997

Gene transfer into marrow repopulating cells: comparison between amphotropic and gibbon ape leukemia virus pseudotyped retroviral vectors in a competitive repopulation assay in baboons.

Many diseases might be treated by gene therapy targeted to the hematopoietic system, but low rates of gene transfer achieved in humans and large animals have limited the application of this technique. We have developed a competitive hematopoietic repopulation assay in baboons to evaluate methods for improving gene transfer and have used this method to compare gene transfer rates for retroviral vectors having an envelope protein (pseudotype) from amphotropic murine retrovirus with similar vectors having an envelope protein derived from gibbon ape leukemia virus (GALV). We hypothesized that vectors with a GALV pseudotype might perform better based on our previous work with cultured human hematopoietic cells. CD34(+) marrow cells from each of four untreated baboons were divided into two equal portions that were cocultivated for 48 hours with packaging cells producing equivalent titers of either amphotropic or GALV pseudotyped vectors containing the neo gene. The vectors contained small sequence differences to allow differentiation of cells genetically marked by the different vectors. Nonadherent and adherent cells from the cultures were infused into animals after they received a myeloablative dose of total body irradiation. Polymerase chain reaction (PCR) analysis for neo gene-specific sequences in colony-forming unit-granulocyte-macrophage from cell populations used for transplant showed gene transfer rates of 2.7%, 7.1%, <15%, and 3.9% with the amphotropic vectors and 7.1%, 11.3%, <15%, and 26.4% with the GALV pseudotyped vector. PCR analysis of peripheral blood and marrow cells after engraftment showed the neo gene to be present in all four animals analyzed at levels between 0.1% and 5%. Overall gene transfer efficiency was higher with the GALVpseudotyped vector than with the amphotropic vectors. Southern blot analysis in one animal confirmed a gene transfer efficiency of between 1% and 5%. The higher gene transfer efficiency with the GALV-pseudotyped vector correlated with higher levels of GALV receptor RNA compared with the amphotropic receptor in CD34(+) hematopoietic cells. These results show that GALV-pseudotyped vectors are capable of transducing baboon marrow repopulating cells and may allow more efficient gene transfer rates for human gene therapy directed at hematopoietic cells. In addition, our data show considerable differences in gene transfer efficiency between individual baboons, suggesting that a competitive repopulation assay will be critical for evaluation of methods designed to improve gene transfer into hematopoietic stem cells.

Animals↗
PubMed1997

Transfer of contaminants in adeno-associated virus vector stocks can mimic transduction and lead to artifactual results.

The potential of adeno-associated virus (AAV) vectors for gene transfer and gene therapy applications is currently being intensively investigated. Although much progress has been made in defining AAV vector biology, inconsistencies remain in the literature regarding the efficiency of AAV transduction in various cell types. In the course of exploring these differences, we have identified a problem associated with the use of AAV vector stocks that results in overestimation of gene transfer efficiencies. We show here that biologically active vector-encoded proteins can contaminate AAV vector stocks, especially cell lysate preparations that have not been further purified, and can be transferred in a virion-independent manner to target cells, a phenomenon called pseudotransduction. This observation is significant because impure cell lysate stocks have been widely employed in the AAV literature, and we demonstrate here that this phenomenon can occur with commonly used reporter proteins such as beta-galactosidase and alkaline phosphatase. We conclude that although there are many potential explanations for apparently conflicting results in the literature, the possibility of pseudotransduction must be considered, especially when cell lysate stocks of AAV vectors have been employed. This artifact can be avoided by further vector purification.

Alkaline Phosphatase↗
PubMed1997

Vestibular inputs to bulbar respiratory interneurons in the cat.

Vestibular inputs to medullary respiratory interneurons were studied in decerebrated and artificially ventilated cats. Extracellular recordings were made from 40 neurons located in the area of pre-Bötzinger complex and activated antidromically from the contralateral ventral respiratory group. Neuronal populations analyzed included inspiratory and expiratory neurons with augmenting, constant and decrementing firing patterns, and a late inspiratory neuron. Seventeen neurons responded to ipsilateral and/or contralateral vestibular nerve electrical stimulation. These responses were observed in all seven cell types. Most neuronal reflex responses consisted of inhibition, while a few consisted of either excitation or a combination of both inhibition and excitation. These results indicate that pre-Bötzinger respiratory interneurons, which may be involved in respiratory rhythmogenesis, also participate in vestibulorespiratory responses.

Animals↗
PubMed1997

Role of nucleus retroambigualis in respiratory reflexes evoked by superior laryngeal and vestibular nerve afferents and in emesis.

An ascending projection from the medullary nucleus retroambigualis (NRA) has recently been described as important for the control of the upper airway during vocalization. We evaluated the importance of this projection in other behaviors by making localized injections of the neurotoxin kainic acid in the NRA in decerebrate cats, most of which were paralyzed and artificially ventilated. In contrast to its importance for vocalization, the NRA is not essential for activation of upper airway musculature during respiration, swallowing, vomiting, or reflexes elicited by superior laryngeal or vestibular nerve afferents. However, kainic acid injections in the NRA and adjacent reticular formation prolonged the inhibitory phrenic motoneuronal response to superior laryngeal nerve stimulation and abolished or reduced abdominal motoneuronal responses during respiration, vomiting, and superior laryngeal nerve stimulation. Thus, of the behaviors we investigated, the importance of the ascending projection from the NRA appears to be limited to vocalization, while descending projections from the NRA region are important in a number of behaviors.

Animals↗
PubMed1997

Optimization of liposome mediated transfection of a neuronal cell line.

A cell line derived from sensory neurons was transfected with high efficiency using cationic liposomes, formulated from 3 beta [N-(N',N'-dimethylaminoethane)carbamoyl]-cholesterol (DC-Chol) and dioleoyl L-alpha-phosphatidylethanolamine (DOPE). This is the first time that cationic liposomes of this type have been reported to transfect a neuronal cell line. We used a reporter gene construct expressing beta-galactosidase under the control of the cytomegalovirus immediate early promoter and routinely observed transfection efficiencies > 40%. Parameters affecting transfection efficiency were examined and the ratio of DNA to liposome proved to be crucial. Liposome formulation procedures and cell transfection protocols devised here will be used as a basis for further in vivo and in vitro work.

Animals↗