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Biomedical subjects

A D Miller

Publications and source records attributed to A D Miller.

At least 109 records · Page 6Linked to original sources
PubMed1996

Prolonged expression of therapeutic levels of human granulocyte colony-stimulating factor in rats following gene transfer to skeletal muscle.

Gene transfer to skeletal muscle was examined as a means of gene therapy for neutropenia. A recombinant retrovirus containing a human granulocyte colony-stimulating factor (G-CSF) gene was introduced into primary human or rat myoblasts, which were then shown to produce biologically active G-CSF. Transplantation of G-CSF-producing rat myoblasts into the muscle of syngeneic rats resulted in a 15-fold increase in absolute neutrophil counts. This increase correlated with detection of circulating human G-CSF protein throughout the 6-month duration of the experiment. These results clearly demonstrate long-term production of therapeutically relevant amounts of a human protein by normal cells in vivo.

Animals↗
PubMed1996

Effects of gamma irradiation on the transduction of dividing and nondividing cells in brain and muscle of rats by adeno-associated virus vectors.

Vectors based on adeno-associated virus (AAV) are under investigation for use in gene therapy applications. Critical aspects of AAV vector biology remain undefined, in particular the intracellular events and activities mediating transduction and determining host cell permissiveness for transduction. Using cultured primary human fibroblasts, we previously showed that AAV vectors preferentially, but not exclusively, transduce cells in the S phase of the cell cycle, and that transduction can be markedly enhanced by pretreatment of target cells with physical and chemical agents that perturb DNA metabolism. In this study, we tested whether similar improvements in AAV vector performance might be achievable in vivo. The adult rat brain and overlying scalp muscle were selected for vector inoculation because of the presence of well-defined populations of dividing, quiescent, and post-mitotic cells, and gamma irradiation was chosen as a reproducible means of inducing DNA repair in these cells. We find that gamma irradiation markedly enhances the transduction of dividing cell populations in the pia-arachnoid and choroid epithelium within the central nervous system, and of mature nondividing muscle cells in the scalp, whereas gamma irradiation did not increase the basal transduction level of post-mitotic neurons in the hippocampus. These data confirm that replicative cellular DNA synthesis is not required for transduction by AAV vectors and show that the mitotic state of target cells is not necessarily predictive of responsiveness to transduction-enhancing treatments. Most importantly, these data demonstrate that target cells can be manipulated in vivo to render them more permissive for AAV vector transduction.

Age Factors↗
PubMed1996

Laparoscopic percutaneous jejunostomy for long term enteral access.

BACKGROUND: Patients suffering from upper gastrointestinal pathology may require jejunal feeding for adequate nutrition. A laparoscopically guided percutaneous jejunostomy offers a minimally invasive means of obtaining such feeding access. METHODS: Laparoscopic jejunostomy was performed in 32 patients. The most common indications were gastroparesis (n = 16), neurological deficits (n = 7), and proximal obstruction (n = 5). The proximal jejunum was affixed to the abdominal wall using intracorporeal and extracorporeal transabdominal sutures, allowing safe insertion of an 18-Fr Silastic dual-lumen tube. RESULTS: Laparoscopic jejunostomy was successfully completed for 28 patients; the procedure was converted to an open operation in four cases. Three of these four were among 14 patients undergoing the procedure who had a history of previous abdominal surgery. Major complications were observed in seven patients, including one reoperation and one death from aspiration pneumonia. Tube feeding was accomplished in all patients; progression to full enteral feeding proceeded without interruption in 20 patients. CONCLUSION: Laparoscopic jejunostomy can be performed with relative safety. Morbidity, though high, is usually related to preexisting disease. Previous abdominal surgery is not necessarily a contraindication to laparoscopic jejunostomy.

Enteral Nutrition↗
PubMed1996

Long-term persistence of canine hematopoietic cells genetically marked by retrovirus vectors.

In 1991 we reported gene transduction into autologous long-term repopulating marrow cells in dogs using amphotropic helper-free retrovirus vectors containing the bacterial neomycin phosphotransferase gene (neo) and the human adenosine deaminase gene (ADA). Two of the dogs are still alive and healthy now more than 5 years after transplantation of transduced autologous marrow cells. In one of the surviving dogs, polymerase chain reaction (PCR) analysis showed the neo and ADA genes to be present in peripheral blood granulocytes and lymphocytes up to the present time. The estimated percentage of neo-positive cells ranged from < 0.001% to 0.1%. ADA mRNA expression was detected by reverse transcriptase PCR (RT-PCR) in granulocytes 63 months after transplantation. The other surviving dog failed to show either persistence or expression of the transduced genes after 50 months. Three additional dogs have been transplanted according to the same transduction protocols and with the same retrovirus vectors, and persistence of the transduced neo gene has been documented in peripheral blood myeloid and lymphoid cells along with G418-resistant colony-forming unit-granulocyte/macrophage (CFU-GM) for now more than 2 years. These findings represent the longest follow-up of retrovirus-mediated gene transduction in any animal species. Long-term transduction efficiency, though, has remained low and will need to be improved for therapeutic application to be possible.

Adenosine Deaminase↗
PubMed1996

Foamy virus vectors.

Human foamy virus (HFV) is a retrovirus of the spumavirus family. We have constructed vectors based on HFV that encode neomycin phosphotransferase and alkaline phosphatase. These vectors are able to transduce a wide variety of vertebrate cells by integration of the vector genome. Unlike vectors based on murine leukemia virus, HFV vectors are not inactivated by human serum, and they transduce stationary-phase cultures more efficiently than murine leukemia virus vectors. These properties, as well as their large packaging capacity, make HFV vectors promising gene transfer vehicles.

3T3 Cells↗
PubMed1996

A novel murine retrovirus identified during testing for helper virus in human gene transfer trials.

An important requirement for the use of retroviral vectors in human gene transfer experiments is the avoidance of human exposure to replication-competent (helper) retroviruses. To meet this requirement, we used a sensitive marker rescue assay for helper virus to screen vector-transduced cells prior to reinfusion into patients. This assay utilized Mus dunni cells harboring a retroviral vector that can be rescued by helper retroviruses. The assay indicated the presence of helper virus in medium exposed to hematopoietic cells from all patients tested, including six patients with various cancers and one patient with Gaucher's disease, whether or not the patient cells had been exposed to retroviral vectors. All of the helper viruses were in a single interference group. We have now shown that treatment of the M. dunni marker rescue assay cells with 5-iodo-2'-deoxyuridine or hydrocortisone can activate production of an apparently identical helper virus, which we have named M. dunni endogenous virus (MDEV). Thus, production of virus in the assays of patient materials was likely due to exposure of the marker rescue assay cells to the hydrocortisone present in the hematopoietic cell growth medium. MDEV does not belong to any of the known murine leukemia virus groups by interference analysis, and we have called the new group multitropic because of the wide range of cells from different species that MDEV can infect.

Animals↗
PubMed1996

Retrovirus packaging cells based on 10A1 murine leukemia virus for production of vectors that use multiple receptors for cell entry.

10A1 murine leukemia virus can enter cells by using either of two different cell surface phosphate transport proteins, the gibbon ape leukemia virus receptor Glvr-1 (Pit-1) or the amphotropic retrovirus receptor Ram-1 (Pit-2). Glvr-1 and Ram-1 are widely expressed in different tissues, but the relative amounts of each are highly variable. We have developed retrovirus packaging cell lines based on 10A1 virus to take advantage of this dual receptor utilization to improve gene transfer rates in somatic cells of animals and humans, in which the relative levels of the two receptors are not always known. Optimization of the Env expression vector allowed the generation of packaging lines that produce helper-free vector titers up to 10(7)/ml. By interference analysis, we found that a 10A1 pseudotype retroviral vector can utilize Ram-1 for efficient entry into mouse, rat, and human cells and can utilize Glvr-1 for entry into mouse and human cells but not for entry into rat cells. The 10A1 pseudotype vector efficiently enters mouse cells by using Glvr-1, while entry into human cells is much less efficient. Thus, the 10A1 pseudotype packaging cells may be advantageous compared with the standard amphotropic packaging cells because vectors produced by the cells can use an additional receptor for cell entry. These packaging cells will also be useful to further explore the complicated pattern of receptor usage conferred by the 10A1 viral surface protein.

Animals↗
PubMed1996

Role of ventral respiratory group bulbospinal expiratory neurons in vestibular-respiratory reflexes.

1. Activation of the vestibular system produces reflex modulation of expiratory muscle activity. The purpose of the present study was to investigate the possible role of bulbospinal expiratory (E) neurons located in the caudal ventral respiratory group (VRG) in mediating vestibulo-respiratory reflexes. Experiments were carried out in decerebrated, paralyzed, and artificially ventilated cats. 2. Electrical stimulation of the vestibular nerve (VN), using short trains of current pulses, elicited bilateral reflex responses on abdominal muscle nerves (ABDNs). This response was not affected by lesions of the cochlear nuclei made by kainic acid injections. The ABDN response typically consisted of a combination of short-latency excitation and long-latency inhibition on the ipsilateral side and, in contrast, a combination of short-latency inhibition and long-latency excitation on the contralateral side. 3. Extracellular recordings were made from 43 caudal VRG bulbospinal E neurons that were activated antidromically from the contralateral upper lumbar spinal cord. More than 80% of these neurons responded to either ipsi- and/or contralateral VN stimulation. The neuronal response consisted of either a combination of excitation and inhibition or only inhibition. The majority of neurons had response patterns appropriate to contribute to the response observed on the contralateral ABDN; however, the latency of the VRG E neuron response was too long to initiate the ABDN response. 4. To further evaluate the contribution of caudal VRG E neurons to the vestibulo-abdominal reflex, ABDN responses were compared before and after sectioning the axons of caudal VRG bulbospinal E neurons where they cross the midline between the obex and first cervical spinal segment. These midsagittal lesions abolished expiratory modulation of ABDN discharge. The lesions also decreased the amplitude of the vestibular-evoked ABDN response but could not abolish the response. The postlesion amplitude was decreased on average to approximately 70% of prelesion values. 5. In conclusion, although the present results indicate that the majority of caudal VRG bulbospinal E neurons respond appropriately to contribute to the vestibulo-abdominal reflex, the reflex largely is unaffected by the removal of caudal VRG E input. The additional descending inputs that are important for mediating the reflex remain to be investigated and may include vestibulospinal and/or reticulospinal tracts.

Abdominal Muscles↗
PubMed1995

T lymphocyte-directed gene therapy for ADA- SCID: initial trial results after 4 years.

In 1990, a clinical trial was started using retroviral-mediated transfer of the adenosine deaminase (ADA) gene into the T cells of two children with severe combined immunodeficiency (ADA- SCID). The number of blood T cells normalized as did many cellular and humoral immune responses. Gene treatment ended after 2 years, but integrated vector and ADA gene expression in T cells persisted. Although many components remain to be perfected, it is concluded here that gene therapy can be a safe and effective addition to treatment for some patients with this severe immunodeficiency disease.

Adenosine Deaminase↗
PubMed1995

Improved transfer of the leukocyte integrin CD18 subunit into hematopoietic cell lines by using retroviral vectors having a gibbon ape leukemia virus envelope.

Leukocyte adherence deficiency (LAD) is an inherited immunodeficiency disease caused by defects in the CD18 leukocyte integrin subunit. Transduction of CD18 into hematopoietic cells from children with LAD represents a potential therapy for this disorder. In an attempt to maximize transfer and expression of CD18, we evaluated retroviral vectors with and without the neomycin selectable marker, with a modified tRNA primer binding site designed to prevent inhibition of gene expression, and with two different viral envelope proteins produced by using the amphotropic retrovirus packaging cell line PA317 or the gibbon ape leukemia virus packaging cell line PG13. The vectors were tested using transducing K562/CD11b cells and LAD Epstein-Barr virus (EBV) B cells and measuring levels of cell-surface CD11/CD18 expression by fluorescence-activated cell sorter analysis. The best results were obtained with vectors made using PG13 packaging cells, for which about 25% of the K562 cells exposed once to the vectors expressed surface CD11b/CD18 and about 25% of the LAD EBV B cells exposed three times over a 3-day period to the vectors expressed surface CD11a/CD18. In contrast, transduction of cells under similar conditions with retroviral vectors produced using PA317 producer cells yielded less than 2% of the K562 cells and less than 4% of the LAD EBV B cells expressing the CD11/CD18 heterodimer on the cell surface. The presence or absence of the neomycin resistance gene or the modified tRNA primer had no effect on CD18 gene transfer rate or expression level. The increase in transduction with PG13 vectors correlated with Northern blotting and reverse transcription-polymerase chain reaction studies that indicated that both K562 cells and the LAD EBV B cells express transcripts for the gibbon ape leukemia virus receptor at higher levels than for the amphotropic virus receptor. These findings indicate that the transduction efficiency of retroviral packaging cell lines correlates with receptor gene expression in the target cells and that vectors made using PG13 cells may be efficacious for gene therapy for LAD and other diseases in which gene transfer to hematopoietic cells is required.

B-Lymphocytes↗
PubMed1995

Multifunctional ventral respiratory group: bulbospinal expiratory neurons play a role in pudendal discharge during vomiting.

Pudendal motoneurons are activated in phasic bursts during the retching and expulsion phases of vomiting. The resulting contraction of the anal and urethral sphincters serves to maintain continence during the large increase in abdominal pressure that occurs during vomiting. We evaluated the contribution of bulbospinal expiratory neurons located in the portion of the ventral respiratory group (VRG) caudal to the obex (nucleus retroambigualis) to the control of pudendal motoneurons during fictive vomiting in decerebrate, paralyzed cats. Pudendal nerve discharge is abolished by cutting the axons of caudal VRG expiratory neurons as they cross the midline between the obex and C1 before descending in the spinal cord. All caudal VRG expiratory neurons that were antidromically activated from the sacral spinal cord, where the pudendal motor pool (nucleus of Onuf) is located, discharged strongly during the end of the expulsion phase of vomiting. However, only a small proportion of these neurons was active in phase with pudendal discharge during the retching phase. The apparent involvement of caudal VRG expiratory neurons in the control of pudendal motoneurons during vomiting is another example of the multifunctional role that can be played by respiratory-related neurons in the mammalian nervous system.

Animals↗
PubMed1995

Vestibular inputs to the lateral tegmental field of the cat: potential role in autonomic control.

The lateral tegmental field (LTF), which is comprised of the lateral reticular formation near the obex, is an important integrative area involved in cardiovascular control and the production of emesis. Using neuroanatomical and electrophysiological techniques, we tested the hypothesis that LTF neurons receive vestibular inputs; the neurons studied included those projecting into the subretrofacial rostral ventrolateral medulla (sRVLM), which contains cells that make direct connections with sympathetic preganglionic neurons. Injections of the anterograde tracer PHA-L into the medial and inferior vestibular nuclei produced labeled terminals in the LTF. Electrical stimulation of the vestibular nerve affected the firing rate of LTF neurons, including approximately one-third of those antidromically activated from the sRVLM. The response latencies ranged from 1.5 to 20 ms, suggesting that the neurons received both direct and polysynaptic vestibular inputs from the vestibular nuclei. The LTF may be involved in the production of vestibulosympathetic reflexes and vestibular-elicited vomiting.

Animals↗
PubMed1995

DNA synthesis and topoisomerase inhibitors increase transduction by adeno-associated virus vectors.

Viral vectors based on adeno-associated virus (AAV) preferentially transduce cells in S phase of the cell cycle. We recently found that DNA-damaging agents increased the transduction of nondividing cells. However, the optimal concentrations were toxic to cells. Here we show that the transduction of normal human fibroblasts by AAV vectors is increased by prior exposure to DNA synthesis inhibitors, such as aphidicolin or hydroxyurea, and topoisomerase inhibitors, such as etoposide or camptothecin. Transduction efficiencies could be increased > 300-fold in stationary cultures at concentrations that did not affect cell viability or proliferative potential. Both S-phase and non-S-phase cells were affected, suggesting that cellular functions other than replicative DNA synthesis may be involved. Applying these methods to gene transfer protocols should improve prospects for gene therapy by AAV vectors.

Aphidicolin↗
PubMed1995

The crystal structure of the lysyl-tRNA synthetase (LysU) from Escherichia coli.

BACKGROUND: Lysyl-tRNA synthetase catalyzes the attachment of the amino acid lysine to the cognate tRNA. The enzyme is a member of the class II amino-acyl-tRNA synthetases; the crystal structures of the seryl- and aspartyl-tRNA synthetases from this class are already known. Lysyl-tRNA synthetase shows extensive sequence homology with aspartyl-tRNA synthetase. In Escherichia coli there are two isoforms of the enzyme, LysS and LysU. Unlike LysS, which is synthesized under normal growth conditions, LysU is the product of a normally silent gene which is overexpressed under extreme physiological conditions (such as heat-shock), and can synthesize a number of adenyl dinucleotides (in particular AppppA). These dinucleotides have been proposed to act as modulators of the heat-shock response and stress response. RESULTS: The crystal structure of E. coli LysU has been determined to 2.8 A resolution, with lysine bound to the active site. The protein is a homodimer, with a rather extended dimer interface spanning the entire length of the molecule. Each monomer consists of two domains: a smaller N-terminal domain which binds the tRNA anticodon, and a larger C-terminal domain with the topology characteristic of the catalytic domain found in class II synthetases. CONCLUSIONS: A comparison of the LysU crystal structure with the structures of seryl- and aspartyl-tRNA synthetases enables a conserved core to be identified. The structural homology with the aspartyl-tRNA synthetase extends to include the anticodon-binding domain. When the active sites of lysyl-, aspartyl- and seryl-tRNA synthetases are compared, a number of catalytically important residues are conserved and a similar extended network of hydrogen bonds can be observed in the amino acid binding pocket in all three structures, although the details may differ. The lysine substrate is involved in an extended network of hydrogen bonds and polar interactions, with the side chain amino group forming a salt bridge with Glu428. The binding of ATP to LysU can be modelled on the basis of the aspartyl-tRNA synthetase-ATP complex, but the tRNA acceptor stem interaction for LysU cannot be easily modelled by similar extrapolation.

Amino Acid Sequence↗
PubMed1995

Sequences within the coding regions of clotting factor VIII and CFTR block transcriptional elongation.

The clotting factor VIII (FVIII) and cystic fibrosis transmembrane conductance regulator (CFTR) cDNAs have dramatically reduced levels of expression compared to clotting factor IX (FIX) and other cDNAs (100 and 1,000-fold lower, respectively), when produced in cells by using an expression vector. Part of the inhibitory signal in the FVIII cDNA has been localized to a 1.2-kb inhibitory sequence (FVIII INS), which decreased steady-state RNA levels from a retroviral vector by 30- to 100-fold. An analysis of RNA degradation indicated that the FVIII INS vector RNA is relatively stable. Nuclear run-on experiments with the FVIII INS vector demonstrated a low signal for FVIII, in contrast to the high signal for a FIX vector. The low signal for FVIII INS was not due to a decrease in transcriptional initiation. Thus, FVIII expression is reduced through a block to transcriptional elongation, as has been found in c-myc and other genes. We show that the inhibitory effect of FVIII INS is orientation dependent with regard to the promoter. In addition, the inhibitory effect is position dependent, because expression of FVIII INS sequence increased when it was moved 1 kb further from the promoter in a retroviral vector. Similar results were observed by using a retroviral vector for expression of the CFTR cDNA. The CFTR retroviral vector produced 1,000-fold decreased steady-state RNA levels, compared to the parent vector. Nuclear run-on analysis with the CFTR vector revealed a block to transcriptional elongation within the CFTR cDNA. The presence of blocks to transcriptional elongation within the FVIII and CFTR cDNAs complicates efforts to produce high levels of these proteins for therapeutic purposes and to develop high-titer retroviral expression vectors for human gene therapy.

3T3 Cells↗
PubMed1995

The effects of human serum and cerebrospinal fluid on retroviral vectors and packaging cell lines.

Human serum is known to inactivate many retroviruses, including murine leukemia viruses (MLV). Exposure of vectors based on MLV to human serum components would presumably decrease the efficiency of gene transfer in vivo. Human serum also lyses xenogeneic cells, which would affect the survival of retroviral vector packaging cells in vivo. The effects of other body fluids, such as cerebrospinal fluid (CSF), on MLV vectors and packaging lines have not been studied. We have found that retroviral vectors packaged in ecotropic, amphotropic, and gibbon ape leukemia virus (GALV) envelope proteins were all inactivated by human sera, and human sera also lysed mouse NIH-3T3 cells and the retroviral vector packaging cells derived from them. Human fibroblasts producing amphotropic vector particles were resistant to lysis, but the particles produced by them were inactivated. In contrast, CSF did not inactivate MLV vectors, nor did it lyse murine retrovirus packaging cells. Our results suggest that exposure to human serum may prevent in vivo gene transfer by MLV vectors and xenogeneic packaging lines, but gene transfer within the central nervous system should be more successful.

3T3 Cells↗