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Biomedical subjects

A Freese

Publications and source records attributed to A Freese.

At least 37 records · Page 2Linked to original sources
PubMed1996

Determination of HLA-B7 T-cell epitopes.

A series of peptides derived from the alpha 1 and alpha 2 regions of the HLA-B7 and HLA-A2 molecules was synthesized with an automatic peptide synthesizer using the FMOC (9-fluorenylmethoxycarbonyl) technique. Peptides were analyzed and purified by reversed-phase high pressure liquid chromatography (HPLC). Peptide purity was > 96%. The effect of B7 peptides on mixed lymphocyte cultures was tested in vivo. The alloresponse in the cultures with B7 peptides was strongly enhanced. The peptides that were most effective inhibited cytotoxic T-lymphocyte (CTL) cytolysis. The data show that the peptides are immunogenic and that they are recognised by both the direct and indirect pathways. Further, the mechanism of peptide recognition was studied. We coupled peptide B7 (residues 62-70 in HLA-B7) and peptide A2 (residues 62-70 in HLA-B2) covalently to fluorescein isothiocyanate (FITC). B7-specific CTLs were incubated with these peptides at 37 degrees C for 90 min and cell fluorescence measured by flow cytometry. The B7 peptide was bound by 60% of the CTLs whereas the A2 peptide, as a negative control, bound only 10%. The molecular size of the ligands to which the peptides bind are being characterized by immunoprecipitation.

Amino Acid Sequence↗
PubMed1996

Hippocampal cell distributions in temporal lobe epilepsy: a comparison between patients with and without an early risk factor.

Neuronal cell distributions were measured for anterior and posterior locations in the hippocampi of epilepsy patients who were seizure-free after temporal lobectomy. Patients were divided into two groups, those with an early risk factor, defined as a neurologic insult occurring in the first 4 years of life, and those with no early risk factor. Early-risk patients had lower hilar cell densities, lower granule cell densities, and fewer granule cells per millimeter, a measured related to total granule cell number, than to early risk patients. Moreover, each risk group had different anteroposterior density gradients for granule cells and hilar cells. These differences in cell distribution may arise from different patterns of cell loss of cell migration in the dentate gyrus during development. In the early-risk group, there was also a distinction between patients with a history of febrile convulsions without CNS infection and patients with a history of meningitis or encephalitis. These two subgroups had similar numbers of granule cells, However, the meningitis/encephalitis subgroup exhibited a wider granule cell layer, suggesting that the granule cell layer was more dispersed. Our results support the hypothesis of a predominantly anterior hippocampal insult in temporal lobe epilepsy (TLE). In nonepileptic hippocampus, the ratio of putatively excitatory granule neurons to putatively inhibitory hilar neurons is highest in the anterior hippocampus. This ratio may explain in part why the anterior hippocampus is more prone to cell loss and seizures.

Adult↗
PubMed1996

Transfection of human lactotroph adenoma cells with an adenovirus vector expressing tyrosine hydroxylase decreases prolactin release.

Pituitary adenomas are common intracranial neoplasms, for which surgery and radiation are usually not curative. In attempting to develop gene therapy as a better approach to treating pituitary adenomas, we chose lactotroph adenomas as a model. The rationale for the use of this model is based on the observation that dopamine agonists decrease prolactin secretion by lactotroph adenomas, and also decrease their size. We transfected primary cultures of human lactotroph adenoma cells with an adenovirus vector containing a cDNA which encodes a human tyrosine hydroxylase, the rate-limiting enzyme in the biosynthesis of dopamine. Transfection induced expression of tyrosine hydroxylase and increased production of dopamine, resulting in the predicted biologic effect of decreased prolactin secretion. These results demonstrate the potential for gene therapy of lactotroph adenomas and perhaps other pituitary adenomas, which are less amenable to pharmacologic treatment than lactotroph adenomas.

Adenoma↗
PubMed1995

An HSV-1 vector expressing tyrosine hydroxylase causes production and release of L-dopa from cultured rat striatal cells.

In this report we demonstrate that a defective herpes simplex virus type one (HSV-1) vector can express enzymatically active tyrosine hydroxylase in cultured striatal cells that are thereby converted into L-DOPA-producing cells. A human tyrosine hydroxylase cDNA (form II) was inserted into an HSV-1 vector (pHSVth) and packaged into virus particles using an HSV-1 strain 17 mutant in the immediate early 3 gene (either ts K or D30EBA) as helper virus. Cultured fibroblasts were infected with pHSVth and 1 day later tyrosine hydroxylase immunoreactivity and tyrosine hydroxylase enzyme activity were observed. The tyrosine hydroxylase enzyme activity directed the production of L-DOPA. pHSVth infection of striatal cells in dissociated cell culture resulted in expression of tyrosine hydroxylase RNA and tyrosine hydroxylase immunoreactivity. Release of L-DOPA and low levels of dopamine were observed from cells in pHSVth-infected striatal cultures. Expression of tyrosine hydroxylase and release of catecholamines were maintained for at least 1 week after infection.

Animals↗
PubMed1994

The application of 5-bromodeoxyuridine in the management of CNS tumors.

A variety of clinical reports have described the application of the bromodeoxyuridine labeling index as an adjunct to conventional pathological examination of CNS tumors. This index has proven useful in predicting the clinical outcome associated with many such tumors. Furthermore, because of its efficacy as a radiosensitizing agent, bromodeoxyuridine (and the closely related iododeoxyuridine) has been used in combination with radiation therapy for malignant glial neoplasms, with some encouraging results. Although most studies suggest that bromodeoxyuridine is safe, there is evidence that this compound does have potential side-effects, including the observation that it is a mutagen and carcinogen in some experimental systems. A number of new alternative approaches for predicting the clinical outcome of CNS tumors has been developed based on an increased understanding of their molecular biology. However, until such approaches are better characterized, the clinical application of bromodeoxyuridine will continue to play an important role in predicting the clinical behavior of many CNS tumors.

Brain Neoplasms↗
PubMed1993

Long-term increases in neurotransmitter release from neuronal cells expressing a constitutively active adenylate cyclase from a herpes simplex virus type 1 vector.

Signal-transduction pathways mediate a wide range of short-term changes in the physiology of neuronal systems from invertebrates to mammals. However, examples of long-term changes in neuronal physiology mediated by these pathways have been limited to invertebrate systems. In this report, long-term changes in the physiology of mammalian neurons were studied by using genetic intervention to cause a long-lasting activation of the cAMP pathway. The catalytic domain of yeast adenylate cyclase (cyr), encoding a constitutive enzyme activity, was expressed in neuronal cells infected with a defective herpes simplex virus vector (pHSVcyr). In PC-12 cells infected with pHSVcyr, increases were seen in cAMP levels, protein kinase A activity, protein phosphorylation, phosphorylation of the tyrosine hydroxylase protein kinase A site (Ser40), and catecholamine release. Infection of sympathetic neurons with pHSVcyr increased cAMP levels, protein phosphorylation, and catecholamine release. Yeast adenylate cyclase immunoreactivity and elevated cAMP levels were localized to the cell bodies of sympathetic neurons. The increase in neurotransmitter release was both Ca(2+)- and activity-dependent and persisted for at least 1 week after infection of the sympathetic neurons, suggesting that sustained physiological activation of the cAMP pathway may mediate long-term changes in the neuronal physiology of mammalian systems.

Adenylyl Cyclases↗
PubMed1993

Superficial siderosis of the central nervous system: magnetic resonance imaging and pathological correlation. Case report.

The authors report a 32-year-old woman who had undergone repair of an occipital encephalocele in infancy and who experienced a 20-year history of progressive hearing loss and intermittent vertigo. After parturition, she developed a rapidly progressive quadriparesis and brain-stem dysfunction associated with persistent intraventricular and subarachnoid hemorrhage. Serial magnetic resonance (MR) images showed progressive deposition of hemosiderin along the surface of the brain, brain stem, and spinal cord, and enhanced thickened membranes at the site of the original encephalocele repair. Posterior fossa exploration disclosed hemorrhagic membranes, which were resected; despite removal of this tissue, the patient deteriorated and died. Postmortem examination confirmed iron-containing pigment along the meninges, cerebral hemispheres, brain stem, spinal cord, and cranial nerves accompanied by atrophy of the superficial cerebellar cortex. It is concluded that superficial siderosis may accompany encephalocele repair. This is believed to be the first report in the literature of superficial siderosis of the central nervous system to correlate in vivo MR images with autopsy results.

Adult↗
PubMed1992

Basic fibroblast growth factor protects striatal neurons in vitro from NMDA-receptor mediated excitotoxicity.

Basic fibroblast growth factor (bFGF) promotes the survival and outgrowth of neurons. In this study the neuroprotective effects of bFGF were examined in 12-18-day-old cultured striatal neurons exposed to glutamic acid, kainic acid (KA), and quinolinic acid (QA), an N-methyl-D-aspartate (NMDA)-receptor agonist. Results showed that preincubation with bFGF (6 pM) from the day of plating significantly increased the survival of striatal neurons treated for 3 h with glutamate (3 mM) or QA (1 mM), but had little effect on KA (1 mM) induced toxicity. Moreover, maximum protection by bFGF against glutamate neurotoxicity was observed in cultures treated as little as 2 h before glutamate exposure. These results show that bFGF markedly protects striatal neurons from NMDA-receptor induced neurotoxicity.

Animals↗
PubMed1992

Biochemical and behavioral recovery in a rodent model of Parkinson's disease following stereotactic implantation of dopamine-containing liposomes.

We have developed and characterized dopamine-containing liposomes which exhibited in vitro sustained release of dopamine for over 40 days. These liposomes were stereotactically implanted into the partially denervated corpus striatum of rats subjected to unilateral lesions of the substantia nigra. In vivo release of dopamine into striatal extracellular fluid was monitored by microdialysis and behavior was assessed by quantifying apomorphine-induced asymmetric rotation. Extracellular dopamine levels in the partially denervated striatum of the dopamine liposome-treated rats were greater than the levels in the lesioned rats which received control liposomes and these levels remained elevated for 25 days. In parallel, those rats which received dopamine liposomes exhibited partial behavioral recovery, with attenuation of asymmetric rotation following systemic apomorphine administration. These results suggest that dopamine-containing liposomes can partially ameliorate the deficits associated with a rodent model of Parkinson's disease, and demonstrate the potential of this technology as a method for the controlled delivery of therapeutic agents into discrete areas of the brain.

Animals↗
PubMed1991

Infection of cultured striatal neurons with a defective HSV-1 vector: implications for gene therapy.

Several neurological diseases which affect the corpus striatum are candidates for gene therapy. We have developed a defective Herpes Simplex Virus (HSV-1) vector system to introduce genes into postmitotic cells, such as neurons. The prototype vector, pHSVlac, contains a transcription unit which places the E. coli Lac Z gene under the control of the HSV-1 immediate early (IE) 4/5 promoter, a constitutive promoter. We now demonstrate that a HSV-1 vector can deliver a gene into striatal neurons. Infection of cultured rat striatal neurons with pHSVlac virus resulted in stable expression of beta-galactosidase for at least two weeks, without cell death. The potential to replace the Lac Z gene with other genes of interest, such as the gene responsible for Huntington's Disease, once it is isolated, may lead to insights about the pathogenesis of this genetic neurodegenerative disease, and may provide a method for performing gene therapy on this disease. Similarly, introduction of the tyrosine hydroxylase gene, which encodes the rate-limiting enzyme in the conversion of tyrosine to dopamine, into striatal neurons might provide a novel gene therapy approach towards treating Parkinson's Disease.

Animals↗
PubMed1990

Characterization and mechanism of glutamate neurotoxicity in primary striatal cultures.

Excitatory amino acids may play a role in the pathogenesis of cell death in neurodegenerative diseases, including Huntington's disease (HD). In an attempt to develop a tissue culture model for HD, the toxicity of glutamate was examined in primary striatal cultures derived from newborn rats. Morphological criteria were used to determine the toxic effects of glutamate in 6-, 12-, and 18-day-old cultures which were examined before and after 1-3 h of exposure to glutamate. Although younger cultures demonstrated little susceptibility to glutamate relative to controls, the number of neurons in older cultures was significantly depleted in the presence of glutamate. Glutamate toxicity was dose-dependent, with an ED50 of approximately 300 microns glutamate, and a maximal effect was observed within 3 h of initial exposure. Affected neurons demonstrated somal swelling within 1 h of glutamate exposure and disruption of neuritic processes and somal integrity within 3 h. Cell death was significantly increased by raising the extracellular calcium concentration and could be decreased by the addition of magnesium to the incubation medium. Moreover, the N-methyl-D-aspartate (NMDA) receptor agonist, quinolinic acid, showed a toxicity profile similar to that of glutamate. The NMDA receptor competitive antagonist, 2-amino-5-phosphonovalerate (APV) significantly reduced toxicity, albeit incompletely. An additional component of glutamate mediated toxicity in striatal cultures could be explained by activation of non-NMDA receptor subtypes. These in vitro studies indicate that glutamate is toxic to a subset of mature striatal neurons in the absence of a glutamatergic afferent input, and that this toxicity is mediated partially by the NMDA receptor, with an additional component due to non-NMDA receptors.

2-Amino-5-phosphonovalerate↗
PubMed1990

The correlation between excitatory amino acid-induced current responses and excitotoxicity in striatal cultures.

Neuronal excitotoxic injury is initiated by activation of specific excitatory amino acid receptors and is mediated by ion flow through associated ion channels. In a companion paper we defined the age dependence of glutamate toxicity in striatal cultures. In this study we examined the correlation between age-dependent changes in the electrophysiologic responses to glutamate and N-methyl-D-aspartate (NMDA) and the development of excitotoxicity. Currents were recorded in voltage-clamped neurons in response to the pressure application of glutamate or NMDA. We found that 1-week-old cultures were resistant to excitotoxic effects of 1 or 3 mM glutamate (Freese et al., companion paper). Neurons from cultures of this age often did not exhibit current responses when exposed to NMDA (10 microM or 100 microM). However, most neurons were responsive to 10 microM glutamate. After 12 days in culture the current responses induced by NMDA were more prevalent and shifted toward higher amplitudes. These changes in NMDA current responses coincided temporally with major increases in excitotoxic sensitivity. However, we then found that excitotoxic susceptibility doubled between 12 and 18 days in culture without any parallel increase in glutamate- or NMDA-induced currents. Additionally, neurons which survived 1 mM glutamate exposure for 3 h were found to exhibit glutamate and NMDA responses. These data show that the expression of excitatory amino acid-induced currents is a prerequisite for excitotoxicity, but that additional factors must also contribute to the full maturation of in vitro neuronal vulnerability.

Animals↗
PubMed1990

Extended levodopa release from a subcutaneously implanted polymer matrix in rats.

It is well recognized that plasma fluctuations resulting from oral levodopa therapy may cause an unstable clinical response in parkinsonian patients. We have therefore developed a slow-release polymer matrix system that can deliver levodopa continuously for extended periods of time (at least 225 days) after subcutaneous implantation in rats. Advantages of this approach include (1) the elimination of levodopa plasma fluctuations and (2) the possibility of reducing the required dose due to constant plasma levels and because the gastrointestinal tract is circumvented. The peripheral implantation of polymer systems containing levodopa, dopamine receptor agonists, or other anti-Parkinson agents may constitute a novel technology of drug delivery to improve the care of patients with Parkinson's disease.

Animals↗
PubMed1990

Infection of cultured central nervous system neurons with a defective herpes simplex virus 1 vector results in stable expression of Escherichia coli beta-galactosidase.

We have developed a defective herpes simplex virus (HSV) vector system that permits the introduction of virtually any gene into mammalian central nervous system neurons. The prototype vector, pHSVlac, contains a transcription unit that places the Escherichia coli lacZ gene under the control of the HSV-1 immediate early 4/5 promoter. pHSVlac was propagated using the HSV-1 temperature-sensitive mutant ts K as helper virus. Infection of rat neurons in primary culture derived from various regions throughout the central nervous system, including spinal cord, cerebellum, thalamus, basal ganglia, hippocampus, occipital cortex, temporal cortex, and frontal cortex, resulted in stable expression of high levels of beta-galactosidase for at least 2 weeks, without cell damage. Since other genes can be expressed from pHSVlac, HSV-1 vectors may prove useful for delivery of genes into central nervous system neurons for studies on nervous system physiology or to perform gene therapy for neurological conditions.

Animals↗
PubMed1990

Kynurenine metabolites of tryptophan: implications for neurologic diseases.

Over the past 2 decades, a number of studies have demonstrated that amino acids act as precursors for the biosynthesis of a variety of neuroactive compounds, including catecholamines and indoleamines. For example, the aromatic amino acid L-tryptophan is a precursor for serotonin biosynthesis. Based on this observed precursor relationship, dietary tryptophan supplementation is used to treat a number of neurologic disorders attributed to alterations in serotoninergic neurotransmission. Recent studies have revealed that, in addition to serotonin, a number of neuroactive compounds, the kynurenines, are metabolities of tryptophan. Of these, perhaps the most important is quinolinic acid, a neurotoxin that acts at the N-methyl-D-aspartate (NMDA) receptor and whose precursor responsiveness to tryptophan far exceeds that of serotonin. In the central nervous system, kynurenines, and in particular quinolinic acid, may modulate excitatory amino acid transmission, and may act as neurotoxic agents implicated in the pathogenesis of several neurologic diseases.

Animals↗