Influence of chronic cocaine on monoamine neurotransmitters in human brain and animal model: preliminary observations.
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Animals↗
Biomedical subjects
J M Wilson
Publications and source records attributed to J M Wilson.
A novel mechanism for achieving transgene persistence in vivo after somatic gene transfer into hepatocytes.
Infusion of hepatocyte-specific DNA-protein complexes into rats leads to transient recombinant gene expression in liver. The eventual deterioration of gene expression is due in part to instability of the targeted DNA. In a previous report, we noted retention of transgene sequences in liver and persistent recombinant gene expression when the animals were subjected to partial hepatectomy following in vivo gene transfer. In an attempt to define the mechanism(s) responsible for persistent gene expression following partial hepatectomy, we characterized the molecular state of the retained, liver-associated transgenes. Southern blot analysis of DNA from liver tissues harvested various times after in vivo gene transfer and partial hepatectomy (10 min to 11 weeks) demonstrated high levels of transgene DNA (100-10,000 copies/cell). The predominant form of this DNA appeared to be episomal based on analyses of uncut DNA or DNA restricted by an endonuclease with one site in the plasmid. Livers from several animals contained a small proportion of transgene sequences of unknown structure. The existence of episomal DNA in liver was confirmed in experiments in which intact plasmid was rescued from total hepatocyte DNA by transformation of bacteria. Both strands of DNA in the liver-associated plasmid retained a bacterial pattern of methylation suggesting that the plasmid had not replicated in the eukaryotic cell. These results are consistent with the hypothesis that the majority of transgene sequences are retained as stabilized plasmids. The specific form of DNA which is transcriptionally active was not identified in these studies. This represents a new mechanism for retaining foreign DNA in eukaryotic cells in vivo and has implications both for the development of somatic gene therapies and the pathogenesis of viral diseases.
Animals↗
Role of CFTR in lysosome acidification.
The role of CFTR in lysosome acidification was examined in CFPAC-1 pancreatic adenocarcinoma cells with the delta F508 mutation that were transduced with a retroviral vector (PLJ-CFPAC) or with the normal CFTR gene (CFTR-CFPAC). Steady-state lysosomal pHi in intact cells was lower in PLJ-CFPAC cells than CFTR-CFPAC cells (3.55 vs 3.80) and was not affected by cAMP or forskolin. Initial rates of ATP-dependent acidification of isolated lysosomes and steady-state ATP-dependent pHi were similar in both cell lines over a range of chloride concentrations and were not altered when cells were exposed to cAMP or to forskolin prior to preparation of lysosomes. These observations suggest that CFTR plays no role in acidification of lysosomes, possibly due to limited permeability of lysosomal membranes to chloride.
Adenocarcinoma↗
Relationships between energy level and insulin secretion in isolated rat islets of Langerhans. Manipulation of [ATP]/[ADP][Pi] by 2-deoxy-D-glucose.
Perifusion of islets with nominally phosphate-free buffer containing increasing concentrations of 2-deoxy-D-glucose (2.5 to 10 mM) produced increments in high alpha-ketoisocaproic acid-induced secretion of insulin beyond those observed in the absence of the sugar analogue. 3-O-methyl-D-glucose, a poorly metabolized sugar, was without effect. Insulin release evoked by 40 mM KCl was not altered by 2-deoxyglucose. The concentration of intracellular inorganic phosphate was lower in islets perifused with 2-deoxyglucose and declined to a lower level after addition of 20 mM alpha-ketoisocaproic acid. The enhancement of alpha-ketoisocaproic acid-induced hormone secretion by 2-deoxyglucose was not seen in islets perifused with medium containing 1.5 mM phosphate; instead a small inhibition was observed. It is postulated that conditions which lower intracellular [Pi] facilitate, either directly or indirectly, hormone release although the mechanism of this effect remains to be elucidated.
Adenine Nucleotides↗
Changes in monoamine transmitter concentration in freshwater mussel tissues.
Freshwater mussels were analyzed for biogenic amine transmitter substances in gill tissue, suprabranchial nerve and blood. Gill tissue from normal pondwater-acclimated mussels contained significant amounts of monoamine neurotransmitter substances. In comparison with the suprabranchial nerve the gill tissue contained 42% of the dopamine, 7% of the serotonin and 490% of norepinephrine. Exposing the animals to deionized water (salt-depleted) resulted in a loss of transmitter substances from gill tissue, but serotonin reduction was modest. The mussel gill tissue content of serotonin and the precursor tryptophan was regulated at nearly constant levels. Serotonin is an important transmitter substance in mussels and the many functions it controls, including sodium transport regulation, would depend on its continued presence.
Animals↗
Hepatocyte-directed gene transfer in vivo leads to transient improvement of hypercholesterolemia in low density lipoprotein receptor-deficient rabbits.
Familial hypercholesterolemia is an inherited disease in humans, caused by a deficiency of low density lipoprotein (LDL) receptors, that we have used as a model for developing liver-directed gene therapies. Our strategy is to reconstitute hepatic LDL receptor expression in vivo by administering a DNA-protein complex that is capable of targeting the delivery of functional LDL receptor genes to hepatocytes. Infusion of this DNA-protein complex into the peripheral circulation of a rabbit animal model for familial hypercholesterolemia resulted in hepatocyte-specific gene transfer and a temporary amelioration of hypercholesterolemia. This noninvasive approach to gene therapy should have applications in the treatment of a wide spectrum of human diseases.
Animals↗
Targeted delivery of antisense oligonucleotides by molecular conjugates.
Antisense oligonucleotides efficiently inhibit gene expression in vitro; however, the successful therapeutic application of this technology in vivo will require the development of improved delivery systems. In this report we describe a technique that efficiently delivers antisense oligonucleotides into cells using molecular conjugates. This technique, which was initially developed for the delivery of eukaryotic genes, is based on the construction of DNA-protein complexes that are recognized by the liver-specific asialoglycoprotein receptor. Binding of poly(L-lysine)-asialoorosomucoid (AsOR) protein conjugates with phosphorothioate antisense oligonucleotides to chloramphenicol acetyltransferase (CAT) led to the formation of 50- to 150-nm toroids. Exposure of the antisense molecular complexes (3 microM oligonucleotide) to NIH 3T3 cells genetically modified to express both the AsOR receptor and CAT, inhibited CAT expression by 54%, which was completely blocked by excess AsOR. Equivalent inhibition of CAT activity with purified oligonucleotide alone was observed at a 30 microM concentration. Furthermore, examination of the cells using indirect immunofluorescence for the presence of CAT protein showed 28% of cells exposed to the molecular conjugates lacked any detectable CAT enzyme. Cells exposed to oligonucleotide alone showed a highly variable staining pattern, and only a few of the cells were completely void of CAT protein. Together these data demonstrate that molecular conjugates provide a highly specific and efficient system for the delivery of antisense oligonucleotides.
3T3 Cells↗
Pulmonary growth and remodeling in infants with high-risk congenital diaphragmatic hernia.
Infants born with congenital diaphragmatic hernia (CDH) have pulmonary hypoplasia, but the pattern of postnatal growth in these lungs has not been documented. The lungs of 21 children dying with CDH were analyzed to determine how the pulmonary morphology changed with age. The patients were stratified into three age groups for ANOVA analysis (less than 8 days, 8 to 21 days, greater than 21 days). Morphometric techniques previously described were used. Lung volume and weight as well as pulmonary artery length and diameter increased with age (P = .04), whereas the number of airway generations was similar for each group. Radial alveolar number also increased, particularly in the contralateral lung (P = .02). The percentage of intraacinar artery muscularization decreased with age (P = .02), while larger intraacinar arteries showed a nonmuscular structure, again particularly in the contralateral lung (P = .004). It is concluded that: (1) significant lung growth does occur postnatally at the alveolar level after CDH repair; and (2) there is postnatal vascular remodelling resulting in larger and less muscular arteries. These changes should contribute to a decrease in pulmonary arterial hypertension over time. However, the time period over which these changes occur exceeds the current limitations of invasive support measures such as extracorporeal membrane oxygenation. Elucidation of the factors responsible for this growth could result in new therapeutic strategies to enhance or accelerate postnatal pulmonary development in infants with CDH.
Arteries↗
Delayed repair and preoperative ECMO does not improve survival in high-risk congenital diaphragmatic hernia.
It has been suggested that delayed repair with preoperative stabilization might improve survival in high-risk (symptomatic within 6 hours of birth) congenital diaphragmatic hernia (CDH). This study compares the results of immediate operation versus delayed repair using extracorporeal membrane oxygenation (ECMO) when necessary. Since we first used ECMO in 1984, 101 high-risk CDH infants have been treated. Prior to 1987, we used immediate repair and postoperative ECMO if necessary. Between 1987 and 1990 we combined delayed operation (24 to 36 hours) with preoperative ECMO as necessary. No infant in this series was excluded from ECMO therapy unless absolute contraindications existed (prematurity, intracranial hemorrhage, or other major anomalies). Fifty-five patients received immediate operation and 46 had delayed repair. The two groups were comparable populations based on gestational age, birth weight, age at onset of symptoms, Apgar scores, best postductal PO2 (BPDPO2), and frequency of antenatal diagnosis. There was no statistically significant difference in overall survival between the two groups. Differences in survival among subpopulations (BPDPO2 greater than 100 or less than 100, antenatal diagnosis, inborn v outborn) also are not significant. The requirement for ECMO was similar in both groups. Survivors in the delayed repair group were ventilated longer and on ECMO longer, but had fewer late deaths (greater than 21 days) and fewer pulmonary sequelae (O2 dependency at discharge) than infants in the immediate repair group (P less than .05).(ABSTRACT TRUNCATED AT 250 WORDS)
Extracorporeal Membrane Oxygenation↗
Schizophrenia scepticism.
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Amyloid beta-Protein Precursor↗
Human cystic fibrosis transmembrane conductance regulator directed to respiratory epithelial cells of transgenic mice.
Human cystic fibrosis transmembrane conductance regulator (CFTR) was expressed in transgenic mice under the control of transcriptional elements derived from the human surfactant protein C (SP-C) gene. The hCFTR mRNA was expressed in lungs and testes: in the lung, we found hCFTR mRNA in bronchiolar and alveolar epithelial cells, and CFTR protein in respiratory epithelial cells. While the level of expression of hCFTR mRNA varied, hCFTR mRNA and protein were detected in pulmonary epithelial cells of several lines. Lung weight, morphology, somatic growth and reproductive capacity were not altered by expression hCFTR in lung and testes of the transgenics. Our findings suggest that hCFTR can be safely transferred to lung epithelial cells for CF therapy.
Animals↗
Submucosal glands are the predominant site of CFTR expression in the human bronchus.
We have used in situ hybridization and immunocytochemistry to characterize the cellular distribution of cystic fibrosis (CF) gene expression in human bronchus. The cystic fibrosis transmembrane conductance regular (CFTR) was primarily localized to cells of submucosal glands in bronchial tissues from non-CF individuals notably in the serous component of the secretory tubules as well as a subpopulation of cells in ducts. Normal distribution of CFTR mRNA was found in CF tissues while expression of CFTR protein was genotype specific, with delta F508 homozygotes demonstrating no detectable protein and compound heterozygotes expressing decreased levels of normally distributed protein. Our data suggest mechanisms whereby defects in CFTR expression could lead to abnormal production of mucus in human lung.
Bronchi↗
Hepatocyte transplantation: development of new systems for liver repopulation and gene therapy.
Following recent advances in molecular and cell biology, development of hepatocyte transplantation has been considerably invigorated. To study the fate of hepatocytes transplanted into the liver, new strategies have included the use of endogenously marked transgenic hepatocytes. Release of peripherally quantifiable marker proteins by transgenic hepatocytes has provided noninvasive ways to determine hepatocyte engraftment and prolonged survival. Further characterization of intrasplenic hepatocyte transplantation has established this to be the most suitable method for targeting hepatocytes to the liver. Advances in gene cloning, retrovirology, and gene transfer technology have provided further tools for accomplishing gene therapy. Practical strategies for ex vivo gene therapy involving hepatocytes have been developed by exploiting these new technologies. The potential value of hepatocyte transplantation in acute hepatic failure has begun to be reexamined. Therefore, hepatocyte transplantation can be predicted to provide new ammunition for continuing our assault on untreatable disorders. Resolution of outstanding issues should accelerate this process.
Animals↗
Ex vivo gene therapy of familial hypercholesterolemia.
Familial hypercholesterolemia (FH) is an autosomal dominant disorder caused by a deficiency in the receptor that clears low density lipoprotein (LDL) from the serum (reviewed in Ref. 1 and 2). Patients with one abnormal LDL receptor allele have moderate elevations in plasma LDL and suffer premature coronary artery disease (CAD). Approximately 5% of all patients under 45 who have had a myocardial infarction carry this trait. Patients with two abnormal LDL receptor genes (homozygous deficient patients) have severe hypercholesterolemia and life-threatening coronary artery disease in childhood. Strategies for treating patients with FH are directed at lowering the plasma level of LDL. In heterozygotes, this is accomplished through the administration of drugs that stimulate the expression of LDL receptor from the normal allele (2). This therapeutic approach is not effective in the treatment of homozygous deficient patients, especially those that retain less than 2% of residual LDL receptor activity. Partial amelioration of hyperlipidemia has been achieved in some homozygous deficient patients by diverting the portal circulation through a portacaval anastomosis (3) and by chronic plasmapheresis therapy (4). A more direct approach has been to correct the deficiency of hepatic LDL receptor by transplanting a liver that expresses normal levels of LDL receptor. Three patients that survived this procedure normalized their serum LDL-cholesterol (5-9). We have used an authentic animal model for FH, the Watanabe Heritable Hyperlipidemic rabbit (WHHL), to develop gene therapies for the homozygous form of FH (10-13). The WHHL rabbit has a mutation in its LDL receptor gene which renders the receptor completely dysfunctional (12) leading to severe hypercholesterolemia, diffuse atherosclerosis, and premature death. The potential efficacy of gene therapy for FH is supported by a series of studies we have performed in the WHHL rabbit in which we have achieved metabolic improvement (14-18). Liver tissue was removed from WHHL rabbits and used to isolate hepatocytes and establish primary cultures. A functional rabbit LDL receptor gene was transduced into a high proportion of hepatocytes using recombinant retroviruses, and the genetically corrected cells were transplanted into the animal from which they were derived. Transplantation of the genetically corrected, autologous hepatocytes was associated with a 30-40% decrease in serum cholesterol that persisted for the duration of the experiment (4 months, Ref. 18). Recombinant derived LDL receptor RNA was detected in liver for at least 6 months. There was no apparent immunological response to the recombinant derived LDL receptor.(ABSTRACT TRUNCATED AT 400 WORDS)
Adolescent↗
Transplantation of genetically modified autologous hepatocytes into nonhuman primates: feasibility and short-term toxicity.
Ex vivo gene therapy directed to the liver is being developed for the treatment of inherited metabolic diseases. Transplantation of hepatocytes that have been transduced with a low-density lipoprotein (LDL) receptor gene is a potential form of therapy for familial hypercholesterolemia (FH). We have demonstrated efficacy of ex vivo gene therapy for familial hypercholesterolemia in a rabbit animal model of this disease. In preparation for human trials, we describe in this report experiments in baboons for documentation of the feasibility and safety of autologous hepatocyte transplantation. Three baboons underwent a partial hepatectomy and their hepatocytes were isolated, cultured, and transduced with a retrovirus containing the human LDL receptor gene. The hepatocytes were harvested and infused into an indwelling catheter that had been placed into the inferior mesenteric vein at the time of liver resection. The baboons tolerated the procedures well and are being maintained and clinically evaluated for an indefinite time period. Follow-up evaluations have ranged from 3 to 8 months. Clinical evaluations have been unremarkable and blood chemistry and hematology determinations have stayed within normal limits.
Animals↗
Hormone secretion by preimplantation embryos in a dynamic in vitro culture system.
Bovine embryos recovered from superovulated donors on Days 8-18 postestrus were cultured in vitro in a tissue perifusion system to quantify hormone secretion. Embryos were cultured for 24 h at 37 degrees C in Ham's F-10 medium supplemented 5% v/v with heat-treated, charcoal-stripped calf serum; 100 IU/ml penicillin; and 100 micrograms/ml streptomycin. The medium was saturated with 5% CO2 in air and perifused at 50 microliters/min (3 ml/h). Estrone (E1) estradiol (E2), progesterone (P4), prostaglandin E2 (PGE2), and prostacyclin (PGI2) were quantified by RIA in 6-h pools of perifusate fractions. Estrone was measurable (pg/h/embryo; mean +/- SE) on Days 13 (10.80 +/- 4.56) and 15 (34.80 +/- 9.80); E2 on Days 11 (36.80), 12 (81.28 +/- 29.80), 13 (11.75 +/- 4.09), 15 (157.20 +/- 112.60), and 16 (30.26 +/- 8.76); and P4 (ng/h/embryo) on Days 13 (0.5-1.0) and 17 (approximately 1.5). PGE2 was secreted by Day 10 bovine embryos during the last 6 h of culture (19-24 h) and throughout culture for Day 11-18 embryos. The rate of PGE2 secretion increased (p less than 0.05) over the previous days(s) at Days 13 and 17. The mean (+/- SE) secretion rates (pg/h/embryo) for the 24-h culture by embryonic ages were as follows: Day 11 (63.39 +/- 14.61), 12 (172.10 +/- 30.90), 13 (3094.08 +/- 283.35), 14 (1633.89 +/- 49.98), 15 (3739.23 +/- 1082.79), 16 (4955.37 +/- 1381.83), 17 (11893.23 +/- 1188.48), and 18 (13827.99 +/- 3587.88).(ABSTRACT TRUNCATED AT 250 WORDS)
6-Ketoprostaglandin F1 alpha↗
Congenital diaphragmatic hernia. Stabilization and repair on ECMO.
Availability of extracorporeal membrane oxygenation (ECMO) support and the potential advantages of delayed repair of congenital diaphragmatic hernia (CDH) have led several centers to delay CDH repair, using ECMO support if necessary. This study reviews the combined experience of five ECMO centers with infants who underwent stabilization with ECMO and repair of CDH while still on ECMO. All infants were symptomatic at birth, with a mean arterial oxygen pressure (PaO2) of 34 mmHg on institution of bypass despite maximal ventilatory support. A total of 42 infants were repaired on ECMO, with 18 (43%) surviving. Seven infants had total absence of the diaphragm, and 28 required a prosthetic patch to close the defect. Only five infants ever achieved a best postductal PaO2 over 100 mmHg before institution of ECMO. Prematurity was a significant risk factor, with no infants younger than 37 weeks of age surviving. Significant hemorrhage on bypass was also a hallmark of a poor outcome, with 10 of the 24 nonsurvivors requiring five thoracotomies and six laparotomies to control bleeding, whereas only one survivor required a thoracotomy to control bleeding. In follow-up, nine of the 18 survivors (50%) have developed recurrent herniation and seven (43%) have significant gastroesophageal reflux. Importantly, five of the 18 survivors were in the extremely high-risk group who never achieved a PaO2 over 100 mmHg or an arterial carbon dioxide pressure (PaCO2) less than 40 mmHg before the institution of ECMO. In conclusion, preoperative stabilization with ECMO and repair on bypass may allow some high-risk infants to survive. Surviving infants will require long-term follow-up because many will require secondary operations.
Extracorporeal Membrane Oxygenation↗
Fast atom bombardment-mass spectrometry for bacterial chemotaxonomy: influence of culture age, growth temperature, gaseous environment and extraction technique.
Extracted phospholipids of Escherichia coli, Proteus mirabilis and Enterobacter cloacae were examined by fast atom bombardment-mass spectrometry which yielded major peaks between m/z 225 and 761. The result of extracting freeze-dried or 'wet' cells showed that freeze-drying may be omitted although weighing of dried cells offers a useful means of standardizing the extraction procedure. Anaerobic growth quantitatively altered the chemical finger-print as a result of increase in ratio of saturated: unsaturated carboxylic acids. Growth temperature also affected profiles over the temperature range 24-45 degrees C. A less drastic influence on mass spectra was culture age, over the range 16-48 h. Comparison of spectra was possible with Pearson's coefficient of linear correlation which yielded the following values: wet and lyophilized cells, r = 0.97; aerobic and anaerobic growth, r = 0.82; 24 degrees C and 45 degrees C, r = 0.76; 16 h and 48 h, r = 0.95. These results show that although quantitative differences do occur between spectra for the same organism prepared in different ways, they are less than interspecies variation, e.g. with E. coli and P. mirabilis, r = 0.46. Any differences which are due preparation method can be overcome by standardization of technique.
Aerobiosis↗