Establishment of a human glioma cell line bearing a homogeneously staining chromosomal region and releasing α- and β-type transforming growth factors

Summary A human glioma cell line (YKG1), which was positively identified for glial fibrillary acidic (GFA) and S-100 proteins, was established from a surgical specimen of a patient with glioblastoma. Chromosome analysis of the cells revealed a homogeneously staining region (HSR) on a marker chromosome. The assay for transforming growth factors (TGFs) in the conditioned medium of the cell line revealed that it contained high levels of - and -type TGFs, which might regulate the growth of glioblastoma and influence on the peritumoral tissues.     

GLAST expression on bullfrog Müller cells is regulated by dark/light

Using immunofluorescence double-labeling, Western blotting and confocal laser scanning microscopy, we investigated if and how the expression of glial high-affinity glutamate/aspartate transporter (GLAST) in bullfrog Müller cells may be regulated by dark/light. Compared with light-adapted retinas, the expression of GLAST in Müller cells was overall up-regulated in retinas dark-adapted for 30 min but declined in retinas dark-adapted for longer (>30 min) periods. The declined expression level of GLAST during prolonged dark adaptation was raised by immersion with 1 mM glutamate. These results suggest that glutamate uptake mediated by GLAST could be regulated dynamically and efficiently in accord with dark/light-induced changes in glutamate release of retinal neurons.     

Expression of basic fibroblast growth factor,nerve growth factor,platelet-derived growth factor and transforming growth factor-β in human brain abscess

We correlated the histopathological findings of six human brain abscesses with the expression of basic fibroblast growth factor (bFGF), nerve growth factor (NGF), platelet-derived growth factor (PDGF) and transforming growth factor-beta (TGF). The clinical courses ranged from 1 month to 1 year and viridans streptoccus was the major pathogen. In early abscesses, we demonstrated strong bFGF and moderate NGF and PDGF immunoreactivities in neutrophils and monocytes/macrophages infiltrating the abscess wall and in the fibrin layer lining the abscess center. In the subacute cases, growth of capillaries and fibroblasts into the fibrin layer and deposition of collagen resulted in the formation of a mesodermal layer between the abscess center and the outer gliotic layer. The proliferative non-neural cells (endothelial cells, fibroblasts and glial cells) expressed mild to strong bFGF, NGF and PDGF immunoreactivities, while strong TGF staining was seen in the extracellular matrix. A loss of growth factor expression and increased fibrosis was seen in the chronic case. These findings suggest that bFGF, NGF, PDGF and TGF produced by the continued influx of leukocytes and by the proliferating non-neural cells may mediate various steps of defense mechanisms and wound healing such as angiogenesis, fibrogenesis and gliosis.Supported by NSC-82-0115-B-006-127 from the National Science Council, Republic of China     

Expression of the enkephalin precursor gene in C6 rat glioma cells: Regulation by β-adrenergic agonists and glucocorticoids

Cultured C6 rat glioma cells contain mRNA coding for preproenkephalin (A), the precursor of methionine- and leucine-enkephalin. The abundance in untreated cells was determined by blot hybridization methods to be 3–6 pg per μg total RNA. Treatment of confluent cells for 12 h with 10 μM (−)-norepinephrine, which activates C6 adenylate cyclase, transiently elevated preproenkephalin mRNA to 3.3 and 7.7 times the control in the absence and presence of the glucocorticoid dexamethasone, respectively. Hydrocortisone and corticosterone also potentiated the effect of norepinephrine. However, glucocorticoids alone did not alter the preproenkephalin mRNA abundance. The effect of norepinephrine + dexamethasone was blocked by the β-adrenergic antagonist propranolol but not by the α-adrenergic antagonist phentolamine. Forskolin, which directly activates adenylate cyclase, similarly elevated the preproenkephalin mRNA abundance; its effect was also potentiated by dexamethasone. C6 cells contain Met-enkephalin-containing protein resembling proenkephalin (apparent M_r 30,000) but little Met-enkephalin, suggesting a low level of proper precursor processing. Treatment with norepinephrine + dexamethasone raised the content of proenkephalin-like protein 11-fold. Thus, preproenkephalin mRNA levels in C6 cells are regulated synergistically by adenosine 3′:5′-cyclic monophosphate and glucocorticoids. These results suggest modes of regulation of proenkephalin biosynthesis in normal rat enkephalinergic cells.     

Antiparkinsonian trophic action of glial cell line-derived neurotrophic factor and transforming growth factor β1 is enhanced after co-infusion in rats

The objective was to analyze functional effects of the combination of GDNF and TGF-β1 in the retrograde model of Parkinsonism in rats, based on the intrastriatal infusion of 6-hydroxydopamine, which leads to protracted and progressive cell death in the substantia nigra. Hemiparkinsonian rats were implanted with osmotic minipumps 2 months after striatal lesion, pumps delivering GDNF alone (10 ng/day), TGF-β1 alone (2 ng/day), or a GDNF and TGF-β1 combination. The findings confirmed that GDNF alone has potent dopaminotrophic effects but they also revealed, for the first time, that GDNF and TGF-β1 co-infusion led to stronger trophic effects relative to the infusion of GDNF alone. TGF-β1 allowed further reducing dopamine receptor hypersensitivity, and potentiated GDNF-mediated effects. This cooperation could be accounted for by the recruitment of GFRα1 on striatal membranes, and by enhanced expression and activation of TH through augmented pSer31TH and pSer40TH. Co-infusion induced striatal sprouting, as revealed by augmentation of p21-Arc, stathmin, and synaptophysin, and led to a reliable recovery of phenotypic expression of TH in surviving nigral neurons. Functional recovery and improvement of TH signal in the nigrostriatal system were long-lasting and sustained, remaining after cessation of trophic infusion.     

Evaluation of serum transforming growth factor β1 and C-reactive protein levels in migraine patients

Background and purposeMigraine is a frequent form of headache. Although many mechanisms describing onset of migraine with and without visual aura have been suggested, the aetiology of migraine headaches is still not clear. Neurogenic inflammation may play a key role in the development of migraine headaches. We evaluated the discriminative power of serum levels of C-reactive protein (CRP) and transforming growth factor beta 1 (TGF-β1) in patients who presented to our clinic with migraine headaches with or without visual aura.Material and methodsWe designed a prospective case-control study of 51 patients with migraine (27 with migraine with aura and 24 with migraine without aura; all had headache) and compared them with 27 healthy subjects during the study period. Demographic and clinical characteristics recorded were age, sex, marital status, occupation, characteristics of headache, laboratory values, and serum CRP and TGF-β1 levels. Statistical analyses used Student t-test, the χ² test, and ANOVA followed by the post-hoc Bonferroni test for multiple comparisons. Receiver operator characteristic (ROC)-curve analysis for CRP and TGF-β1 was also conducted.ResultsThere was no difference between the groups in terms of demographic characteristics, marital status, and socioeconomic status. Statistically, white blood cell levels, serum glucose levels, triglyceride levels, high-density lipoprotein levels, and serum CRP and TGF-β1 were significantly higher in patients with migraine (p < 0.05). The ROC curve results in this study identified that CRP and TGF-β1 may discriminate patients who have different types of migraine headache.ConclusionsThis study suggests that serum CRP and TGF-β1 levels may be diagnostic factors to differentiate migraine patients with and without aura. These findings show that neurogenic inflammation may have a role in the aetiology of migraines.     

Induced secretion of β-hexosaminidase by human brain endothelial cells: A novel approach in Sandhoff disease?

Sandhoff disease is an autosomal recessive lysosomal disorder due to mutations in the β-hexosaminidase β-chain gene, resulting in β-hexosaminidases A (αβ) and B (ββ) deficiency and GM2 ganglioside accumulation in the brain. In this study, our aim was to demonstrate that transduction of cerebral endothelial cells cultured in two-chamber culture inserts with a lentiviral vector encoding the hexosaminidases α and β chains could induce a vectorial secretion of hexosaminidases. Therefore, the human cerebral endothelial cell line hCMEC/D3 was infected with the bicistronic vector from the apical compartment, and β-hexosaminidase activity was measured in transduced cells and in deficient fibroblasts co-cultured in the basal (i.e. brain) compartment. Induced β-hexosaminidase secretion by transduced hCMEC/D3 cells was sufficient to allow for a 70–90% restoration of β-hexosaminidase activity in deficient fibroblasts. On the basis of these in vitro data, we propose that brain endothelium be considered as a novel therapeutic target in Sandhoff disease.     

Benfotiamine prevents increased β-amyloid production in HEK cells induced by high glucose

Objective To determine whether high glucose enhances β-amyloid (Aβ) production in HEK293 Swedish mutant (APPsw) cells with Aβ precursor protein (APP) overexpression, and whether under this condition benfotiamine reduces the increased Aβ production. Methods HEK293 APPsw cells were cultured with different concentrations of glucose for different times. TheAβ content in the supernatant was determined by ELISA. To investigate the mechanism by which benfotiamine reduced Aβ production, glycogen synthase kinase-3 (GSK-3) activity and expression were measured after the cells were cultured with 5.5 g/L glucose for 12 h. Results With 1.0, 3.0, 4.5, 5.5, 6.5, 7.5, 8.5, or 10.5 g/L glucose, Aβ production by HEK293 APPsw cells was highest in the presence of 5.5 g/L glucose for 6 and 12 h. The difference in Aβ content between 5.5 and 1.0 g/L was most marked after incubation for 12 h. Benfotiamine at 20 and 40 μg/mL significantly reduced Aβ production in cells incubated with 5.5 g/L glucose for 12 h. Moreover, 40 μg/mL benfotiamine significantly enhanced the ratio of phosphorylated GSK-3 to total GSK-3, together with consistent down-regulation of GSK-3 activity. Conclusion High glucose increases Aβ production by HEK293 APPsw cells while benfotiamine prevents this increase. This is correlated with the modulation of GSK-3 activity.     

Sodium-dependent glutamate transport in Müller glial cells: regulation by phorbol esters.

The regulation of the Na(+)-dependent high affinity glutamate/aspartate transporter system expressed in cultured Müller glia cells from chick retina was studied. Treatment of the cells with the Ca(2+)/diacylglycerol dependent protein kinase C (PKC) activator, phorbol 12-tetradecanoil-13-acetate (TPA) produced a decrease in [(3)H]D-aspartate uptake which was reversed by staurosporine and partially by H7 [1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochoride], two PKC inhibitors. Long-term treatment with TPA resulted in a drastic decrease in the uptake activity, correlated with a substantial fall in the expression of the transporter protein. These findings suggest that PKC is involved in transport modulation at two different levels: phosphorylation and transporter expression in retinal Müller glial cells.     

Gangliosides inhibit the release of interleukin-1β in amyloid β-protein-treated human monocytic cells

Amyloid-beta protein (A beta) is known to induce microglial activation with concomitant release of cytokines. Gangliosides have documented neuritogenic and neurotrophic properties. We determined the effects of A beta on the release of interleukin-1beta (IL-1beta) from the human monocytic cell line, THP-1 cells. A beta 1-42 significantly induced the release of IL-1beta from the cells. A beta 1-40, A beta 40-1, A beta 1-38, and A beta precursor protein (beta-APP) analogs also released a small amount of IL-1beta. A beta 1-42-activated cells demonstrated approx an 18-fold higher IL-1beta release than that for control cells or A beta 1-40 (soluble; S) treated cells. The release of IL-1beta from A beta 1-42-activated cells was significantly inhibited (33-48% of activated cells; p < 0.05 for the control value) by addition of gangliosides, suggesting that gangliosides inhibit the continuous cycle of the IL-1beta production in THP-1 cells.     

Iron homeostasis in astrocytes and microglia is differentially regulated by TNF-α and TGF-β1

Abnormal iron homeostasis is increasingly thought to contribute to the pathogenesis of several neurodegenerative disorders. We have previously reported impaired iron homeostasis in a mouse model of spinal cord injury and in a mouse model of amyotrophic lateral sclerosis. Both these disorders are associated with CNS inflammation. However, what effect inflammation, and in particular, inflammatory cytokines have on iron homeostasis in CNS glia remains largely unknown. Here we report that the proinflammatory cytokine TNF-α, and the anti-inflammatory cytokine TGF-β1 affect iron homeostasis in astrocytes and microglia in distinct ways. Treatment of astrocytes in vitro with TNF-α induced the expression of the iron importer "divalent iron transporter 1" (DMT1) and suppressed the expression of the iron exporter ferroportin (FPN). However, TGF-β1 had no effect on DMT1 expression but increased the expression of FPN in astrocytes. In microglia, on the other hand, both cytokines caused induction of DMT1 and suppression of FPN expression. Iron influx and efflux assays in vitro confirmed that iron homeostasis in astrocytes and microglia is differentially regulated by these cytokines. In particular, TNF-α caused an increase in iron uptake and retention by both astrocytes and microglia, while TGF-β1 promoted iron efflux from astrocytes but caused iron retention in microglia. These data suggest that these two cytokines, which are expressed in CNS inflammation in injury and disease, can have profound and divergent effects on iron homeostasis in astrocytes and microglia.     

Increase of ryanodine receptors by dopamine D1 receptors is negatively regulated by γ-aminobutyric acid type B receptors in primary cultures of mouse cerebral cortical neurons

Although upregulation of ryanodine receptor (RyR)-1 and -2 is mediated through the activation of dopamine D1 receptors (D1DRs) in the development of psychostimulant-induced place preference, little is known about how such increased expressions of RyRs are negatively regulated. This study investigated negative regulatory mechanisms of increase of RyR-1 and -2 expression by D1DR stimulation with its full agonist, SKF82958 or A 68930, using cultures of mouse cerebral cortical neurons. Sustained exposure to SKF82958 or A 68930 of the neurons increased RyR-1 and -2 proteins in a dose- and time-dependent-manner. The SKF82958-induced increases of RyR-1 and -2 proteins were significantly suppressed by SCH23390 (a selective D1DR antagonist). In addition, the SKF82958- or A 68930-induced increases of RyR-1 and -2 proteins were completely abolished by baclofen (a selective γ-aminobutyric acid type B [GABA(B)] receptor agonist), whereas muscimol (an agonist specific to GABA(A) receptors) had no effect. SKF82958 or A 68930 significantly increased intracellular cAMP level, which was completely suppressed by baclofen. Furthermore, sustained exposure to phorbol 12,13-dibutyrate, a protein kinase C activator, did not change the expression of RyR-1 or -2 proteins. Immunohistochemical study showed colocalizaton of immunoreactivities for three types of proteins, D1DRs and GABA(B) receptor R1 and R2 subunits in the same neuronal bodies, suggesting that the neurochemical changes induced by the activation of D1DRs and GABA(B) receptors occur in the same neurons. These results indicate that RyR-1 and -2 expression facilitated by D1DR stimulation are negatively regulated by GABA(B) receptor via suppression of cAMP production.     

Effect on peripheral nerve regeneration by transgene in vivo with human insulin-like growth factor-1

INTRODUCTION In 1950s, the development of microsurgery have greatly improved the quality in repairing peripheral nerve injury. Especially that more than 10 neurotrophic factors (NTFs) have been known in recent 30 years with the continuous exploration of t…     

Induction of glutathione S-transferase,placental type in T9 glioma cells by dibutyryladenosine 3′,5′-cyclic monophosphate and modification of its expression by naturally occurring isothiocyanates

Summary The effect of dibutyryladenosine 3,5-cyclic monophosphate (dibutyryl cAMP) on the expression of glutathione S-transferase placental type (GST-P) was examined in rat glioma cell line using an immunohistochemical technique. Cultured T9 glioma cells were negative for GST-P activity under normal conditions. However, treatment with 1 mM dibutyryl cAMP produced GST-P expression in about 50% of the cells, as well as some morphological changes. The expression of GST-P was increased with addition of dibutyryl cAMP together with 1 g/ml allyl isothiocyanate (AITC) or 0.1 g/ml benzyl isothiocyanate (BITC). With these combinated treatments, almost all cultured cells showed a strong positive reaction for GST-P, although AITC or BITC alone elicited GST-P in only 5% of the cultured cells. The results of the present study indicate that dibutyryl cAMP causes functional as well as morphological differentiation of T9 glioma cells.     

Effect on peripheral nerve vivo with human insulin-like regeneration by transgene in growth factor-1

BACKGROUND: Human insulin-like growth factor (hIGF-1) has been successful in treating peripheral nerve injury, but it is still unclear whether hIGF-1 after transgene in vivo has the effect on promoting the regeneration of peripheral nerve. OBJECTIVE: To observe the effect of hIGF-1 on the regeneration of peripheral nerve by transgene in vivo with electrophysiology, histological morphology and ultromicro morphology. DESIGN: A univariate design. SETTINGS: Jilin Institute of Surgery, China-Japan Friendship Hospital Affiliated to Jilin University; School of Basic Medical Sciences, Jilin University. MATERIALS: Thirty male adult Wistar rats of grade Ⅱ, weighing 200-250 g, were provided by the Animal Experimental Center of Jilin University [certification number: SCXK-(Ji)20030001]. The rats were raised in the environment at the temperature of 25 ℃ and humidity of 70%. All the rats were randomly divided into hIGF-1-treated group, treatment control group and blank control group, 10 rats in each group. Positive liposomes (mass concentration of 2 g/L) and pcDNA3.1 (mass concentration of 1 g/L) were purchased from Beijing Yuanpinghao Company; pcDNAhIGF-1 (mass concentration of 1 g/L) was provided by Dr. Shen from the School of Public Health of Jilin University. The liposomes were mixed with plasmids with the mass ratio of 1.5 to 10.Operative microscope was made by Jiangsu Zhenjiang Microsurgical Instrument Factory; EMB-5304K electromyogram (EMG) evoked potential meter by Nihon Kohden Corporation. HPIAS-1 000 high-acuity color pathological imaging analytical system (Japan) and JEM-1200EX transmission electron microscope (Japan) were also used. METHODS: The experiments were carried out in Jilin Institute of Surgery from April to June in 2004. ① All the rats were anesthetized, and the right sciatic nerve was exposed, and it was clipped with a clip at 5 mm below the piriform muscle for 3 times, 10 s for each time. The pressed width was 3 mm, and formed as membrane under operating microscope (×6). Rats in the hIGF-1-treated group were subepineurially injected with the mixture of pcDNAhIGF-1 and positive liposomes (10 μL) immediately, those in the treatment control group were injected with the mixture of pcDNA3.1, positive liposomes and distilled water (10 μL), and those in the blank control group were not given any injection. ② The sciatic nerve functional indexes (SFI) were measured within 56 days postoperatively according to the methods used by Shen et al. ISFI=0 was taken as normal, and ISFI=-100 as completely damaged. EMG evoked potential meter was used to record the electrophysiological changes of the regenerated nerve fibers. The indexes of histological morphology in 5 randomly selected sights were determined with the color pathological imaging analytical system, and the ultrostructures of the regenerated nerve fibers were also observed. MAIN OUTCOME MEASURES: ① Comparison of the SFI within 56 days postoperatively; ② Comparison of the electrophysiology, histological morphology and ultrastructure of the regenerated nerve fibers 56 days postoperatively. RESULTS: All the 30 Wistar rats were involved in the analysis of results. ① SFI: The SFI values were gradually increased as time prolonged in all the three groups, and the changes were more obvious after 24 days, the SFI values recovered better at each time point in the hIGF-1-treated group than in the other two groups. ② Eelectrophysiological results of right sciatic nerve: The latency of motor evoked potential (MEP) was close between the treatment control group and the blank control group [(2.55±0.36), (2.65±0.55) ms, P > 0.05], but higher in the hIGF-1-treated group [(2.14±0.22) ms] than in the blank control group (P < 0.01). The amplitude and conduction velocity of MEP in the treatment control group [(6.67±0.69) mV, (29.57±4.06) m/s] were close to those in the blank control group [(6.60±0.59) mV, (29.22±3.20) m/s, P > 0.05], but those in the hIGF-1-treated group [(7.81±0.84) mV, (36.91±4.37) m/s] were larger or faster than those in the blank control group (P < 0.01). ③ Results of the pathological image analysis of the regenerated nerve fibers: The axonal diameter, thickness of myelin sheath of the regenerated nerve fiber and the number of myelinated nerve fiber in the treatment control group [(2.28±0.33) μm, (1.08±0.18) μm2, (71.80±8.25) fibers] were close to those in the blank control group [(2.18±0.29) μm, (1.03±0.15) μm2, (68.60±8.55) fibers] (P > 0.05), and those in the hIGF-1-treated group [(3.03±0.35) μm, (1.65±0.24) μm2, (88.20±8.82) fibers] were obviously larger or more than those in the blank control group (P < 0.01). ④ Ultrastructure of the regenerated nerve fibers of sciatic nerve: In the hIGF-1-treated group, the regenerated fibers of sciatic nerve were more and mature, manifested by thicker nerve fibers, thicker and evener myelin sheath, which were better than those in the other two groups. CONCLUSION: The results of the quantitative parameters of the electrophysiology, gross histological morphology and ultrostructural changes in the process of repairing damaged peripheral nerve indicate that transgene in vivo with hIGF-1 can promote the neural regeneration after peripheral nerve injury.     

Glial-derived transforming growth factor β1(TGF-β1):a key factor in multiple sclerosis neuroinflammation

正Glial cell activation and neuroinflammation in multiple sclerosis (MS):MS is an irreversible and progressive central nervous system (CNS) disease which originates in the autoimmune attack of lymphocytes against CNS myelin.This specialized membrane,synthesized by oligodendrocytes (OL) in the CNS,provides metabolic support to axons and allows for     

How is firing activity of substantia nigra cells regulated? Relevance of pattern‐code in the basal ganglia

The current model of the basal ganglia (BG) assumes that neurons use a firing rate renewal code for movement computing under normal and pathological conditions. Here, we report nonrenewal firing (neuronal firing is influenced by its own previous activity) in cells of the anesthetized rat's substantia nigra (SN). Both compensatory (short interspike intervals (ISIs) are followed by long ISIs and vice versa) and persistent (short and long ISIs cluster for long time periods) nonrenewal activity was found in 52.6% and 33.8% of SN cells, respectively. A compensatory pattern was found in 77.7% of DA cells, but in only 9.8% of GABA-cells. Conversely, a persistent pattern was observed in 74.6% of GABAergic cells and in only 9.9% of DA cells. These findings indicate two types of nonrenewal firing pattern codes specifically present in SN dopaminergic and GABAergic neurons. Disruption of these patterns may play a role in the pathophysiology of basal ganglia disorders such as Parkinson's disease and dyskinesias.