The cost of delaying treatment in multiple sclerosis: what is lost is not regained.
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Humans↗
Biomedical subjects
C T Bever
Publications and source records attributed to C T Bever.
Glatiramer acetate blocks interleukin-1-dependent nuclear factor-kappaB activation and RANTES expression in human U-251 MG astroglial cells.
RANTES is a basic 8-kDa polypeptide of the C-C chemokine subfamily with strong chemoattractant activity for T lymphocytes and monocytes/macrophages that are implicated in the pathogenesis of multiple sclerosis (MS) lesions. Glatiramer acetate is a drug recently approved for the treatment of MS. We therefore investigated the effect of glatiramer acetate on RANTES expression in glial cells in vitro. Treatment of human U-251 MG astroglial cells with glatiramer acetate blocks IL-1beta-induced RANTES chemokine production in a dose- and time-dependent manner. Glatiramer acetate also decreased steady-state levels of RANTES mRNA in these cells, which was attributable to reduced transcription, as assessed by nuclear run-on assays. In addition, we showed that NF-kappaB may be the transcriptional activator responsible for the IL-1beta-mediated RANTES gene expression in this system. Our data indicated that the IL-1beta-induced increase in RANTES was associated with an increase in in vitro nuclear extract binding activity specific for the NF-kappaB site in the promoter region of the RANTES gene. The increases in RANTES mRNA and protein expression were suppressed by the NF-kappaB inhibitors gliotoxin, isohelenin, and pyrrolidine dithiocarbamate (PDTC). Furthermore, we demonstrated that the increase in NF-kappaB DNA-binding activity was prevented by pretreatment with glatiramer acetate or the NF-kappaB inhibitors. Our results suggest that glatiramer acetate may inhibit IL-1beta-stimulated RANTES expression in human glial cells by blocking NF-kappaB activation, thus identifying part of the molecular basis for its anti-inflammatory and immunosuppressive effects in demyelinating diseases.
Astrocytes↗
Th1 cytokines stimulate RANTES chemokine secretion by human astroglial cells depending on de novo transcription.
Beta-chemokines induce the directional migration of monocytes and T lymphocytes that are implicated in the pathogenesis of multiple sclerosis (MS) lesions. RANTES is a member of the beta-chemokine family that has been detected in the lesions of MS patients. However, the cellular sources of RANTES message and the molecular basis for the regulation of its production in MS lesions are not well understood. Glial cells may be a major source of RANTES in vivo and have been shown to produce RANTES in vitro. Thus, the objective of this study was to establish a model system for studying the regulation of RANTES expression by cytokines in cultured human glial cells, and to determine the mechanism involved in the process. We show that the Th1 cytokines TNF-alpha and IL-1beta independently induce RANTES mRNA and chemokine levels in human U-251 MG astroglial cells, and that these effects are time- and concentration-dependent. In addition, we demonstrate that both cytokines increased the rate of transcription of the RANTES gene, as estimated by in vitro nuclear transcript elongation assays. The transcriptional activity in TNF-alpha-treated U-251 MG cells started to increase at 2 h and peaked at 8 h, with levels more than 14 times greater than controls. We further show that NF-kappaB may play a critical role in the up-regulation of human RANTES gene expression in this system. Gel shift assays revealed an induction of in vitro nuclear extract binding activity to the NF-kappaB element of RANTES in cells incubated with the Th1 cytokines. These observations suggest that human astroglia, within diseased brain, may be stimulated to produce RANTES chemokine in response to TNF-alpha and IL-1beta, and that this effect of the Th1 cytokines is attributed to increase of transcription.
Astrocytes↗
Glatiramer acetate inhibition of tumor necrosis factor-alpha-induced RANTES expression and release from U-251 MG human astrocytic cells.
Glatiramer acetate is an approved drug for the treatment of multiple sclerosis (MS). RANTES is a beta-family chemokine that manifests chemoattractant activity for T lymphocytes and monocytes/macrophages implicated in the pathogenesis of MS lesions. However, the effect of glatiramer acetate on the regulation of RANTES secretion in glial cells is unknown. In the present study, we demonstrate for the first time that treatment of human U-251 MG astrocytic cells with glatiramer acetate blocks tumor necrosis factor-alpha (TNF-alpha)-induced RANTES mRNA and protein in a dose- and time-dependent manner. This effect is attributed to inhibition of transcription and a 40% decrease in transcript stability. Furthermore, our electrophoretic mobility shift assays of nuclear extracts from TNF-alpha-treated cells reveal an increase in DNA-binding activity specific for the nuclear factor-kappa B (NF-kappaB) binding site, in the 5'-flanking promoter region of the human RANTES gene, and that this increase in NF-kappaB binding activity is prevented by pretreatment with glatiramer acetate or the NF-kappaB inhibitors. These findings suggest that glatiramer acetate may exert its therapeutic effect in MS partially through inhibiting NF-kappaB activation and chemokine production.
Astrocytes↗
Induction of RANTES chemokine expression in human astrocytic cells is dependent upon activation of NF-kappaB transcription factor.
RANTES is a C-C (beta)-family chemokine that is implicated in the migration of peripheral blood leukocytes to brain lesions in multiple sclerosis (MS), an inflammatory demyelinating disease of the central nervous system (CNS). Glial cells are active participants in the inflammatory response in the CNS, and they have been shown to respond to and produce a number of cytokines and chemokines in vivo and in vitro. Recently, we have shown inducibility of RANTES gene expression by TNF-alpha in human astrocytic cells. Therefore, the goal of the current study was to investigate the transcription activating factor involved in the process. We found that the induction of RANTES mRNA and protein by TNF-alpha in human astrocytic cells is associated with increased NF-kappaB DNA-binding activity. p65 and p50 were determined to be the components of the activated NF-kappaB transcription factor complex by supershift assay. In addition, the blockade of NF-kappaB activation by three known NF-kappaB inhibitors markedly reduced the TNF-alpha-induced RANTES expression at the mRNA and protein levels. Furthermore, the reduction in NF-kappaB binding activity to the promoter of the human RANTES gene caused by the NF-kappaB inhibitors parallels a decrease in RANTES expression in these cells. Our data suggest that NF-kappaB may mediate the induction of RANTES gene expression, in human glial cells, through its cognate cis-acting element.
Astrocytes↗
Comparison of RANTES chemokine induction by Th1 cytokines in human astroglial cell lines.
Multiple sclerosis (MS) is a primary inflammatory demyelinating disease of the human central nervous system, characterized by accumulation of mononuclear cells of hematogenous origin. RANTES is a C-C (beta)-chemokine family member with strong chemoattractant activity for lymphocytes and monocytes that are implicated in the pathogenesis of MS lesions. However, the cellular sources of RANTES message and the regulation of its secretion within diseased brain are poorly understood. Therefore, we carried out this study to compare the effect of Th1 cytokines on the induction of RANTES in different human astrocytic cell lines. IFN-gamma alone had little effect on RANTES production in both U-373MG and U-105MG cells, while TNF-alpha or IL-1beta alone had differential effects in the two cell lines. Low levels of RANTES chemokine were detected in culture supernatants from U-373MG cells. By contrast, TNF-alpha or IL-1beta dramatically increased RANTES secretion in U-105MG cells. Interestingly, different combination treatments of cells with the three cytokines synergistically induced RANTES release from both U-373MG and U-105MG cells. Consistent with these results, we found similar expression patterns for RANTES at the comparable steady-state mRNA levels in both cell lines. Furthermore, we showed that U-105MG cells treated with TNF-alpha and IL-1beta alone or in combination markedly induced increases in the rate of transcription of the RANTES chemokine gene. Our results indicate that these cell lines may be good model systems for studying the regulation of RANTES expression by cytokines in human glial cells.
Astrocytes↗
Mechanisms underlying the synergistic effect of Th1 cytokines on RANTES chemokine production by human glial cells.
RANTES is a beta-family chemokine with potent chemoattractant activity for lymphocytes and monocytes that are implicated in the pathogenesis of multiple sclerosis (MS) lesions. Glial cells have been shown to produce RANTES in response to stimulation with Th1 cytokines (IFN-gamma, TNF-alpha, and IL-1beta) in vitro, and they may be a major source of RANTES production within diseased brain. This study was undertaken to investigate the mechanism underlying the effect of the Th1 cytokines on the induction of RANTES in a model system for human astroglia. We show that IFN-gamma has a synergistic effect with TNF-alpha or IL-1beta on RANTES mRNA and chemokine production in this system. We further show that the combination treatment of IFN-gamma and TNF-alpha, or IFN-gamma and IL-1beta induced 3-fold higher levels of RANTES gene transcription than seen with either TNF-alpha or IL-1beta alone, as measured by in vitro nuclear transcript elongation assays. In addition, we found that IFN-gamma decreased the rate of degradation of RANTES mRNA caused by TNF-alpha or IL-1beta. The t(1/2) of RANTES mRNA was 25+/-1 h in the presence of both IFN-gamma and TNF-alpha, as compared to a t(1/2) of 15+/-1 h in the presence of TNF-alpha alone. This 10 h difference represents an approximate 70% increment in RANTES mRNA half-life. Thus, these results suggest that both increased RANTES gene transcription and increased RANTES mRNA stability may account for the synergistic effect of Th1 cytokines on the up-regulation of RANTES expression in human astroglial cells.
Astrocytoma↗
Matrix metalloproteinases in multiple sclerosis: targets of therapy or markers of injury?
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Adjuvants, Immunologic↗
Inactivation gating and 4-AP sensitivity in human brain Kv1.4 potassium channel.
Voltage-gated K(+) channels vary in sensitivity to block by 4-aminopyridine (4-AP) over a 1000-fold range. Most K(+) channel phenotypes with leucine at the fourth position (L4) in the leucine heptad repeat region, spanning the S4-S5 linker, exhibit low 4-AP sensitivity, while channels with phenylalanine exhibit high sensitivity. Mutational analysis on delayed rectifier type K(+) channels demonstrate increased 4-AP sensitivity upon mutation of the L4 heptad leucine to phenylalanine. This mutation can also influence inactivation gating, which is known to compete with 4-AP in rapidly inactivating A-type K(+) channels. Here, in a rapidly inactivating human brain Kv1.4 channel, we demonstrate a 400-fold increase in 4-AP sensitivity following substitution of L4 with phenylalanine. Accompanying this mutation is a slowing of inactivation, an acceleration of deactivation, and depolarizing shifts in the voltage dependence of activation and steady-state inactivation. To test the relative role of fast inactivation in modulating 4-AP block, N-terminal deletions of the fast inactivation gate were carried out in both channels. These deletions produced no change in 4-AP sensitivity in the mutant channel and approximately a six-fold increase in the wild type channel. These results support the view that changes at L4 which increase 4-AP sensitivity are largely due to 4-AP binding and may, in part, arise from alterations in channel conformation. Primarily, this study demonstrates that the fast inactivation gate is not a critical determinant of 4-AP sensitivity in Kv1.4 channels.
4-Aminopyridine↗
Multiple Sclerosis: Symptomatic Treatment.
Therapy for multiple sclerosis (MS) that prevents exacerbation of the disease and slows the progression of disability has not diminished the importance of treating symptoms. Because the new agents are not curative and rarely reverse existing deficits, many patients under treatment have or will have persistent symptoms. Many neurologic symptoms are seen in patients with MS, but it is important to recognize that some nonneurologic symptoms, such as pain, fatigue, and mood disturbance, are common and may cause significant disability. The first and most important step in the management of symptoms is to discuss the symptoms with the patient on an ongoing basis. The second step is to recognize treatable symptoms and to apply the appropriate strategies for management. There have been promising results with experimental agents, primarily potassium channel blockers, that may improve function in demyelin-ated fiber pathways and that offer the possibility of treatment for a range of symptoms. At present, the management of symptoms varies, depending on the symptom, and it involves the coordinated application of a range of treatment approaches including medication, lifestyle changes, rehabilitation, and, in some cases, surgery.
Journal Article↗
Glatiramer acetate blocks the activation of THP-1 cells by interferon-gamma.
Glatiramer acetate (previously known as copolymer 1) is a synthetic copolymer of four amino acids that has been approved for use in the treatment of multiple sclerosis. It has been shown to suppress myelin antigen specific T cell activation by competing with these antigens at the major histocompatibility complex class II binding site and by inducing antigen specific suppressor T cells. In this study we investigated the effects of glatiramer acetate on the human monocytic cell line, THP-1, activated by lipopolysaccharide and interferon-gamma as a model for macrophages. At non-toxic concentrations of glatiramer acetate there were dose dependent reductions in the percentage of cells expressing human leukocyte DR and DQ antigen as well as in mean fluorescence intensity by flow cytometry. Production of tumor necrosis factor-alpha and the lysosomal cysteine proteinase cathepsin B were markedly inhibited, but production of interleukin-1 increased. These results suggest that glatiramer acetate might alter macrophage effector function and suggest that further studies in human monocytes and macrophages are warranted.
Cathepsin B↗
Modulation of interferon gamma induced increases in cathepsin B in THP-1 cells by adrenergic agonists and antagonists.
In order to investigate the possible modulation of macrophage function by the autonomic nervous system, the effect of adrenergic agonists and antagonists on interferon (IFN)-gamma-induced increases in cathepsin B (CB) in a macrophage-like cell line was studied. It has been shown previously that IFN-gamma induces increased CB activity in phorbol myristate acetate (PMA)-primed THP-1 cells. Isoproterenol (ISO) (10 micrometers), a mixed beta-receptor agonist, increased the induction of CB activity in the cells but norepinephrine (10 micrometers) and epinephrine (10 micrometers), the alpha and beta receptor agonists, had little effect. The addition of the mixed alpha-receptor antagonist phentolamine (10 micrometers) had no effect on ISO induced increases but the mixed beta-receptor antagonist propranolol (10 micrometers) and the selective beta1-receptor antagonist atenolol produced significant inhibition. These results suggest that the activation of beta-receptors could be involved in the induction of CB activity in macrophages and provide a possible mechanism for the regulation of macrophage effector function by the autonomic nervous system. Dibutyryl cAMP (1 mm) alone also induced increases in CB in THP-1 cells, and H-89 or HA1004 abrogated the effect of dibutyryl cAMP, suggesting that the effect of ISO on CB could be through the elevation of cAMP and the activation of cAMP-dependent protein kinases.
Adrenergic Agonists↗
Effects of IFN-beta on human cerebral blood flow distribution.
The effect of interferon-beta1b (IFN-beta) on human cerebral blood flow distribution was examined in five multiple sclerosis patients using functional brain single-photon emission tomography (SPECT). Of nine regions of interest studied, only the basal ganglia exhibited a significant change (increase) in relative photon emission intensity (i.e., relative blood flow) when comparing SPECT scans obtained 6 h after s.c. IFN-beta injection with scans obtained at the same time of day (noon) 30 h after IFN-beta injection (IFN-beta-free day). The increase in relative blood flow to the basal ganglia following IFN-beta injection correlated positively with changes in mean arterial pressure (MAP). Additional studies will be required to determine the relevance of these observations for IFN-beta-induced central nervous system side effects.
Adult↗
Effect of propranolol and IFN-beta on the induction of MHC class II expression and cytokine production by IFN-gamma IN THP-1 human monocytic cells.
This study was undertaken to investigate the effects of propranolol, IFN-beta, and the protein kinase modulators on IFN-gamma induction of MHC class II antigen expression and cytokine production in THP-1 human monocytic cells. IFN-gamma induced expression of HLA-DR and DQ molecules and secretion of the monokines IL-1 beta and TNF-alpha in THP-1 cells in a time and dose-dependent manner. The effect of INF-gamma on class II HLA antigens was dose-dependently inhibited by IFN-beta. H-7, phloretin, staurosporine as well as GF 109203X are selective enzyme inhibitors of protein kinase C (PKC), down-regulating IFN-gamma induced MHC class II expression and cytokine production. Stimulators of PKC, like PMA, replaced IFN-gamma in the induction of monokines in THP-1 cells, whereas the addition of HA 1004 or arachidonic acid to the culture had no effect on IFN-gamma mediated changes. Blocking of phospholipase D (PLD)-derived diacylglycerol (DAG) formation by propranolol abrogated IFN-gamma increased HLA class II expression and IL-1 beta secretion, but had little effect on IFN-gamma induced TNF-alpha production. These findings appear to suggest that PLD-derived phosphatidate is not the primary source of DAG production in IFN-gamma-induced TNF-alpha secretion, but may be necessary for IFN-gamma-mediated MHC class II induction and IL-1 beta production in human monocytes, whereas phospholipase A2 may not be required for IFN-gamma activation of PKC in the process.
1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
Neurologic symptoms following Pfiesteria exposure: case report and literature review.
Although the recently identified dinoflagellate, Pfiesteria piscicida, may have neurotoxic effects on humans, the precise nature of the neurologic symptoms associated with varying levels of exposure is unknown. Toward this end, we review the neurologic symptoms of three Pfiesteria-exposed laboratory workers reported to data and compare them to the evaluation of an exposed waterman from Maryland. The occupational exposure of a Maryland waterman appears to produce a mild, reversible encephalopathy which predominantly affects functions associated with the frontal and temporal lobes. A comprehensive neurologic examination is recommended for all Pfiesteria piscicida and morphologically related organism-exposed, symptomatic persons.
Adult↗
Interferon-gamma induced increases in intracellular cathepsin B activity in THP-1 cells are dependent on RNA transcription.
Interferon-gamma (IFN-gamma) treatment of human macrophages induces increased intracellular levels of cathepsin B (CB), a lysosomal cysteine proteinase which is implicated in inflammatory tissue injury. To determine the mechanism of the increase, we studied the macrophage-like cell line, THP-1. Dose and time dependent increases in intracellular CB were seen when cells primed with phorbol ester (PMA) were cultured with IFN-gamma. To determine whether protein synthesis was required for the increase, PMA primed cells were cultured in the presence of IFN-gamma and cycloheximide: The expected increase was inhibited. To determine whether RNA synthesis was required for the IFN-gamma induced increases, PMA primed cells were cultured in the presence of IFN-gamma and actinomycin D. Again the expected increases were not seen. Direct measurement of CB mRNA levels showed increases in cells not treated with inhibitors. These results suggest that the IFN-gamma induced increases in THP-1 cell CB are dependent on RNA and protein synthesis.
Cathepsin B↗
Endotoxin induces increased intracellular cathepsin B activity in THP-1 cells.
Macrophage cathepsin B (CB) is implicated in tissue injury in inflammatory diseases. Lipopolysaccharide (LPS) is an activator of macrophages whose effect on CB is unknown. This study was undertaken to investigate the potential of macrophages as a source of increased CB and to determine if exposure to LPS might stimulate CB levels. As a model we chose the macrophage-like tumor line, THP-1. Incubation with LPS led to a time and dose-dependent increase in CB activity. LPS potentiated interferon-gamma (IFN-gamma)-induced elevations of CB and led to an additive increase in CB activity. Pretreatment of the cells with LPS not only caused a marked stimulation of CB activity over that seen with IFN-gamma alone, but also decreased the concentration and exposure time to the cytokine necessary to achieve maximum induction of the enzyme. The LPS and IFN-gamma induced CB increases were abolished by cycloheximide or actinomycin D in the cultures, indicating that the increases in CB required increased RNA transcription and de novo protein synthesis. Direct measurement of CB mRNA showed increases. These data indicate that although LPS alone appears to induce the production of CB in THP-1 cells, it augments IFN-gamma induced increases, suggesting that two signals are necessary for maximum CB induction in this system.
Cathepsin B↗
Interferon beta-1b serum levels in multiple sclerosis patients following subcutaneous administration.
Recombinant interferon beta-1b (rIFNbeta) reduces the frequency of exacerbations in relapsing-remitting MS when administered subcutaneously on alternate days. However, the pharmacokinetics of rIFNbeta are not well understood and there are scant data on the detectability of rIFNbeta in the serum of MS patients following subcutaneous administration. Moreover, existing assays for detecting IFNbeta are biologic, time-consuming, and require handling of infectious agents. We developed and standardized an ELISA specific for measuring rIFNbeta with a detection range of 40 to 1,000 IU/ml. The specificity of our ELISA was confirmed by the lack of cross-reactivity with other cytokines, including IFNalpha, IFNgamma, IFN Consensus-1, and TNFalpha. We screened serum from 34 MS patients drawn within 12-36 hours of treatment: 15 patients taking 8 MIU, four patients taking 1.6 MIU, and 15 patients taking placebo. Eleven of the 15 patients in the 8-MIU treatment group had measurable rIFNbeta serum levels ranging from 120 to 475 MIU/ml. Two of four patients in the 1.6-MIU treatment group, but none of the placebo group, had detectable serum rIFNbeta levels. A small prospective time-course study was carried out in four MS patients receiving rIFNbeta. Serial blood samples were obtained prior to and 4, 8, 24, and 48 hours after rIFNbeta injection. A peak serum rIFNbeta level was observed between 8 and 24 hours after rIFNbeta injection and tended to decline to near preinjection levels at 48 hours postinjection. These results are consistent with the rationale of alternate-day, subcutaneous administration of rIFN beta. In addition, the ELISA described might be a useful tool to study the pharmacokinetics and the relationship of rIFN beta serum levels to clinical efficacy.
Animals↗