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Biomedical subjects

J S Hong

Publications and source records attributed to J S Hong.

At least 37 records · Page 2Linked to original sources
PubMed2000

Effective gene transfer into regenerating sciatic nerves by adenoviral vectors: potentials for gene therapy of peripheral nerve injury.

Replication defective adenoviral vectors have been demonstrated as an effective method for delivering genes into a variety of cell types and tissues both in vivo and in vitro. Transfecting genes into neuronal cells has proven to be difficult because of their lack of cell division. Since the major problem in neurological disease is the degeneration of the terminally differentiated neuronal cells, the adenoviral vector's ability to transfer genes into differentiated post-mitotic cells makes them advantageous for a gene delivery system for the nervous system. Here we showed that a replication defective recombinant adenovirus carrying the lacZ gene could infect the neuronal stem cells and even the differentiated neuronal cells derived from the central nervous system. The lacZ gene delivered into the neuronal cells was expressed efficiently. In addition, the recombinant virus also infected Schwann cells in intact and injured nerves in vivo. The expression of the lacZ gene lasted for 5 weeks, within which nerve regeneration is accomplished in the rat. Adenoviral vectors might thus be used to modulate Schwann cell gene expression for treating peripheral nerve injury or peripheral neuropathy.

Adenoviridae↗
PubMed2000

Time dependency of the action of nitric oxide in lipopolysaccharide-interferon-gamma-induced neuronal cell death in murine primary neuron-glia co-cultures.

We investigated the time-dependency of the action of nitric oxide (NO) on glia-mediated neuronal cell death. Cortical neuron-glia co-cultures were treated with lipopolysaccharide and interferon gamma (LPS/IFNgamma). The production of NO was first detectable 9 h after the exposure to LPS/IFNgamma and increased for up to 48 h. A significant neuronal cell death was observed 36-48 h after treatment with LPS/IFNgamma. The NO generated at the initial stage of NO synthesis (about 12 h) following exposure to LPS/IFNgamma was found to be critical for LPS/IFNgamma-induced neurotoxicity. Furthermore, the rate of NO production at the initial stage of NO synthesis was correlated linearly with the extent of neuronal cell death. These findings suggest that the maximal rate of NO synthesis, instead of the accumulated NO(2)(-) level, is a sensitive index for predicting endotoxin-induced cytotoxicity.

Animals↗
PubMed2000

Immune modulatory effects of neural cell adhesion molecules on lipopolysaccharide-induced nitric oxide production by cultured glia.

Activation of glial cells often occurs at sites of neuronal injury or death and where there is disruption of communication between glia and neurons. We have previously reported that neurons exert an inhibitory influence on LPS-stimulated nitric oxide (NO) production in glial cells. We hypothesized that neural cell adhesion molecules (NCAM) might mediate this inhibitory effect, and this study was designed to elucidate the role of NCAM on lipopolysaccharide (LPS)-induced NO production. We found that soluble NCAMs reduced LPS-stimulated NO production by cultured glia. A monoclonal antibody that recognizes the third immunoglobulin (Ig) domain and can mimic the functions of NCAMs reduced LPS-stimulated NO production, whereas another antibody that binds to other regions of the NCAM did not modulate NO production. Using a 10-amino acid peptide from the third Ig domain of the NCAM, a peptide fragment within the region recognized by the NCAM antibody, mimics the effect of the molecule in reducing NO production. This study demonstrated that NCAMs could modulate LPS-stimulated NO production, most likely via interaction between NCAMs. These results suggest that neuron-glia interactions via NCAMs play an important role in regulating the activities of glial cells in the brain.

Adjuvants, Immunologic↗
PubMed2000

Regional difference in susceptibility to lipopolysaccharide-induced neurotoxicity in the rat brain: role of microglia.

Inflammation in the brain has been increasingly associated with the development of a number of neurological diseases. The hallmark of neuroinflammation is the activation of microglia, the resident brain immune cells. Injection of bacterial endotoxin lipopolysaccharide (LPS) into the hippocampus, cortex, or substantia nigra of adult rats produced neurodegeneration only in the substantia nigra. Although LPS appeared to impact upon mesencephalic neurons in general, an extensive loss of dopaminergic neurons was observed. Analysis of the abundance of microglia revealed that the substantia nigra had the highest density of microglia. When mixed neuron-glia cultures derived from the rat hippocampus, cortex, or mesencephalon were treated with LPS, mesencephalic cultures became sensitive to LPS at a concentration as low as 10 ng/ml and responded in a dose-dependent manner with the production of inflammatory factors and a loss of dopaminergic and other neurons. In contrast, hippocampal or cortical cultures remained insensitive to LPS treatment at concentrations as high as 10 microg/ml. Consistent with in vivo observations, mesencephalic cultures had fourfold to eightfold more microglia than cultures from other regions. The positive correlation between abundance of microglia and sensitivity to LPS-induced neurotoxicity was further supported by the observation that supplementation with enriched microglia derived from mesencephalon or cortex rendered LPS-insensitive cortical neuron-glia cultures sensitive to LPS-induced neurotoxicity. These data indicate that the region-specific differential susceptibility of neurons to LPS is attributable to differences in the number of microglia present within the system and may reflect levels of inflammation-related factors produced by these cells.

Animals↗
PubMed2000

Post-transcriptional inhibition of lipopolysaccharide-induced expression of inducible nitric oxide synthase by Gö6976 in murine microglia.

Glia in the brain respond to various toxins with an increased expression of inducible nitric oxide synthase (iNOS) and an increased production of nitric oxide (NO). Here, we report that lipopolysaccharide (LPS)-induced expression of iNOS was down-regulated post-transcriptionally through the destabilization of iNOS mRNA by the indolocarbazole compound, Gö6976, in murine microglia. This Gö6976 effect is specific for iNOS since tumor necrosis factor alpha was unaffected by the compound. Interestingly, the post-transcriptional effects ascribed to Gö6976 were not observed with other inhibitors of protein kinase A, C (PKC), G, or protein tyrosine kinases. Instead, these kinases appear to affect the iNOS/NO system at the transcriptional level. In the past, Gö6976 has been reported to be a rather specific inhibitor of PKC in vitro. Results from our experiments, through prolonged treatment with phorbol esters and with the various PKC inhibitors including phorbol ester-insensitive PKC isotype inhibitor, suggest that the Gö6976-mediated post-transcriptional regulation of iNOS gene expression and NO production in microglia is not mediated through its reputed effects on PKC activity. Since the effects of various neurotoxins and certain neurodegenerative diseases may be manifested through alterations in the iNOS/NO system, post-transcriptional control of this system may represent a novel strategy for therapeutic intervention.

Animals↗
PubMed2000

The indolocarbazole Gö6976 protects neurons from lipopolysaccharide/interferon-gamma-induced cytotoxicity in murine neuron/glia co-cultures.

The expression of inducible nitric oxide synthase (iNOS) and the production of nitric oxide (NO) after exposure to endotoxins has been implicated in immune-mediated neurotoxicity. The indolocarbazole compound Gö6976, which has been described as a selective protein kinase C (PKC) inhibitor in vitro, rescued neurons from lipopolysaccharide/interferon-gamma (LPS/IFNgamma)- or interleukin-1alpha/tumor necrosis alpha/IFNgamma (IL-1alpha/TNFalpha/IFNgamma)-induced cytotoxicity in murine primary neuron-glia co-cultures. Other compounds known to inhibit PKC, Ro31-8220, GF109203X, Gö7874, H7, staurosporine and H89, failed to rescue neurons from the LPS/IFNgamma-induced cytotoxicity. These results suggest that the neuroprotection by Gö6976 from the LPS/IFNgamma-induced neuronal cell death is not mediated through its reputed effects on PKC activity. The neuroprotection paralleled the inhibition of iNOS gene expression and NO production. However, further analyses correlating NO production with the extent of neurotoxicity suggested that additional mechanism(s) besides the inhibition of the iNOS/NO system may be responsible for the neuroprotective effects of Gö6976. An understanding of the mechanism underlying the neuroprotective effect of Gö6976 may provide key insights into potential interventions for immune-mediated neurodegenerative diseases.

Animals↗
PubMed2000

A novel effect of an opioid receptor antagonist, naloxone, on the production of reactive oxygen species by microglia: a study by electron paramagnetic resonance spectroscopy.

Microglia as the first line of defensive cells in the brain produce free radicals including superoxide and nitric oxide (NO), contributing to neurodegeneration. An opioid receptor antagonist, naloxone, has been considered pharmacologically beneficial to endotoxin shock, experimental cerebral ischemia, and spinal cord injury. However, the mechanisms underlying these beneficial effects of naloxone are still not clear. This study explores the effects of naloxone on the production of superoxide and NO by the murine microglial cell line, BV2, stimulated with lipopolysaccharide (LPS) as measured by electron paramagnetic resonance (EPR). The production of superoxide triggered by phobol-12-myristate-13-acetate (PMA) resulted in superoxide dismutase (SOD)-inhibitable, catalase-uninhibitable 5,5-dimethyl-1-pyrroline N-oxide (DMPO) hydroxyl radical adduct formation. LPS enhanced the production of superoxide and triggered the formation of non-heme iron-nitrosyl complex. Cells pre-treated with naloxone showed significant reduction of superoxide production by 35%. However, it could not significantly reduce the formation of non-heme iron-nitrosyl complex and nitrite. Taken together, the results expand our understanding of the neuroprotective effects of naloxone as it decreases superoxide production by microglia.

Animals↗
PubMed2000

Changes of hippocampal Cu/Zn-superoxide dismutase after kainate treatment in the rat.

In order to evaluate the putative role of Cu,Zn-superoxide dismutase (SOD-1) in the antioxidant defense mechanism during the neurodegenerative process, we examined the level of mRNA, the specific activity and immunocytochemical distribution for SOD-1 in the rat hippocampus after systemic injection of kainic acid (KA). Hippocampal SOD-1 mRNA levels were significantly increased by the seizure intensity 3 and 7 days after KA. These enhanced mRNA levels for SOD-1 were consistent with the increased specific activities for SOD-1, suggesting that the superoxide radical generated in neurotoxic lesion, induced SOD-1 mRNA. The CA1 and CA3 neurons lost their SOD-1-like immunoreactivity, whereas SOD-1-positive glia-like cells mainly proliferated throughout the CA1 sector and had an intense immunoreactivity at 3 and 7 days after KA. This immunocytochemical distribution for SOD-1-positive non-neuronal elements was similar to that for glial fibrillary acidic protein (GFAP)-positive cells. Each immunoreactivity for SOD-1-positive non-neuronal cell or GFAP in the layers of CA1 and CA3 disappeared 3 and 7 days after a maximal stage 5 seizure. On the other hand, activated microglial cells as selectively marked with the lectin occurred in the areas affected by KA-induced lesion. Double-labeling immunocytochemical analysis demonstrated the co-localization of SOD-1-positive glia-like cells and reactive astrocytes as labeled by GFAP or S-100 protein immunoreactivity. This finding suggested that the mobilization of astroglial cells for the synthesis of SOD-1 protein is a response to the KA insult designed to decrease the neurotoxicity induced by oxygen-derived free radicals. Therefore, these alterations might reflect the regulatory role of SOD-1 against oxygen-derived free radical-induced neuronal degeneration after systemic KA administration.

Animals↗
PubMed2000

Influence of neurons on lipopolysaccharide-stimulated production of nitric oxide and tumor necrosis factor-alpha by cultured glia.

Cerebral inflammation often originates in a region where neuronal death occurs and thereafter slowly spreads outward. This study aimed to elucidate the roles of neurons in modulating the production of inflammatory factors stimulated by the bacterial endotoxin lipopolysaccharide (LPS). Culturing neurons with mixed glia reduced nitrite and tumor necrosis factor-alpha (TNF-alpha) production compared to cultures with only mixed glia, and shifted the dose-response curve to the right. The decreased nitrite and TNF-alpha production were not due to the cytotoxicity of LPS. Immunocytochemical analysis of glia-neuron co-cultures revealed the morphological changes in the activated microglia. Culturing PC12 cells with rat mixed-glia also reduced nitrite production. The influence of neurons on glial inflammation was partly due to the cell-cell contacts between neurons and glia via neural cell adhesion molecules (NCAM) because NCAM significantly reduced LPS-stimulated nitrite production. These results demonstrate that neurons reduce the production of inflammatory factors by glia. Since cerebral inflammation is important in many neurological disorders, this study might provide insight about the role of glia-neuron interactions in inflammatory responses in the brain.

Animals↗
PubMed2000

Reduction by naloxone of lipopolysaccharide-induced neurotoxicity in mouse cortical neuron-glia co-cultures.

An inflammatory response in the CNS mediated by activation of microglia is a key event in the early stages of the development of neurodegenerative diseases. Using mouse cortical mixed glia cultures, we have previously demonstrated that the bacterial endotoxin lipopolysaccharide induces the activation of microglia and the production of proinflammatory factors. Naloxone, an opioid receptor antagonist, inhibits the lipopolysaccharide-induced activation of microglia and the production of proinflammatory factors. Using neuron-glia co-cultures, we extended our study to determine if naloxone has a neuroprotective effect against lipopolysaccharide-induced neuronal damage and analysed the underlying mechanism of action for its potential neuroprotective effect. Pretreatment of cultures with naloxone (1 microM) followed by treatment with lipopolysaccharide significantly inhibited the lipopolysaccharide-induced production of nitric oxide and the release of tumor necrosis factor-alpha, and significantly reduced the lipopolysaccharide-induced damage to neurons. More importantly, both naloxone and its opioid-receptor ineffective enantiomer (+)-naloxone were equally effective in inhibiting the lipopolysaccharide-induced generation of proinflammatory factors and the activation of microglia, as well as in the protection of neurons. These results indicate that the neuroprotective effect of naloxone is mediated by its inhibition of microglial activity and may be unrelated to its binding to the classical opioid receptors.

Animals↗
PubMed2000

High concentrations of extracellular potassium enhance bacterial endotoxin lipopolysaccharide-induced neurotoxicity in glia-neuron mixed cultures.

A sudden increase in extracellular potassium ions (K(+)) often occurs in cerebral ischemia and after brain trauma. This increase of extracellular K(+) constitutes the basis for spreading depression across the cerebral cortex, resulting in the expansion of neuronal death after ischemic and traumatic brain injuries. Besides spreading depression, it has become clear that cerebral inflammation also is a key factor contributing to secondary brain injury in acute neurological disorders. Experiments to validate the relationship between elevated levels of extracellular K(+) and inflammation have not been studied. This study aims to elucidate the roles of high concentrations of extracellular K(+) in bacterial endotoxin lipopolysaccharide-induced production of inflammatory factors. Increased concentration of KCl in the medium (20mM) significantly enhanced neurotoxicity by lipopolysaccharide in glia-neuron mixed cultures. To delineate the underlying mechanisms of increased neurotoxicity, the effects of high extracellular K(+) were examined by using mixed glial cultures. KCl at 20mM significantly enhanced nitrite, an index for nitric oxide, production by about twofold, and was pronounced from 24 to 48h, depending on the concentration of KCl. Besides nitric oxide production of tumor necrosis factor-alpha was also enhanced. The augmentative effects of high KCl on the production of inflammatory factors were probably due to the further activation of microglia, since high KCl also enhanced the production of tumor necrosis factor-alpha in microglia-enriched cultures. The increased production of nitrite by high K(+) was eliminated through use of a K(+)-blocker. Taken together, the results show that increases of extracellular K(+) concentrations in spreading depression augment lipopolysaccharide-elicited neurotoxicity, because production of inflammatory factors such as nitric oxide and tumor necrosis factor-alpha are potentiated. Since spreading depression and cerebral inflammation are important in acute neurological disorders, the present results suggest a biochemical mechanism: elevated extracellular K(+) concentrations augment glial inflammatory responses, and thus the neurotoxicity.

Animals↗
PubMed2000

Naloxone protects rat dopaminergic neurons against inflammatory damage through inhibition of microglia activation and superoxide generation.

Degeneration of dopaminergicrgic neurons in the substantia nigra of the brain is a hallmark of Parkinson's disease and inflammation and oxidative stress are closely associated with the pathogenesis of degenerative neurological disorders. Treatment of rat mesencephalic mixed neuron-glia cultures with lipopolysaccharide (LPS)-activated microglia, resident immune cells of the brain, to release proinflammatory and neurotoxic factors tumor necrosis factor-alpha, interleukin-1beta, nitric oxide, and superoxide and subsequently caused damage to midbrain neurons, including dopaminergic neurons. The LPS-induced degeneration of the midbrain neurons was significantly reduced by cotreatment with naloxone, an opioid receptor antagonist. This study focused on understanding the mechanism of action for the protective effect of naloxone on dopaminergic neurons because of relevance to Parkinson's disease. Both naloxone and its opioid receptor inactive stereoisomer (+)-naloxone protected the dopaminergic neurons with equal potency. Naloxone inhibited LPS-induced activation of microglia and release of proinflammatory factors, and inhibition of microglia generation of superoxide free radical best correlated with the neuroprotective effect of naloxone isomers. To further delineate the site of action, naloxone was found to partially inhibit the binding of [(3)H]LPS to cell membranes, whereas it failed to prevent damage to dopaminergic neurons by peroxynitrite, a product of nitric oxide and superoxide. These results suggest that naloxone at least in part interferes with the binding of LPS to cell membranes to inhibit microglia activation and protect dopaminergic neurons as well as other neurons in the midbrain cultures from inflammatory damage.

Animals↗
PubMed2000

Systemic infusion of naloxone reduces degeneration of rat substantia nigral dopaminergic neurons induced by intranigral injection of lipopolysaccharide.

A massive degeneration of dopamine-containing neurons in the substantia nigra (SN) in the midbrain is characteristic of Parkinson's disease. Inflammation in the brain has long been speculated to play a role in the pathogenesis of this neurological disorder. Recently, we reported that treatment of primary rat mesencephalic mixed neuron-glia cultures with lipopolysaccharide (LPS) led to the activation of microglia, resident immune cells of the brain, and subsequent death of dopaminergic neurons. The LPS-induced degeneration of dopaminergic neurons was significantly attenuated by the opiate receptor antagonist (-)-naloxone and its inactive isomer (+)-naloxone, with equal potency, through an inhibition of microglial activation and their production of neurotoxic factors. In this study, injection of LPS into the rat SN led to the activation of microglia and degeneration of dopaminergic neurons: microglial activation was observed as early as 6 h and loss of dopaminergic neurons was detected 3 days after the LPS injection. Furthermore, the LPS-induced loss of dopaminergic neurons in the SN was time- and LPS concentration-dependent. Systemic infusion of either (-)-naloxone or (+)-naloxone inhibited the LPS-induced activation of microglia and significantly reduced the LPS-induced loss of dopaminergic neurons in the SN. These in vivo results combined with our cell culture observations confirmed that naloxone protects dopaminergic neurons against inflammation-mediated degeneration through inhibition of microglial activation and suggest that naloxone would have therapeutic efficacy in the treatment of inflammation-related neurological disorders. In addition, the inflammation-mediated degeneration of dopaminergic neurons in the rat SN resulting from the targeted injection of LPS may serve as a useful model to gain further insights into the pathogenesis of Parkinson's disease.

Animals↗
PubMed1999

Integrating signals from IFN-gamma and IL-4 by B cells: positive and negative effects on CD40 ligand-induced proliferation, survival, and division-linked isotype switching to IgG1, IgE, and IgG2a.

IL-4 and IFN-gamma each have potent effects on B cell responses as well as strong mutual antagonism. Here we have examined the quantitative effects of these cytokines on CD40 ligand-induced B cell proliferation, cell survival, and division-linked isotype switching. Both IL-4 (strongly) and IFN-gamma (weakly) enhanced the number of B cells found in culture by reducing the average time cells take to enter the first division cycle and by promoting B cell survival. When added in combination, the net effect of IL-4 and IFN-gamma on time to division and survival was a response intermediate between that of the two cytokines alone, indicating a partial antagonism of IL-4 by IFN-gamma. By modulating both time to division and cell survival, these small effects of IFN-gamma are amplified and give rise to large reductions in cell number in the presence of IL-4. At higher concentrations, IFN-gamma had minor inhibitory effects on IL-4-induced isotype switching to IgG1 and greater effects on IgE. A reciprocal relation was observed between the ability to inhibit IgE at late cell divisions vs induction of IgG2a. In contrast, IL-4 did not prevent switching to IgG2a induced by IFN-gamma alone. Therefore, antagonism between IFN-gamma and IL-4 is observed at multiple levels and over different concentration ranges, resulting in complex net outcomes. The evolutionary significance of this complexity is discussed.

Adjuvants, Immunologic↗
PubMed1999

Long-term increase of Sp-1 transcription factors in the hippocampus after kainic acid treatment.

Systemic administration of kainic acid (KA), a glutamate receptor agonist, causes robust seizures and has been used as an excellent rodent model for human temporal lobe epilepsy. Recently, we have demonstrated that a single injection of KA increases the steady-state levels of proenkephalin (PENK) mRNA in the rat hippocampus for at least one year. However, the molecular mechanisms underlying this long-term increase in PENK mRNA levels have not been clearly defined. To determine the possible involvement of the Sp-1 transcription factors in this regulation, electrophoresis mobility-shift assays were used to study the expression of Sp-1 factors in the hippocampus after KA treatment. The results showed that there are long-lasting increases in Sp-1 DNA-binding activity. The Sp-1 DNA-binding complexes were only competed by the non-radioactive Sp-1 element and not by ENKCRE2, AP-1 or CRE elements, indicating the specificity of Sp-1 DNA-binding activity. Since the expression of Sp-1 parallels the time course of long-lasting increase in the expression of PENK mRNA and mossy fiber sprouting after KA treatment, we hypothesize that the increase in Sp-1 activity may be associated with the long-term changes in the plasticity of hippocampal function after KA-induced seizures.

Animals↗
PubMed1999

Dextromethorphan modulates the AP-1 DNA-binding activity induced by kainic acid.

We have recently reported that dextromethorphan attenuates the neurotoxicity induced by kainic acid in a dose-related fashion. Pretreatments with dextromethorphan (50 mg/kg, p.o. x2) significantly reduced the activator protein-1 DNA-binding activity and the Fos-related antigen-immunoreactive protein induced by kainic acid (10 mg/kg, i.p.) in the CA1, but not the CA3 or the dentate gyrus sector of the rat hippocampus. Paradoxically, dextromethorphan itself caused an elevated activator protein-1 DNA-binding activity and Fos-related antigen-immunoreactive protein in the CA1 region which lasted for at least 4 days. The results suggest that the CA1 area is the critical site for mediating the putative neuroprotective effect induced by dextromethorphan.

Animals↗
PubMed1999

Heightened transcription for enzymes involved in norepinephrine biosynthesis in the rat locus coeruleus by immobilization stress.

BACKGROUND: The locus coeruleus (LC), a target for CRH neurons, is critically involved in responses to stress. Various physiological stresses increase norepinephrine turnover, tyrosine hydroxylase (TH) enzymatic activity, protein and mRNA levels in LC cell bodies and terminals; however, the effect of stress on other enzymes involved in norepinephrine biosynthesis in the LC is unknown. METHODS: Rats were exposed to single (2 hour) or repeated (2 hour daily) immobilization stress (IMO). Recombinant rat dopamine b-hydroxylase (DBH) cDNA was expressed in E. coli and used to generate antisera for immunohistochemistry and immunoblots in LC. Northern blots were used to assess changes in mRNA levels for TH, DBH, and GTP cyclohydrolase I (GTPCH) in the LC in response to the stress. Conditions were found to isolate nuclei from LC and to use them for run-on assays of transcription. RESULTS: Repeated stress elevated the DBH immunoreactive protein levels in LC. Parallel increases in TH, DBH and GTPCH mRNA levels of about 300% to 400% over control levels were observed with single IMO, and remained at similar levels after repeated IMO. This effect was transcriptionally mediated, and even 30 min of a single IMO significantly increased the relative rate of transcription. CONCLUSIONS: This study is the first to reveal transcriptional activation of the genes encoding catecholamine biosynthetic enzymes in the LC by stress. In addition to TH, changes in DBH and GTPCH gene expression may also contribute to the development of stress-triggered affective disorders.

Analysis of Variance↗