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1.
Psychopharmacology (Berl) ; 234(9-10): 1573-1586, 2017 05.
Article in English | MEDLINE | ID: mdl-28243714

ABSTRACT

RATIONALE: Smoking is the leading cause of preventable death in the USA, but quit attempts result in withdrawal-induced cognitive dysfunction and predicts relapse. Greater understanding of the neural mechanism(s) underlying these cognitive deficits is required to develop targeted treatments to aid quit attempts. OBJECTIVES: We examined nicotine withdrawal-induced inattention in mice lacking the α7 nicotinic acetylcholine receptor (nAChR) using the five-choice continuous performance test (5C-CPT). METHODS: Mice were trained in the 5C-CPT prior to osmotic minipump implantation containing saline or nicotine. Experiment 1 used 40 mg kg-1 day-1 nicotine treatment and tested C57BL/6 mice 4, 28, and 52 h after pump removal. Experiment 2 used 14 and 40 mg kg-1 day-1 nicotine treatment in α7 nAChR knockout (KO) and wildtype (WT) littermates tested 4 h after pump removal. Subsets of WT mice were killed before and after pump removal to assess changes in receptor expression associated with nicotine administration and withdrawal. RESULTS: Nicotine withdrawal impaired attention in the 5C-CPT, driven by response inhibition and target detection deficits. The overall attentional deficit was absent in α7 nAChR KO mice despite response disinhibition in these mice. Synaptosomal glutamate mGluR5 and dopamine D4 receptor expression were reduced during chronic nicotine but increased during withdrawal, potentially contributing to cognitive deficits. CONCLUSIONS: The α7 nAChR may underlie nicotine withdrawal-induced deficits in target detection but is not required for response disinhibition deficits. Alterations to the glutamatergic and dopaminergic pathways may also contribute to withdrawal-induced attentional deficits, providing novel targets to alleviate the cognitive symptoms of withdrawal during quit attempts.


Subject(s)
Attention/physiology , Nicotine/administration & dosage , Nicotine/adverse effects , Psychomotor Performance/physiology , Substance Withdrawal Syndrome/metabolism , alpha7 Nicotinic Acetylcholine Receptor/deficiency , Animals , Attention/drug effects , Choice Behavior/drug effects , Choice Behavior/physiology , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Psychomotor Performance/drug effects , Substance Withdrawal Syndrome/psychology , Synaptosomes/drug effects , Synaptosomes/metabolism , alpha7 Nicotinic Acetylcholine Receptor/agonists
2.
Transl Psychiatry ; 4: e400, 2014 Jun 17.
Article in English | MEDLINE | ID: mdl-24937094

ABSTRACT

Autism spectrum disorders (ASDs) now affect 1-2% of the children born in the United States. Hundreds of genetic, metabolic and environmental factors are known to increase the risk of ASD. Similar factors are known to influence the risk of schizophrenia and bipolar disorder; however, a unifying mechanistic explanation has remained elusive. Here we used the maternal immune activation (MIA) mouse model of neurodevelopmental and neuropsychiatric disorders to study the effects of a single dose of the antipurinergic drug suramin on the behavior and metabolism of adult animals. We found that disturbances in social behavior, novelty preference and metabolism are not permanent but are treatable with antipurinergic therapy (APT) in this model of ASD and schizophrenia. A single dose of suramin (20 mg kg(-1) intraperitoneally (i.p.)) given to 6-month-old adults restored normal social behavior, novelty preference and metabolism. Comprehensive metabolomic analysis identified purine metabolism as the key regulatory pathway. Correction of purine metabolism normalized 17 of 18 metabolic pathways that were disturbed in the MIA model. Two days after treatment, the suramin concentration in the plasma and brainstem was 7.64 µM pmol µl(-1) (±0.50) and 5.15 pmol mg(-1) (±0.49), respectively. These data show good uptake of suramin into the central nervous system at the level of the brainstem. Most of the improvements associated with APT were lost after 5 weeks of drug washout, consistent with the 1-week plasma half-life of suramin in mice. Our results show that purine metabolism is a master regulator of behavior and metabolism in the MIA model, and that single-dose APT with suramin acutely reverses these abnormalities, even in adults.


Subject(s)
Behavior, Animal/drug effects , Child Development Disorders, Pervasive/drug therapy , Metabolic Networks and Pathways/drug effects , Purinergic Antagonists/pharmacology , Purines/metabolism , Social Behavior , Suramin/pharmacokinetics , Animals , Brain/drug effects , Brain/metabolism , Brain Stem/drug effects , Brain Stem/metabolism , Child Development Disorders, Pervasive/chemically induced , Disease Models, Animal , Male , Metabolomics , Mice , Mice, Inbred C57BL , Purinergic Antagonists/administration & dosage , Purinergic Antagonists/pharmacokinetics , Random Allocation , Suramin/administration & dosage , Suramin/pharmacology
3.
Neurology ; 65(9): 1493-5, 2005 Nov 08.
Article in English | MEDLINE | ID: mdl-16177225

ABSTRACT

Described are six patients with Alpers syndrome from four unrelated families. Affected individuals harbored the following combinations of POLG mutations: 1) A467T/W1020X, 2) W748S-E1143G/G848S, 3) A467T/A467T, and 4) A467T/G848S. Homozygosity for the A467T allele in one patient was associated with a later age at onset. Mitochondrial respiratory chain studies in skeletal muscle were normal in each case. Nine combinations of mutant POLG alleles that cause Alpers syndrome are summarized.


Subject(s)
DNA-Directed DNA Polymerase/genetics , Diffuse Cerebral Sclerosis of Schilder/enzymology , Diffuse Cerebral Sclerosis of Schilder/genetics , Genetic Predisposition to Disease/genetics , Mutation/genetics , Age of Onset , Child , DNA Mutational Analysis , DNA Polymerase gamma , Developmental Disabilities/enzymology , Developmental Disabilities/genetics , Diffuse Cerebral Sclerosis of Schilder/physiopathology , Electron Transport/genetics , Female , Gene Frequency , Genetic Testing , Genetic Variation/genetics , Homozygote , Humans , Infant , Liver Diseases/enzymology , Liver Diseases/genetics , Male , Mitochondria/enzymology , Mitochondria/genetics , Muscle, Skeletal/enzymology , Muscle, Skeletal/physiopathology , Status Epilepticus/enzymology , Status Epilepticus/genetics
4.
Biomed Microdevices ; 7(4): 331-9, 2005 Dec.
Article in English | MEDLINE | ID: mdl-16404511

ABSTRACT

Currently, pathologists rely on labor-intensive microscopic examination of tumor cells using staining techniques originally devised in the 1880s that depend heavily on specimen preparation and that can give false readings. Emerging BioMicroNanotechnologies (Gourley, 2005) have the potential to provide accurate, realtime, high throughput screening of tumor cells without invasive chemical reagents. These techniques are critical to advancing early detection, diagnosis, and treatment of disease. Using a new technique to rapidly assess the properties of cells flown through a nanolaser semiconductor device, we discovered a method to rapidly assess the respiratory health of a single mammalian cell. The key discovery was the elucidation of biophotonic differences in normal and transformed (cancer) mouse liver cells by using intracellular mitochondria as biomarkers for disease. This technique holds promise for detecting cancer at a very early stage and could nearly eliminate delays in diagnosis and treatment.


Subject(s)
Flow Cytometry/instrumentation , Liver Neoplasms/pathology , Microfluidic Analytical Techniques/instrumentation , Microscopy, Confocal/instrumentation , Mitochondria/pathology , Nanotechnology/instrumentation , Nephelometry and Turbidimetry/instrumentation , Animals , Cell Line, Tumor , Computer Systems , Equipment Design , Equipment Failure Analysis , Feasibility Studies , Flow Cytometry/methods , Mice , Microfluidic Analytical Techniques/methods , Microscopy, Confocal/methods , Nanotechnology/methods , Nephelometry and Turbidimetry/methods , Reproducibility of Results , Sensitivity and Specificity
5.
Atherosclerosis ; 177(1): 183-8, 2004 Nov.
Article in English | MEDLINE | ID: mdl-15488882

ABSTRACT

The mechanism of the muscle toxicity associated with lipid-lowering therapy remains obscure. Pathological and biochemical findings in patients with statin myotoxicity suggest impaired fatty acid oxidation. Exhaled gas analysis can be used to assess substrate utilization including fatty acid oxidation. In order to determine if muscle toxicity due to lipid-lowering therapy might be related to abnormalities in lipid oxidation, exhaled gas analysis was performed in the fasted state on 11 patients subsequent to statin-associated myositis reactions. Results were compared to those of 16 normal controls who were measured both on and off statin therapy. Post-myositis patients showed a depressed anaerobic threshold (AT) (P=0.009) compared to controls while age-adjusted maximal oxygen consumption (VO2max) and ventilatory efficiency (VE/VCO2) were not significantly different. The fasting respiratory exchange ratio (RER) of post-myositis patients off statins was abnormally increased (P=0.00001) as was their S1-slope (P=0.023). Controls demonstrated a significant increase in their RER while taking statins consistent with decreased lipid oxidation (P <0.00001). These findings suggest that abnormal lipid oxidation in certain patients may predispose them to the myotoxicity caused by lipid-lowering therapies.


Subject(s)
Anticholesteremic Agents/adverse effects , Fatty Acids/metabolism , Heart/physiopathology , Lung/physiopathology , Myositis/chemically induced , Myositis/physiopathology , Aged , Aged, 80 and over , Breath Tests , Exercise Test , Female , Humans , Male , Middle Aged , Oxidation-Reduction
7.
Muscle Nerve ; 24(7): 916-24, 2001 Jul.
Article in English | MEDLINE | ID: mdl-11410919

ABSTRACT

Serial measurements of nerve conduction velocities and amplitudes were performed in 27 patients with congenital lactic acidemia over 1 year of sodium dichloroacetate (DCA) administration. Patients were treated with oral thiamine (100 mg) and DCA (initial dose of 50 mg/kg) daily. Nerve conduction velocity and response amplitude were measured in the median, radial, tibial, and sural nerves at 0, 3, 6, and 12 months, and plasma DCA pharmacokinetics were measured at 3 and 12 months. Baseline electrophysiologic parameters in this population were generally below normal but as a group were within 2 standard deviations of normal means. Although symptoms of neuropathy were reported by only three patients or their families, nerve conduction declined in 12 patients with normal baseline studies, and worsening of nerve conduction occurred in the two who had abnormalities at baseline. Peripheral neuropathy appears to be a common side effect during chronic DCA treatment, even with coadministration of oral thiamine. Nerve conduction should be monitored during DCA treatment.


Subject(s)
Dichloroacetic Acid/adverse effects , MELAS Syndrome/drug therapy , MELAS Syndrome/physiopathology , Median Neuropathy/chemically induced , Neural Conduction/drug effects , Action Potentials/drug effects , Action Potentials/physiology , Adolescent , Adult , Child , Child, Preschool , Electromyography , Female , Humans , Infant , MELAS Syndrome/diagnosis , Male , Median Neuropathy/diagnosis , Neural Conduction/physiology , Neurons, Afferent/physiology , Radial Neuropathy/chemically induced , Radial Neuropathy/diagnosis , Tibial Neuropathy/chemically induced , Tibial Neuropathy/diagnosis
8.
Arch Neurol ; 58(5): 811-4, 2001 May.
Article in English | MEDLINE | ID: mdl-11346377

ABSTRACT

BACKGROUND: Autosomal recessive inborn errors of metabolism often present as life-threatening disease in infancy and have adverse effects on the nervous system. Parents are usually heterozygotes. This is true of most disorders of fatty acid oxidation, which are rare and present with hypoketotic hypoglycemia. However, the gene for medium-chain acyl coenzyme A dehydrogenase (MCAD) deficiency is common in white people, raising the possibility that a parent may be homozygous. OBJECTIVE: To document the occurrence of MCAD deficiency in a 12-month-old boy and his father, both of whom were homozygous for the A985G mutation. DESIGN: Clinical observations and definitive biochemical testing. SETTING: Children's hospital and university laboratory. PARTICIPANTS: One child and one adult. INTERVENTIONS: Diagnosis and treatment. MAIN OUTCOME MEASURES: Clinical outcome; analysis results of plasma and urine for carnitine and organic acids. RESULTS: An infant admitted with an acute illness requiring intensive care was found to have carnitine deficiency and dicarboxylic aciduria; MCAD deficiency was diagnosed by assay of his DNA for the common mutation. Test results of the father revealed him also to be homozygous. CONCLUSION: In MCAD deficiency, as opposed to the usual rare autosomal recessive metabolic disease, a parent may also be an affected homozygote.


Subject(s)
Fatty Acid Desaturases/deficiency , Acyl-CoA Dehydrogenase , Adult , Base Sequence/genetics , Homozygote , Humans , Infant , Male , Metabolism, Inborn Errors/genetics , Mutation/genetics
9.
J Child Neurol ; 16(2): 136-8, 2001 Feb.
Article in English | MEDLINE | ID: mdl-11292221

ABSTRACT

We report a patient with mitochondrial DNA depletion, partial complex II and IV deficiencies, and 3-methylglutaconic aciduria. Complex II deficiency has not been previously observed in mitochondrial DNA depletion syndromes. The observation of 3-methylglutaconic and 3-methylglutaric acidurias may be a useful indicator of a defect in respiratory chain function caused by mitochondrial DNA depletion.


Subject(s)
DNA, Mitochondrial/analysis , Glutarates/urine , Meglutol/analogs & derivatives , Meglutol/urine , Mitochondrial Encephalomyopathies/genetics , Mitochondrial Encephalomyopathies/urine , Biopsy , Blotting, Southern , Child, Preschool , Humans , Infant , Male , Mitochondrial Encephalomyopathies/complications , Muscle, Skeletal/pathology
10.
Hum Reprod ; 15 Suppl 2: 44-56, 2000 Jul.
Article in English | MEDLINE | ID: mdl-11041512

ABSTRACT

Mitochondrial disease can lead to clinical abnormalities in any organ system. Both inherited and spontaneous disorders are known. The spontaneous forms can occur as a mitochondrial DNA (mtDNA) mutation early in embryogenesis or, later in life, as somatic mutations that accumulate with age. The inherited forms may arise from any of >100 characterized mutations in mtDNA or from >200 nuclear gene defects that affect proteins required for mitochondrial function. Most dividing cells survive and interact normally despite their mitochondrial defects. Thus post-mitotic, terminally differentiated cells are preferentially affected in mitochondrial disease. This review emphasizes cellular metabolic co-operation and the structural and biochemical diversity of mitochondria as the framework for understanding the clinical spectrum of mitochondrial disease. The principles of the mitochondrial clinical assessment scale I (MCAS-I) are presented to assist in the development of diagnostic spectra of mitochondrial disease.


Subject(s)
DNA, Mitochondrial/genetics , Mitochondria/metabolism , Mitochondrial Myopathies/metabolism , Animals , Child , Child, Preschool , DNA, Mitochondrial/metabolism , Humans , Mitochondria/genetics , Mitochondria/ultrastructure , Mitochondrial Myopathies/diagnosis , Mitochondrial Myopathies/genetics
11.
J Child Neurol ; 15(6): 357-61, 2000 Jun.
Article in English | MEDLINE | ID: mdl-10868777

ABSTRACT

We report a family with a heterogeneous group of neurologic disorders associated with the mitochondrial DNA G8363A transfer ribonucleic acid (RNA)Lys mutation. The phenotype of one child in the family was consistent with autism. During his second year of life, he lost previously acquired language skills and developed marked hyperactivity with toe-walking, abnormal reciprocal social interaction, stereotyped mannerisms, restricted interests, self-injurious behavior, and seizures. Brain magnetic resonance imaging (MRI) and repeated serum lactate studies were normal. His older sister developed signs of Leigh syndrome with progressive ataxia, myoclonus, seizures, and cognitive regression. Her laboratory studies revealed increased MRI T2-weighted signal in the putamen and posterior medulla, elevated lactate in serum and cerebrospinal fluid, and absence of cytochrome c oxidase staining in muscle histochemistry. Molecular analysis in her revealed the G8363A mutation of the mitochondrial transfer RNA(Lys) gene in blood (82% mutant mitochondrial DNA) and muscle (86%). The proportions of mutant mitochondrial DNA from her brother with autism were lower (blood 60%, muscle 61%). It is likely that the origin of his autism phenotype is the pathogenic G8363A mitochondrial DNA mutation. This observation suggests that certain mitochondrial point mutations could be the basis for autism in some individuals.


Subject(s)
Autistic Disorder/genetics , DNA, Mitochondrial/genetics , Leigh Disease/genetics , Point Mutation , RNA, Transfer, Lys/genetics , Adult , Autistic Disorder/physiopathology , Autistic Disorder/psychology , Child , Child, Preschool , DNA Mutational Analysis , Epilepsy/genetics , Female , Genotype , Humans , Learning Disabilities/genetics , Male , Muscle, Skeletal/pathology , Pedigree , Phenotype , Severity of Illness Index
12.
Mol Genet Metab ; 69(1): 64-8, 2000 Jan.
Article in English | MEDLINE | ID: mdl-10655159

ABSTRACT

A patient with 2-oxoadipic aciduria and 2-aminoadipic aciduria presented at 2 years of age with manifestations typical of organic acidemia, episodes of ketosis and acidosis, progressive to coma. This resolved and the key metabolites disappeared from the urine and blood. At 9 years of age she developed typical Kearns-Sayre syndrome with complete heart block, retinopathy, and ophthalmoplegia. Southern blot revealed a deletion in the mitochondrial genome.


Subject(s)
Adipates/urine , Kearns-Sayre Syndrome/urine , 2-Aminoadipic Acid/blood , 2-Aminoadipic Acid/urine , Adipates/blood , Adult , Black People/genetics , Child , Child, Preschool , Coma/blood , Coma/genetics , Coma/urine , DNA, Mitochondrial/blood , DNA, Mitochondrial/genetics , Female , Humans , Hydrogen-Ion Concentration , Kearns-Sayre Syndrome/blood , Kearns-Sayre Syndrome/genetics , Ketosis/blood , Ketosis/genetics , Ketosis/urine , Macular Degeneration/blood , Macular Degeneration/genetics , Macular Degeneration/urine , Male , Sequence Deletion/genetics
13.
Eur J Pediatr ; 159 Suppl 3: S219-26, 2000 Dec.
Article in English | MEDLINE | ID: mdl-11216904

ABSTRACT

UNLABELLED: Over 100 pathogenic point mutations and 200 deletions, insertions, and rearrangements have been identified since the first mitochondrial DNA mutations were described in 1988. About 60% of the point mutations affect mitochondrial tRNAs, 35% affect polypeptide subunits of the respiratory chain, and 5% affect mitochondrial ribosomal RNAs. The clinical phenotypes of mitochondrial tRNA disease span the spectrum of all known oxidative phosphorylation disorders and include MELAS, MERRF, Leigh syndrome, PEO, deafness, diabetes, sideroblastic anemia, myoclonus, skeletal myopathy, cardiomyopathy, and renal tubular acidosis. Mutations in respiratory chain proteins encoded by mtDNA result in phenotypes ranging from exercise intolerance to blindness, ataxia, dystonia, dementia, and Leigh syndrome. CONCLUSION: The primary disorders of oxidative phosphorylation are commonly associated with a delayed age of onset, organ selectivity, and an episodic, progressive course. Organ-specific, non-ATP related functions of mitochondria are discussed as important considerations in evaluating the pathogenesis of mitochondrial disease.


Subject(s)
DNA, Mitochondrial/genetics , Mitochondrial Myopathies/genetics , Humans , Penetrance , Point Mutation/genetics , RNA, Transfer/genetics
14.
Clin Chem ; 45(10): 1725-33, 1999 Oct.
Article in English | MEDLINE | ID: mdl-10508117

ABSTRACT

BACKGROUND: The mitochondrial DNA polymerase gamma is the principal polymerase required for mitochondrial DNA replication. Primary or secondary deficiencies in the activity of DNA polymerase gamma may lead to mitochondrial DNA depletion. We describe a sensitive and robust clinical assay for this enzyme. METHODS: The assay was performed on mitochondria isolated from skeletal muscle biopsies. High-molecular weight polynucleotide reaction products were captured on ion-exchange paper, examined qualitatively by autoradiography, and quantified by scintillation counting. RESULTS: Kinetic analysis of DNA polymerase gamma by this method showed a K(m) for dTTP of 1.43 micromol/L and a K(i) for azidothymidine triphosphate of 0.861 micromol/L. The assay was linear from 0.1 to 2 microgram of mitochondrial protein. The detection limit was 30 units (30 fmol dTMP incorporated in 30 min). The linear dynamic range was three orders of magnitude; 30-30 000 units. Imprecision (CV) was 6.4% within day and 12% between days. Application of this assay to a mixed population of 38 patients referred for evaluation of mitochondrial disease revealed a distribution with a range of 0-2506 U/microgram, reflecting extensive biologic variation among patients with neuromuscular disease. CONCLUSION: This assay provides a useful adjunct to current laboratory methods for the evaluation of patients with suspected mitochondrial DNA depletion syndromes.


Subject(s)
DNA, Mitochondrial/analysis , DNA-Directed DNA Polymerase/analysis , Animals , Autoradiography , Cattle , Chromatography, Ion Exchange , Chromatography, Paper , DNA Polymerase gamma , Humans , Mitochondria, Heart/chemistry , Muscle, Skeletal/chemistry , Reproducibility of Results , Sensitivity and Specificity
15.
Ann Neurol ; 45(1): 54-8, 1999 Jan.
Article in English | MEDLINE | ID: mdl-9894877

ABSTRACT

Deficiency of mitochondrial DNA polymerase gamma activity was found in a patient with mtDNA depletion and Alpers' syndrome. Metabolic evaluation revealed fasting hypoglycemia, dicarboxylic aciduria, and reduced activity of the electron transport chain in skeletal muscle. The patient died in early childhood of fulminant hepatic failure, refractory epilepsy, lactic acidemia, and coma. mtDNA content was 30% of normal in skeletal muscle and 25% in the liver. The activity of mtDNA polymerase gamma was undetectable.


Subject(s)
DNA, Mitochondrial/metabolism , DNA-Directed DNA Polymerase/deficiency , Diffuse Cerebral Sclerosis of Schilder/enzymology , Diffuse Cerebral Sclerosis of Schilder/genetics , Ataxia/etiology , Biopsy , Cerebellar Cortex/enzymology , Cerebellar Cortex/pathology , DNA Mutational Analysis , DNA Polymerase gamma , DNA, Mitochondrial/analysis , DNA-Directed DNA Polymerase/metabolism , Diffuse Cerebral Sclerosis of Schilder/complications , Electroencephalography , Electron Transport , Epilepsy/diagnosis , Epilepsy/etiology , Fatal Outcome , Humans , Infant , Liver Failure/etiology , Liver Failure/genetics , Liver Failure/pathology , Magnetic Resonance Imaging , Male , Mitochondria/enzymology , Mitochondria/genetics , Muscle, Skeletal/enzymology , Muscle, Skeletal/pathology
16.
Exp Cell Res ; 238(1): 51-62, 1998 Jan 10.
Article in English | MEDLINE | ID: mdl-9457056

ABSTRACT

The aim of this study was to demonstrate that the induction of growth arrest in human glioblastoma multiforme (GBM) cell lines by retrovirus-mediated transduction of growth control genes was dependent upon the integrity of specific endogenous control pathways. We assessed the status of the endogenous p16INK4A, p21CIP1, pRb, or p53 genes in eight GBM lines. As expected, we found varied combinations of gene defects. The outcome of transducing five of these cell lines with p16INK4A, p21CIP1, pRb, or p53 genes was not entirely predictable. The growth-inhibitory effects mediated by the transfer of the gene encoding p16 was dependent on the presence of the pRb protein, but was independent of p53 status. p21, a broadly active CDK inhibitor and a strong inducer of growth arrest, was not a universal growth suppressor in the group of glioblastoma cell lines analyzed. The suppression of GBM cell proliferation by viruses encoding pRb or p53 was generally predictable and appeared to be independent of the status of either p16 or p21. Suppression of cell growth was assessed by a colony formation assay, by observance of alterations in morphology, and by cell viability staining for trypan blue exclusion. Our findings suggest that to accomplish the suppression of GBM cell proliferation by the transduction of these cell-cycle control genes, the status of endogenous cell-cycle control genes must be taken into account.


Subject(s)
Carrier Proteins/metabolism , Cell Cycle Proteins , Cell Cycle/physiology , Cyclin-Dependent Kinase Inhibitor p16 , Retinoblastoma Protein/metabolism , Tumor Suppressor Protein p53/metabolism , Tumor Suppressor Proteins , Carrier Proteins/biosynthesis , Cell Division , Cyclin-Dependent Kinase Inhibitor p15 , Cyclin-Dependent Kinase Inhibitor p21 , Cyclins/biosynthesis , Cyclins/metabolism , Enzyme Inhibitors/metabolism , Glioblastoma , Humans , Kinetics , Recombinant Proteins/metabolism , Retinoblastoma Protein/biosynthesis , Retroviridae , Transfection , Tumor Cells, Cultured , Tumor Suppressor Protein p53/biosynthesis
17.
J Virol ; 70(8): 5701-5, 1996 Aug.
Article in English | MEDLINE | ID: mdl-8764092

ABSTRACT

We describe the construction and characterization of retroviral vectors and packaging plasmids that produce helper-free retrovirus with titers of 1 X 10(6) to 5 X 10(6) within 48 h. These vectors contain the immediate early region of the human cytomegalovirus enhancer-promoter fused to the Moloney murine leukemia virus long terminal repeat at the TATA box in the 5' U3 region, yielding the pCL promoter. By selecting vectors designed to express genes from one of four promoters (dihydrofolate reductase, Rous sarcoma virus, long terminal repeat, or cytomegalovirus), the pCL system permits the investigator to control the level of gene expression in target cells over a 100-fold range, while maintaining uniformly high titers of virus from transiently transfected producer cells. The pCL packaging plasmids lack a packaging signal (delta-psi) and include an added safety modification that renders them self-inactivating through the deletion of the 3' U3 enhancer. Ecotropic, amphotropic (4070A), and amphotropic-mink cell focus-forming hybrid (10A1) envelope constructions have been prepared and tested, permitting flexible selection of vector pseudotype in accordance with experimental needs. Vector supernatants are free of helper virus and are of sufficiently high titer within 2 days of transient transfection in 293 cells to permit infection of more than 50% of randomly cycling target cells in culture. We demonstrated the efficacy of these vectors by using them to transfer three potent cell cycle control genes (the p16(INK4A), p53, and Rb1 genes) into human glioblastoma cells.


Subject(s)
DNA, Recombinant/genetics , DNA, Viral/genetics , Genetic Vectors , Retroviridae/genetics , Humans
18.
Hum Gene Ther ; 6(5): 591-601, 1995 May.
Article in English | MEDLINE | ID: mdl-7578396

ABSTRACT

We evaluated the effects of different doses of interleukin-2 (IL-2)-transduced fibroblasts in the treatment of colorectal carcinoma in the CT-26 murine tumor model. Immunization with a mixture of irradiated tumor cells and IL-2-transduced fibroblasts (100 units of IL-2/24 hr) induced significantly greater protection against a live tumor challenge compared to irradiated tumor cells alone (22/35, 65% vs. 10/30, 33%, p < 0.02). Protective effects were observed with doses of IL-2-transduced fibroblasts secreting from 5 to 100 units of IL-2/24 hr. Parallel experiments in nude mice produced no protection, indicating that the effects of immunization were mediated by a T-cell-dependent mechanism. In animals with established tumors, complete tumor remissions were observed following immunization with a mixture of irradiated tumor cells and IL-2-transduced fibroblasts secreting 100 units of IL-2/24 hr, but not after immunization with irradiated tumor cells alone (7/16 vs. 0/11 complete remissions, p < 0.02). Fibroblasts secreting higher doses of IL-2 were ineffective in generating systemic immunity, but were required to prevent tumor implantation. A statistically significant difference in the prevention of tumor implantation was observed between groups inoculated with a mixture of live tumor cells and IL-2-transduced fibroblasts (1,750 units of IL-2/24 hr) compared to control fibroblasts (6/8 vs. 0/12, p < 0.001). Similar results were observed in nude mice, suggesting that the implantation rejection response is mediated in part by cells other than thymus-derived T cells. Our data support the utility of IL-2-transduced fibroblasts and indicate that the level of IL-2 expression is an important variable in activating different effector components of antitumor immune responses in IL-2 gene therapy.


Subject(s)
Colorectal Neoplasms/therapy , Gene Transfer Techniques , Genetic Therapy/methods , Interleukin-2/genetics , T-Lymphocytes/immunology , Adenocarcinoma/pathology , Animals , Cell Line , Colorectal Neoplasms/immunology , Colorectal Neoplasms/pathology , Cytotoxicity, Immunologic , Fibroblasts , Gene Expression , Genetic Vectors , Humans , Immunity, Cellular , Interleukin-2/biosynthesis , Mice , Mice, Inbred BALB C , Mice, Nude , Neoplasm Transplantation , Retroviridae/genetics
19.
Proc Natl Acad Sci U S A ; 89(22): 10892-5, 1992 Nov 15.
Article in English | MEDLINE | ID: mdl-1332058

ABSTRACT

We have explored the use of primary myoblasts as a somatic tissue for gene therapy of acquired and inherited diseases where systemic delivery of a gene product may have therapeutic effects. Mouse primary myoblasts were infected with replication-defective retroviruses expressing canine factor IX cDNA under the control of a mouse muscle creatine kinase enhancer and human cytomegalovirus promoter. The infected myoblasts were injected into the hindlegs of recipient mice and levels of secreted factor IX protein were monitored in the plasma. We report sustained expression of factor IX protein for over 6 months without any apparent adverse effect on the recipient mice.


Subject(s)
Factor IX/biosynthesis , Factor IX/genetics , Genetic Therapy , Muscles/transplantation , Animals , Cell Line , Cells, Cultured , Creatine Kinase/genetics , Cytomegalovirus/genetics , Dogs , Enhancer Elements, Genetic , Gene Expression , Genetic Vectors , Humans , Kinetics , Mice , Mice, Nude , Promoter Regions, Genetic , Time Factors
20.
Curr Opin Biotechnol ; 3(5): 540-7, 1992 Oct.
Article in English | MEDLINE | ID: mdl-1368938

ABSTRACT

Retroviral vectors provide a safe and efficient method of introducing genes of therapeutic interest into dividing cells. The principle limitation of these vectors in the past has been poor gene expression in vivo. This problem has been overcome recently through the use of tissue-specific enhancers in commonly used retroviral vectors. In this review we discuss both the relevant biology and some of the practical applications of retroviral vectors in gene therapy.


Subject(s)
Genetic Vectors , Retroviridae/genetics , Animals , Biotechnology , Gene Expression , Genetic Engineering , Genetic Therapy , Humans , Safety
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