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Biomedical subjects

C R Freed

Publications and source records attributed to C R Freed.

At least 37 records · Page 2Linked to original sources
PubMed1998

Brain contains inhibiting factors specific to the large T-antigen gene.

SV40 large T-antigen (LTa) gene-induced immortalized rat dopamine-producing nerve cells (IRB3AN27), which produce LTa protein and divide in vitro, do not divide and do not produce LTa protein when transplanted into striatum of adult rats. This suggests the presence of LTa gene-inhibiting factors in brain. Here we report that rat brain soluble fraction (SF) contains factors which specifically inhibit LTa gene activity in vitro. The brain SF inhibited LTa protein levels and the growth of IRB3AN27 cells and 2RSG cells (LTa gene-induced immortalized rat parotid acinar cells) in vitro, but it stimulated the growth of spontaneously immortalized human parotid acinar cells (2HPC8) and had no effect on the proliferation of murine neuroblastoma cells (NBP2) and rat glioma cells (C-6) in culture. In contrast, the liver SF inhibited the growth of all cell lines tested at varying degrees and thus lacked specificity with respect to LTa gene activity. The presence of specific LTa gene-inhibiting factors in the brain and general tumor growth-inhibiting factors in the liver may provide some of the mechanisms of protection against in vivo carcinogenesis.

Animals↗
PubMed1998

Efficacy of grafted immortalized dopamine neurons in an animal model of parkinsonism: a review.

Dopamine (DA) deficiency is one of the primary lesions in the pathogenesis of Parkinson disease (PD). Because of long-term toxicity of L-DOPA therapy, the grafting of fetal mesencephalic tissue containing dopamine neurons or homogeneous populations of DA neurons into striatum appears to be rational. Fetal tissue transplants have many problems which include legal (in some countries), ethical, paucity of tissue availability, heterogenicity of cell populations, and the presence of antigen-presenting cells that are responsible for rejection of allogeneic grafts. In order to resolve the above problems, we have established immortalized DA neurons from fetal rat mesencephalon by inserting the large T-antigen (LTa) gene of the SV40 virus into the cells. A clone of DA neurons (1RB3AN27) was isolated, characterized, and tested in 6-hydroxydopamine (6-OHDA)-lesioned rats (a model of PD). These cells divided with a doubling time of about 26 h, expressed the LTa gene, and contained the tyrosine hydroxylase and dopamine transporter proteins and their respective mRNAs, which became elevated upon differentiation. These cells were nontumorigenic and nonimmunogenic and improved the symptoms of neurological deficits (methamphetamine-induced rotation) in 6-OHDA-lesioned rats. The differentiated DA neurons were more effective than undifferentiated ones. These studies suggest that immortalized DA neurons generated in vitro by LTa gene insertion may be used in transplant therapy without fear of tumor formation or rejection.

Animals↗
PubMed1998

Somatic cell cloned transgenic bovine neurons for transplantation in parkinsonian rats.

Parkinson's disease symptoms can be improved by transplanting fetal dopamine cells into the putamen of parkinsonian patients. Because the supply of human donor tissue is limited and variable, an alternative and genetically modifiable non-human source of tissue would be valuable. We have generated cloned transgenic bovine embryos, 42% of which developed beyond 40 days. Dopamine cells collected from the ventral mesencephalon of the cloned fetuses 42 to 50 days post-conception survived transplantation into immunosuppressed parkinsonian rats and cells from cloned and wild-type embryos improved motor performance. Somatic cell cloning can efficiently produce transgenic animal tissue for treating parkinsonism.

Animals↗
PubMed1997

Inhibition of proliferation and expression of T-antigen in SV40 large T-antigen gene-induced immortalized cells following transplantations.

Rat dopamine-producing nerve cells (1RB3AN27) and rat parotid acinar cells (2RSG) were immortalized by insertion of simian virus 40 (SV40) large T-antigen gene (LTa). Both of these cells divided and produced nuclear LTa in vitro. In order to assess the relationship between cell proliferation and expression of LTa in vivo, immortalized dopamine-producing nerve cells and parotid cells were grafted into the striatum and parotid gland of adult Sprague-Dawley rats, respectively. Grafted cells exhibited nuclear LTa at 1 day but not at 7 and 30 days after transplantation. At 30 days after transplantation, no tumor was found, and there was no evidence of cell division as determined by H and E staining. When the striatal areas containing the grafts were cultured, these cells did not express LTa at 4 days after plating; however, after 3 weeks, when most host cells were eliminated, the cultured grafted cells expressed LTa. After 3 months of culturing, only cells exhibiting LTa were present. These cells had the same morphology and divided with the same doubling time as 1RB3AN27 cells before grafting. Results suggest the presence of a LTa-inhibiting factor in vivo, and support the hypothesis that the expression of LTa is directly linked with proliferation of immortalized cells.

Animals↗
PubMed1997

GDNF improves survival and reduces apoptosis in human embryonic dopaminergic neurons in vitro.

Dopamine cell death is the primary problem limiting the value of neurotransplantation in human patients with Parkinson's disease. To address this problem, we added glial cell line-derived neurotrophic factor (GDNF) to cultures of embryonic dopaminergic neurons obtained from human and from Bonnet monkey (Macaca radiata) in an effort to reduce apoptotic cell death and improve overall cell survival. Tissue from three human embryos, 7-8 weeks post-conception, and one 9-week post-conception monkey embryo were dissociated and cultured in F-12 media with 5% human placental serum. GDNF (10 ng/ml) in human cultures nearly doubled dopamine neuron survival and reduced the rate of apoptosis from 6% to 3%. In monkey cultures, GDNF also enhanced dopamine neuron survival and reduced the apoptotic rate. We conclude that GDNF improves the survival of primate embryonic dopamine neurons in culture by reducing apoptosis.

Animals↗
PubMed1997

Flecainide overdose: is cardiopulmonary support the treatment?

Flecainide toxicity can impair cardiac function and precipitate circulatory collapse, which in turn depresses clearance and redistribution of flecainide. Treatment directed at improving cardiac function is often ineffective in the presence of persistently increased flecainide levels. We report a novel approach to severe flecainide overdose using peripheral cardiopulmonary bypass support (CBS) to maintain perfusion of the liver, thereby allowing clearance of the drug. CBS was initiated to resuscitate a young woman who had ingested flecainide in a suicide attempt. The patient had an agonal rhythm, no effective blood pressure, and a flecainide level of 5.4 micrograms/mL (therapeutic range, .2 to 1.0 microgram/mL). During 10 hours of CBS, the flecainide level decreased to 1.4 micrograms/mL, a half-life of 6 hours. Effective cardiac rhythm and blood pressure returned. CBS successfully supported this patient until the flecainide level decreased as a result of redistribution and normal clearance mechanisms. Unfortunately, because of severe neurologic damage sustained at the time of overdose, the patient died 4 days after admission.

Adult↗
PubMed1997

Preliminary voice and speech analysis following fetal dopamine transplants in 5 individuals with Parkinson disease.

A surgical procedure involving transplantation of fetal dopamine cells into the striatum of persons with advanced Parkinson disease (PD) has recently been performed in an attempt to alleviate Parkinsonian and drug-dose related symptoms (e.g., the "on-off" phenomena). Improvements in limb motor and neurological function, as well as less severe and shorter on-off episodes have been reported following fetal cell transplant (FCT) surgery. Acoustic, electroglottographic, and perceptual measures were analyzed pre- and post-surgery to determine if phonotory and articulatory function were affected by this relatively new form of treatment. In addition, speech and motor exam measures were compared to determine if similar directional changes across motor systems were apparent. Findings suggest that FCT surgery did not systematically influence voice and speech production. Also, it appears that FCT surgery may differentially affect phonatory, articulatory, and limb motor systems. Findings are discussed relative to these differential effects.

Aged↗
PubMed1997

Intrastriatal grafting of Cos cells stably expressing human aromatic L-amino acid decarboxylase: neurochemical effects.

To study the possibility that increasing striatal activity of aromatic L-amino acid decarboxylase (AADC; EC 4.1.1.28) can increase dopamine production in dopamine denervated striatum in response to L-3,4-dihydroxy-phenylalanine (L-DOPA) administration, we grafted Cos cells stably expressing the human AADC gene (Cos-haadc cells) into 6-hydroxydopamine denervated rat striatum. Before grafting, the catalytic activity of the enzyme was assessed in vitro via the generation of 14CO2 from L-[14C]DOPA. The Km value for L-DOPA in intact and disrupted cells was 0.60 and 0.56 mM, respectively. The cofactor, pyridoxal 5-phosphate, enhanced enzymatic activity with maximal effect at 0.1 mM. The pH optimum for enzyme activity was 6.8. Grafting Cos-haadc cells into denervated rat striatum enhanced striatal dopamine levels measured after systemic administration of L-DOPA. When measured 2 h after L-DOPA administration, the mean dopamine level in the striata of Cos-haadc-grafted animals was 2 micrograms/g of tissue, representing 31% of normal striatal dopamine concentration. The mean dopamine concentration in the striata grafted with untransfected Cos cells (Cos-ut cells) was 1 microgram/g. At 6-8 h after L-DOPA administration, striatal dopamine content in the Cos-haadc-grafted animals was 0.67 microgram/g of tissue weight, representing 9% of intact striatum dopamine content. By contrast, the average dopamine content in the Cos-ut-grafted animals was undetectable. These findings demonstrate that enhancing striatal AADC activity can improve dopamine bioformation in response to systemically administered L-DOPA.

Animals↗
PubMed1996

Growth factors rescue embryonic dopamine neurons from programmed cell death.

Poor survival of embryonic dopamine neurons is a primary problem limiting the value of neurotransplantation for Parkinson's disease. Several neurotrophic factors have been shown to promote dopamine neuron survival when used individually in culture. We have found that two peptides, insulin-like growth factor-I (IGF-I) and basic fibroblast growth factor (bFGF), have additive effects on cell survival when used in combination. These growth factors reduced the number of dopamine cells undergoing apoptotic cell death. The neurotrophic factors induced proliferation of astrocytes but not dopamine neurons. When cell proliferation was blocked by cytosine arabinoside, the beneficial effects of IGF-I and bFGF were abolished, suggesting that effects of the growth factors were mediated, at least in part, by factors associated with glia. These results indicate that growth factors in combination may prove useful for enhancing dopamine neuron survival for neurotransplantation.

Animals↗
PubMed1996

Characterization and transplantation of two neuronal cell lines with dopaminergic properties.

Immortalized rat mesencephalic cells (1RB3AN27) produced dopamine (DA) at a level that was higher than produced by undifferentiated or differentiated murine neuroblastoma cells (NBP2) in culture. Treatment of 1RB3AN27 and NBP2 cells with a cAMP stimulating agent increased tyrosine hydroxylase (TH) activity and the intensity of immunostaining for the DA transporter protein (DAT). 1RB3AN27 cells were labelled with primary antibodies to neuron specific enolase (NSE) and nestin and exhibited very little or no labeling with anti-glial fibrillary acidic protein (GFAP). 1RB3AN27 cells exhibited beta- and alpha-adrenoreceptors, and prostaglandin E1 receptors, all of which were linked to adenylate cyclase (AC). Dopamine receptor (D1) and cholinergic muscarinic receptors linked to AC were not detectable. The levels of PKC alpha and PKC beta isoforms were higher than those of PKC gamma and PKC delta in 1RB3AN27 cells. The 1RB3AN27 cells were more effective in reducing the rate of methamphetamine-induced turning in rats with unilateral 6-OHDA lesion of the nigrostriatal system than differentiated NBP2 cells. The grafted 1RB3AN27 were viable as determined by DiI labelling, but they did not divide and did not produce T-antigen protein; however, when these grafted cells were cultured in vitro, they resumed production of T-antigen and proliferated after the primary glia cells and neurons of host brain died due to maturation and subsequent degeneration. Examination of H&E stained sections of the grafted sites revealed no evidence of infiltration of inflammatory cells in the grafted area suggesting that these cells were not immunogenic. They also did not form tumors.

3,4-Dihydroxyphenylacetic Acid↗
PubMed1996

Conditioned medium from aged monkey fibroblasts stably expressing GDNF and BDNF improves survival of embryonic dopamine neurons in vitro.

Fibroblasts derived from the cerebral cortex of an aged Bonnet monkey (Macaca radiata) were utilized to express recombinant cDNAs encoding rat glial-cell-line-derived neurotrophic factor (GDNF) and human prepro brain-derived neurotrophic factor (BDNF) by lipofection. The cells showed stable expression and secretion of biologically active proteins. Conditioned medium from fibroblasts expressing BDNF or GDNF increased the number of surviving mesencephalic tyrosine-hydroxylase-immunoreactive neurons after 7 days in culture. The trophic effects of BDNF and GDNF were examined at two different plating densities of embryonic mesencephalic cells. At 50 000 cells/cm2 plating density, treatment of the mesencephalic cultures with BDNF-conditioned medium increased the number of tyrosine-hydroxylase-immunoreactive neurons by about 40% compared with vector-transfected control. At the same plating density, GDNF-conditioned medium increased the number of surviving tyrosine-hydroxylase-immunoreactive neurons above the vector-transfected control by 30%. When the tissue was plated at a higher density, viz., 75 000 cells/cm2, the number of tyrosine-hydroxylase-immunoreactive neurons increased by 41% with BDNF-conditioned medium, and by 56% with GDNF-conditioned medium above vector-transfected controls. Conditioned medium from cells secreting GDNF was also found to reduce the number of apoptotic tyrosine-hydroxylase-immunoreactive cells by 50%.

Aging↗
PubMed1996

Insulin-like growth factor binding proteins in fetal rat mesencephalic cultures: regulation by fibroblast growth factor and insulin-like growth factor I.

In rat ventral mesencephalic cultures, IGF-I and bovine fibroblast growth factor (bFGF) act cooperatively to support the survival of dopaminergic neurons. To determine the potential role of IGFBPs in modulating the actions of IGF-I in the ventral mesencephalon, we identified the IGFBPs present in ventral mesencephalic cultures and examined their regulation by IGF-I and bFGF. In the absence of added growth factors, the major binding protein secreted from these cultures was IGFBP-2. Small amounts of IGFFBP-3 and IGFBP-4 were also detected. Addition of bFGF to the cultures increased the amounts of IGFBP-3 and IGFBP-4 released from the cells by 4.4 +/- 2.6 -fold (P < 0.1) and 11.5 +/- 3.5 -fold (P < 0.05), respectively. IGF-I, itself, had little effect on the production of IGFBPs, but when added together with bFGF increased the levels of IGFBP-3 and IGFBP-4 by 12.4 +/- 5.1 -fold (P < 0.05) and 27.4 +/- 5.3 -fold (P < 0.02), respectively. The stimulatory effect of bFGF and IGF-I on IGFBP production was apparent after a 2- to 3-day exposure of the mesencephalic cultures to the peptides. IGFBP-4, the most abundant IGFBP present in the cultures after 7 days of growth factor treatment, was immunocyto-chemically localized primarily to neurons, of which a subset were dopaminergic neurons. The addition of purified rat IGFBP-4 to the cultures in the absence of added growth factors had no effect on the survival of dopaminergic neurons, but when added with IGF-I potentiated the effect of IGF-I on neuronal survival. We propose that the up-regulation of IGFBP-4 by IGF-I and bFGF may serve to localize IGF-I to sites of action in the nervous system and thereby potentiate the neurotrophic actions of IGF-I.

Amino Acid Sequence↗
PubMed1995

GDNF reduces apoptosis in dopaminergic neurons in vitro.

To explore the mechanism by which glial cell line-derived neurotrophic factor (GDNF) improves cell survival, we measured the apoptotic rate of dopamine neurons incubated with GDNF. Cultures were prepared from embryonic day 15 rat mesencephalon in medium with 5% human placental serum. GDNF reduced the rate of apoptosis in dopamine neurons from 5% to 2%. By contrast, GDNF had no effect on apoptoisis in astrocytes from embryonic mesencephalon or from neonatal cortex. Co-cultures with embryonic striatum as well as with combinations of growth factors were also tested for effects on dopamine neuron survival. Neuronal survival was maximal during co-culture with striatal cells with or without added growth factors. We conclude that GDNF inhibits apoptotic cell death in dopamine neurons.

Analysis of Variance↗
PubMed1995

Electrophysiology of ventromedial striatal neurons during movement.

The firing rate of ventromedial striatal cells was studied in rats trained to run in place on a rotating turntable treadmill. Animals were trained to run clockwise and counter-clockwise as they propelled a turntable for a water reward. After a period of training, Parylene C coated stainless steel wire electrodes were chronically implanted for single unit recording. Nearly all ventromedial striatal cells increased their firing rate concomitant with locomotion in the treadmill (32 of 36 cells). The magnitude of this response was influenced by the order of testing and by the direction of circling relative to the side of brain being recorded. The increase in firing rate was greater during the first test of the day and was greater when animals circled contralateral to the side of the recording electrode than when they circled ipsilateral to the recorded side.

Action Potentials↗
PubMed1995

Ascorbic acid concentration in the lateral hypothalamus is related to plasma osmolality.

Microdialysis was used to measure extracellular ascorbic and uric acid concentrations in the lateral hypothalamus of water-restricted rats as they drank distilled water or 1.5% NaCl. Other water-restricted rats, not implanted with microdialysis probes, were decapitated 2 h after beginning to drink these fluids. Rats were inverted and their blood was collected for measurements of plasma osmolality and percent hematocrit. Results showed that drinking distilled water produced a significant increase in the ascorbic acid concentration but not in the uric acid concentration. Drinking 1.5% NaCl produced a significant decrease in the uric acid concentration but not in the ascorbic acid concentration. Drinking distilled water decreased mean osmolality from 306.0 to 291.5 mOsm/kg, whereas drinking 1.5% NaCl maintained mean osmolality at water-restricted levels. These results indicate that the extracellular fluid concentration of ascorbic acid in the lateral hypothalamus rises in response to a fall in plasma osmolality.

Animals↗
PubMed1995

Implantation of fetal tissue for the management of Parkinson's disease: a technical note.

A relatively simple method for implanting fetal tissue within the human basal ganglia is described. Stereotactic techniques are used to deposit 14 to 16 strands of fetal mesencephalic tissue in a linear array within the putamen bilaterally. To eliminate the need for targeting each needle pass independently, we have devised a novel rotating template/micromanipulator apparatus that can be mounted on a Cosman-Roberts-Wells stereotactic frame. The template contains two parallel columns of nine holes, all spaced at 4-mm intervals. It can be aligned to the longitudinal axis of the putamen and targeted with coordinates for the center point of the putamen. Surgery is performed on an awake patient. Short-term morbidity (4-6 wk) in the form of variability of Parkinsonian signs, particularly "off" symptoms and mild confusion, appears to be the rule with simultaneous bilateral implants.

Equipment Design↗