Biomedical subjects
Publications and source records attributed to A D Miller.
Advances in the understanding of molecular biology of human disease and the development of efficient gene transfer techniques have resulted in practical approaches to human gene therapy, with new techniques being developed at an increasing rate. The first trials have now begun in humans and initial results are positive.
The behavior of propriobulbar respiratory neurons was studied during fictive vomiting in decerebrate, paralyzed, artificially ventilated cats. Fictive vomiting was identified by a characteristic series of synchronous phrenic and abdominal nerve bursts, induced by electrical stimulation of abdominal vagal afferents and/or i.v. infusion of emetic drugs. Data were obtained from inspiratory neurons having decrementing (I-DEC) or constant (I-CON) discharge patterns and expiratory decrementing (E-DEC) neurons located in the Bötzinger complex and adjacent rostral ventral respiratory group. These neurons are known to make excitatory (I-CON) and inhibitory (I-DEC, E-DEC) connections with a variety of medullary respiratory neurons. During fictive vomiting: 8 of 14 I-DEC neurons fired in phase with synchronous bursts of phrenic and abdominal nerve discharge; the other 6 were silent. Of 12 I-CON neurons, 5 fired in phase with phrenic and abdominal bursts; 7 were silent. All (6) E-DEC neurons were either silent or fired weakly between bursts of phrenic and abdominal discharges. The possible roles of I-DEC and I-CON neurons in actively reorganizing the behavior of other respiratory neurons during fictive vomiting are discussed. In particular, the firing of many I-DEC neurons was found to be appropriate to inhibit inspiratory, and two types of expiratory, bulbospinal neurons during fictive vomiting.
Gene transfer into vascular smooth muscle cells in animals was examined by using recombinant retroviral vectors containing an Escherichia coli beta-galactosidase gene or a human adenosine deaminase (adenosine aminohydrolase, EC 3.5.4.4) gene. Direct gene transfer by infusion of virus into rat carotid arteries was not observed. However, gene transfer by infection of smooth muscle cells in culture and seeding of the transduced cells onto arteries that had been denuded of endothelial cells was successful. Potentially therapeutic levels of human adenosine deaminase activity were detected over 6 months of observation, indicating the utility of vascular smooth muscle cells for gene therapy in humans.
The impact of introducing laparoscopy as part of the overall gastrointestinal endoscopy case load performed by residents was reviewed. During 1990, there was a significant increase (56.9%) in the number of flexible diagnostic endoscopic procedures performed compared with 1989. When the total number of laparoscopic procedures was considered, the increase was 117%. Residents participated in the "surgeon's" position in 59% of the therapeutic laparoscopic procedures and as either surgeon or "first assistant" in 86% of all therapeutic laparoscopic procedures and 94% of all diagnostic laparoscopic procedures. Complication rates for diagnostic laparoscopic procedures were low in 1989 (0.03%) and 1990 (0.2%). Complication rates for therapeutic laparoscopic procedures were also low (4%). There was no difference in the complication rate for cases in which residents were in the surgeon's position (4%) versus cases in which they were not (4%). Introduction of laparoscopic procedures into a surgical residency program can be done safely, especially in cases in which an established program in endoscopy exists.
We studied the membrane potential changes of 11 phrenic motoneurons (PMs), located in the C5 portion of the cervical spinal cord, in decerebrate, paralyzed and artificially ventilated cats. Intracellular chloride iontophoresis into PMs could reverse the waves of inhibitory postsynaptic potentials (IPSPs) causing membrane hyperpolarization during expiration. In all PMs, we revealed, in addition to the previously described late expiratory augmenting wave of IPSPs, an early expiratory decrementing wave of IPSPs. Moreover, in three PMs, recorded in two cats exhibiting slow respiratory rhythms, an additional wave of IPSPs was interposed between the early and late ones. The possible origins and roles of these three different waves of inhibition are discussed.
Chinese hamster ovary (CHO) cells are resistant to infection by all of the major classes of murine retroviruses and are partially resistant to infection by gibbon ape leukemia virus. Treatment of CHO cells with the glycosylation inhibitor tunicamycin rendered these cells susceptible to infection by retroviral vectors with ecotropic, xenotropic, and amphotropic host ranges and increased the titer of gibbon ape leukemia virus pseudotyped vectors 10-fold. Vectors having a polytropic host range did not infect CHO cells in the presence or absence of tunicamycin, showing that the effect of tunicamycin was specific and related to the pseudotype of the vector. We present evidence for three mechanisms of resistance to infection: lack of viral receptors on CHO cells, the presence of nonfunctional receptors which can be made functional by treatment with tunicamycin, and the secretion of a protein factor that blocks retroviral infection of CHO cells. Several criteria indicate that the secreted inhibitor is not an interferon, and secretion of this factor was not detected in several other cell lines that were examined.
In decerebrate, paralyzed cats, phrenic (PHR) and lumbar abdominal (ABD) nerve discharges during both neural respiration and fictive vomiting (FV) were subjected to spectral and coherence analyses. During respiration, PHR discharge exhibited high-frequency oscillation (HFO), manifested as a narrow spectral peak (range 57-90 Hz) in autospectra and left-right coherence spectra. During FV, the following occurred: 1) the HFO peak disappeared and was replaced by a broad peak with higher modal frequency (range 84-120 Hz), indicating elimination of inputs from the medullary inspiratory pattern generator. 2) Left-right PHR coherence spectra had no distinct peaks, indicating that correlations between opposite PHR discharges were now not frequency specific. 3) Although ABD and PHR autospectra were similar, PHR-ABD coherences were near zero, indicating lack of common inputs on a short time scale. 4) Nonzero coherences between ABD nerves were confined to ipsilateral pairs. Thus coherence analysis indicates that the outputs of the vomiting pattern generator are temporally dispersed on a short time scale and are not necessarily common to different motoneuron populations.
1. The patterns of membrane potential changes of phrenic motoneurons were compared during fictive vomiting, fictive coughing, and fictive swallowing in decerebrate, paralyzed cats. These fictive behaviors were identified by motor nerve discharge patterns similar to those recorded from the muscles of nonparalyzed animals. Phrenic motoneurons (n = 54) were identified by antidromic activation from the thoracic phrenic nerve. Intracellular recordings were obtained from 27 motoneurons during fictive vomiting, 40 during fictive coughing, and 27 during fictive swallowing. Sixteen motoneurons were recorded during both fictive coughing and fictive swallowing, eight during both fictive coughing and fictive vomiting, and two during both fictive vomiting and fictive swallowing. Seven motoneurons were studied during all three behaviors. 2. Fictive vomiting, typically evoked by electrical stimulation of abdominal vagal afferents, was characterized by a series of bursts of coactivation of phrenic and abdominal motor nerves, culminating in an expulsion phase in which abdominal discharge was prolonged both with respect to phrenic discharge and to abdominal discharge during the preceding retching phase. During fictive vomiting, phrenic motoneurons depolarized abruptly, and the amplitude of depolarization was significantly greater than during control inspirations. They then repolarized slowly throughout the phrenic burst, rapidly repolarizing at the end of each phrenic burst during retching and reaching a level similar to that observed during expiration. During the expulsion phase, the pattern was initially the same. However, after the cessation of phrenic discharge, the membrane potential repolarized slowly until the end of the abdominal burst, exhibiting greater synaptic noise than during expiration. One phrenic motoneuron, presumably innervating the periesophageal region of the diaphragm, received a strong hyperpolarization just before the onset of the emetic episode and fired for shorter periods during fictive vomiting than did other phrenic motoneurons.(ABSTRACT TRUNCATED AT 250 WORDS)
Amplification of the HER-2 (c-erbB-2) gene and overexpression of the p185HER-2 gene product is found in approximately one-third of primary human breast and ovarian cancers and is associated with a poor clinical outcome of early relapse and death. The HER-2 gene encodes a cell-surface growth factor receptor with intrinsic tyrosine kinase activity. Wild-type human HER-2 has been shown to act as a potent oncogene when over-expressed in mouse fibroblasts. Recent data suggest that the mechanism by which HER-2 mediates transformation requires the interaction of the epidermal growth factor (EGF) receptor. To test whether overexpression of normal human HER-2 can transform cells independently of the EGF receptor, we have introduced multiple copies of HER-2 into the EGF receptor-negative cell line, NR6, and have performed assays for both transformation and tumorigenicity. Engineered NR6 cells that overexpress the HER-2 gene product display a highly transformed and tumorigenic phenotype as compared with control cells. Additionally, a monoclonal antibody to the extracellular domain of the HER-2 receptor is able to inhibit the proliferation of the overexpressing cells in vitro as well as tumor growth in vivo. This study provides clear evidence that HER-2-mediated transformation can be achieved independently of the EGF receptor.
The locations of serotonin-3 (5-HT3) receptors involved in initiating vomiting (emesis) were assessed by cutting visceral afferents or lesioning the area postrema. The 5-HT3 receptor agonists phenylbiguanide (PBG) and 2-methyl-5-HT were shown to induce vomiting and related prodromal signs (e.g., licking, swallowing) in nonoperated cats. Two-methyl-5-HT, but not PBG, also usually produced defecation and sometimes urination. Most studies were conducted using PBG, which induced vomiting in 40/49 (82%) cats at doses of 8.0 mg/kg i.p. or less (thresholds ranged from 2-8 mg/kg, median 5 mg/kg). Latencies to the first episode ranged from 4 to 21 min (median 7.5 min). PBG-induced vomiting was blocked by the 5-HT3 receptor antagonist MDL 72222. Lesions of the area postrema had no apparent effect on vomiting induced by PBG or by electrical stimulation of abdominal vagal afferents. In contrast, the threshold of PBG-induced vomiting was increased by supradiaphragmatic vagotomy and greatly increased by splanchnic nerve section. Thus, abdominal visceral afferents, but not the area postrema, play an important role in mediating vomiting induced by i.p. injection of the 5-HT3 receptor agonist PBG. The mechanisms by which vomiting is induced by PBG as compared to the cancer chemotherapeutic drug cisplatin are discussed.
Amphotropic helper-free retrovirus vectors containing the bacterial neomycin phosphotransferase gene (neo) and the human adenosine deaminase gene (adenosine aminohydrolase, EC 3.5.4.4; ADA) were used to transduce canine marrow cells. In one approach, dogs were treated for 7 days with recombinant human granulocyte colony-stimulating factor to stimulate hematopoietic cell division. Bone marrow cells were collected and transduced by 24 hours of cocultivation on vector-producing cells followed by incubation in a vector-containing long-term marrow culture system for 4 days. Transduced autologous marrow (0.4 to 1.0 x 10(8) cells/kg) was infused into dogs administered otherwise lethal total body irradiation (TBI) of 920 cGy. Two of four dogs engrafted, and their marrows showed intermittently between 1% and 11% G418-resistant colony-forming unit granulocyte-macrophage (CFU-GM) colonies for up to 2 years after transplantation. In a different experimental approach, autologous marrow, obtained at the time of the PB neutrophil nadir 7 days after a single cyclophosphamide injection (40 mg/kg intravenously), was cocultivated for 24 hours on vector-producing cells and infused at doses of 0.06 to 0.18 x 10(8) cells/kg into dogs administered 920 cGy TBI. One of three dogs engrafted, and the marrow showed intermittently 1% to 10% G418-resistant CFU-GM colonies for at least 2 years. Culture results were confirmed by polymerase chain reaction (PCR) showing the presence of the neo gene in marrow cells, peripheral blood (PB) granulocytes, and PB and lymph node lymphocytes. Dilution experiments indicated that up to 10% of marrow, lymph node, and PB cells contained the neo gene, consistent with the culture results. Samples harboring the neo gene also contained the gene for human ADA. However, repeated analyses of PB and marrow cells for human ADA gene expression by starch gel electrophoresis were negative. PB samples of all dogs were free of helper virus, and no long-term side effects from the transduction were observed.
A decline in the T-cell population usually marks the onset of progressive immunological disease in individuals infected with the human immunodeficiency virus (HIV). Because CD4+ cells help to coordinate efficient immune responses, some of the defects in the immune function in advanced cases of AIDS may be explained by the disappearance of these cells. Therefore, an understanding of the mechanisms used by HIV to induce the reduction of CD4+ cells is important. Here we use a Moloney murine leukaemia virus-based retroviral vector in order to express the nef gene of HIV-1 in three lymphocytic cell lines expressing CD4. In all cases we find that cell-surface CD4 expression is inversely related to nef expression. However, nef does not alter steady-state levels of CD4 RNA or CD4 protein. Also, nef can downregulate a CD4 triple mutant (Ser----Ala) that is neither phosphorylated nor down-regulated by phorbol esters, indicating that nef is acting by a different mechanism.
Genetically engineered fibroblasts have been successfully used to produce therapeutic proteins in animals, but sustained production of the proteins has not been achieved. This limits the potential of fibroblast-mediated gene therapy in humans. We have studied the phenomenon of decreased production in rats by using retroviral vectors carrying genes encoding human adenosine deaminase and neomycin phosphotransferase. While transplanted skin fibroblasts containing vector sequences persisted at constant levels for at least 8.5 mo, vector expression decreased by greater than 1500-fold after 1 mo. Cellular or antibody-mediated immune responses were not detected in transplanted animals, and expression could not be restored in fibroblasts recultivated from the grafts. This phenomenon is reminiscent of sequence-specific gene inactivation observed in other cell types. Because genetic manipulation and expression of foreign proteins did not affect survival of the transplanted cells, effective long-term therapy may be possible with the use of alternative gene regulatory elements.
Explore the source record for details and available documents.
The development of liver-directed gene therapy protocols depends upon the ability to transfer genes into a large number of liver cells such that the genes are expressed persistently. We used a retroviral vector to transfer the gene for neomycin phosphotransferase (neo) into mouse liver cells in vivo. Direct injection of the retrovirus preparation into mitotically active (regenerating) liver parenchyma resulted in efficient gene transfer, with neo sequences detectable in the livers of every animal tested 10 weeks to 6 months later. The neo gene was expressed for at least 3 months. This methodology may eventually be applicable to the treatment of human disease.
Retroviral vectors carrying the neomycin phosphotransferase (neo) gene have been shown to confer G418 resistance to canine keratinocytes at relatively high frequency. To investigate the usefulness of keratinocytes as potential target cells for gene therapy, we used a retroviral vector (LASN) that contains both human adenosine deaminase (hADA) and neo genes. We show here that LASN-transduced canine keratinocytes expressed high levels of hADA, a human protein of therapeutic relevance. Selection of LASN-transduced keratinocytes in medium containing G418 resulted in a population of cells that expressed even higher levels of hADA, about 80-fold higher than the endogenous canine ADA level. However, the G418-selected cells had a reduced proliferative potential and altered morphology indicative of terminal differentiation. To test whether L-histidinol is more beneficial for selection of keratinocytes than G418, we constructed two retroviral vectors that contain both the neo and the histidinol dehydrogenase (hisD) genes. Cocultivation of primary keratinocytes with lethally irradiated PA317 retrovirus packaging cells that produce these vectors gave rise to 12-53% drug-resistant colonies in either G418 or L-histidinol. In contrast to G418, selection of transduced keratinocytes in L-histidinol had no apparent effect on the proliferative potential or morphology of drug-resistant cells containing the vectors. Given the utility of this selection system, two hisD-based generic constructs containing cloning sites for cDNA expression from either the retroviral promoter or from an internal human cytomegalovirus immediate early promoter were constructed. Our results suggest that hisD will be a useful selectable marker for use in studies of keratinocyte differentiation and for transfer of genes into keratinocytes for the purposes of gene therapy.
The host range of retroviruses is determined primarily by the presence of specific receptors on target cells which are recognized by the retroviral envelope glycoprotein. Somatic cell hybrids have been used to determine the chromosomal locations of several retroviral receptors in mice prior to their molecular cloning. Here we report that by using human-Chinese hamster somatic cell hybrids and a retroviral vector, we have mapped the receptor for the amphotropic murine leukemia virus to the pericentromeric region of human chromosome 8.