Growth Hormone Improves Cognitive Function After Experimental Stroke.

BACKGROUND AND PURPOSE: Cognitive impairment is a common outcome for stroke survivors. Growth hormone (GH) could represent a potential therapeutic option as this peptide hormone has been shown to improve cognition in various clinical conditions. In this study, we evaluated the effects of peripheral administration of GH at 48 hours poststroke for 28 days on cognitive function and the underlying mechanisms. METHODS: Experimental stroke was induced by photothrombotic occlusion in young adult mice. We assessed the associative memory cognitive domain using mouse touchscreen platform for paired-associate learning task. We also evaluated neural tissue loss, neurotrophic factors, and markers of neuroplasticity and cerebrovascular remodeling using biochemical and histology analyses. RESULTS: Our results show that GH-treated stroked mice made a significant improvement on the paired-associate learning task relative to non-GH-treated mice at the end of the study. Furthermore, we observed reduction of neural tissue loss in GH-treated stroked mice. We identified that GH treatment resulted in significantly higher levels of neurotrophic factors (IGF-1 [insulin-like growth factor-1] and VEGF [vascular endothelial growth factor]) in both the circulatory and peri-infarct regions. GH treatment in stroked mice not only promoted protein levels and density of presynaptic marker (SYN-1 [synapsin-1]) and marker of myelination (MBP [myelin basic protein]) but also increased the density and area coverage of 2 major vasculature markers (CD31 and collagen-IV), within the peri-infarct region. CONCLUSIONS: These findings provide compelling preclinical evidence for the usage of GH as a potential therapeutic tool in the recovery phase of patients after stroke.

Characterization of White Matter Injury in a Rat Model of Chronic Cerebral Hypoperfusion.

BACKGROUND AND PURPOSE: Chronic cerebral hypoperfusion can lead to ischemic white matter injury resulting in vascular dementia. To characterize white matter injury in vascular dementia, we investigated disintegration of diverse white matter components using a rat model of chronic cerebral hypoperfusion. METHODS: Chronic cerebral hypoperfusion was modeled in Wistar rats by permanent occlusion of the bilateral common carotid arteries. We performed cognitive behavioral tests, including the water maze task, odor discrimination task, and novel object test; histological investigation of neuroinflammation, oligodendrocytes, myelin basic protein, and nodal or paranodal proteins at the nodes of Ranvier; and serial diffusion tensor imaging. Cilostazol was administered to protect against white matter injury. RESULTS: Diverse cognitive impairments were induced by chronic cerebral hypoperfusion. Disintegration of white matter was characterized by neuroinflammation, loss of oligodendrocytes, attenuation of myelin density, structural derangement at the nodes of Ranvier, and disintegration of white matter tracts. Cilostazol protected against cognitive impairments and white matter disintegration. CONCLUSIONS: White matter injury induced by chronic cerebral hypoperfusion can be characterized by disintegration of diverse white matter components. Cilostazol might be a therapeutic strategy against white matter disintegration in patients with vascular dementia.

Zinc Protoporphyrin Attenuates White Matter Injury after Intracerebral Hemorrhage.

Intracerebral hemorrhage (ICH)-induced white matter injury has not been well studied. The objective of this study was to examine the effect of zinc protoporphyrin (ZnPP) on white matter injury induced by ICH. This study was divided into two parts. In the first part, rats received either a needle insertion (sham) or 100 µl autologous blood into the right basal ganglia. The rats were euthanized at 1, 3, 7, 14, or 28 days later for myelin basic protein (MBP) measurement. In the second part, rats had intracerebral infusion of 100 µl autologous blood, and an intraperitoneal osmotic mini-pump was implanted immediately after ICH to deliver vehicle or ZnPP (1 nmol/h), a heme oxygenase inhibitor, for up to 14 days. Rats were euthanized at day 28 for MBP staining. The number of MBP-labeled fiber bundles and their area were determined. The time-course showed that the white matter was lost in the ipsilateral basal ganglia from day 1 to day 28 after ICH. The number of MBP-labeled bundles and their area were significantly lower 2 weeks after ICH compared with sham-operated rats (p < 0.05). Systemic treatment with ZnPP attenuated the loss of MBP-labeled bundles (p < 0.01) and area (p < 0.01). In conclusion, marked white matter injury occurs after ICH. ZnPP reduces white matter injury, suggesting a role of heme degradation products in ICH-induced white matter damage.

Brain-derived neurotrophic factor administration mediated oligodendrocyte differentiation and myelin formation in subcortical ischemic stroke.

BACKGROUND AND PURPOSE: Translational research is beginning to reveal the importance of trophic factors as a therapy for cellular brain repair. The purpose of this study was to analyze whether brain-derived neurotrophic factor (BDNF) administration could mediate oligodendrogenesis and remyelination after white matter injury in subcortical stroke. METHODS: Ischemia was induced in rats by injection of endothelin-1. At 24 hours, 0.4 µg/kg of BDNF or saline was intravenously administered to the treatment and control groups, respectively. Functional evaluation, MRI, and fiber tract integrity on tractography images were analyzed. Proliferation (KI-67) and white matter repair markers (A2B5, 2',3'-cyclic-nucleotide 3'-phosphodiesterase [CNPase], adenomatous polyposis coli [APC], platelet-derived growth factor receptor alpha [PDGFR-α], oligodendrocyte marker O4 [O4], oligodendrocyte transcription factor [Olig-2], and myelin basic protein [MBP]) were analyzed at 7 and 28 days. RESULTS: The BDNF-treated animals showed less functional deficit at 28 days after treatment than the controls (P<0.05). Although T2-MRI did not show differences in lesion size at 7 and 28 days between groups, diffusion tensor imaging tractography analysis revealed significantly better tract connectivity at 28 days in the BDNF group than in the controls (P<0.05). Increased proliferation of oligodendrocyte progenitors was observed in treated animals at 7 days (P<0.05). Finally, the levels of white matter repair markers (A2B5, CNPase, and O4 at 7 days; Olig-2 and MBP at 28 days) were higher in the BDNF group than in the controls (P<0.05). CONCLUSIONS: BDNF administration exerted better functional outcome, oligodendrogenesis, remyelination, and fiber connectivity than controls in rats subjected to subcortical damage in ischemic stroke.

Effects of dietary folic acid on the expression of myelin basic protein in the brain and spinal cord of pregnant and lactating rats.

BACKGROUND/AIMS: This study investigated the effects of dietary folic acid on the expression of myelin basic protein (MBP) in the maternal brain and spinal cord during pregnancy and lactation. METHODS: Female Sprague-Dawley rats were fed either a folic-acid-supplemented diet (FS, 8 mg/kg diet) or a folic-acid-deficient diet (FD, 0 mg/kg diet) from 2 weeks prior to mating until the end of lactation. The expressions of MBP were analyzed using Western blot analysis and immunohistochemistry, and myelin oligodendrocyte glycoprotein (MOG), and neuronal nuclear antigen by immunohistochemistry. RESULTS: The cerebrocortical expression of MBP was 87% higher at day 20 of pregnancy than before pregnancy in FS animals (p < 0.05) but did not change significantly in FD animals. No significant change was observed in the hippocampus or spinal cord in either dietary treatment group. The cerebrocortical MOG and NeuN expressions were significantly lower in FD animals than in FS animals before pregnancy and increased at day 20 of pregnancy but did not differ with the dietary folic acid level. CONCLUSIONS: Folic acid deficiency did not increase the expression level of MBP in the cerebral cortex during pregnancy, suggesting that folate intake during pregnancy plays an important role in the maintenance of myelin.

Effects of quetiapine on behavioral changes and expression of myelin proteins in a chronic alcohol dependence rat model.

BACKGROUND: As an atypical antipsychotic drug, quetiapine had been approved for bipolar disorder and for adjunctive therapy in major depressive disorder and schizophrenia. Recently quetiapine has been suggested to be a promising pharmacotherapy for alcohol dependence. This study was performed to determine the effects of quetiapine in rats chronically exposed to ethanol. METHODS: Rats were exposed to ethanol solution (10 %; v/v) for 6 weeks. Saline or one of three doses of quetiapine (10, 20 or 40 mg/kg/day) was given by oral gavage while ethanol exposure for the next 14 weeks. Performance of learning and memory and withdrawal signs were evaluated. Then immunohistochemistry, western blot, quantitative real-time-PCR and transmission electron microscopy were performed to determine the effects of quetiapine on alterations of brain white matter markers (myelin basic protein, MBP; proteolipid protein, PLP) and morphology caused by chronic ethanol exposure. RESULTS: Quetiapine treatment significantly alleviated withdrawal signs in the ethanol exposed rats. Chronic ethanol exposure reduced Y-type electric maze scores and the protein/mRNA expression levels of MBP and PLP in the prefrontal cortex and hippocampus, and these effects were reversed by quetiapine treatment. Similar ultrastructure morphological changes were observed. CONCLUSIONS: Chronic quetiapine treatment alleviated the damage induced by chronic ethanol exposure with regard to learning and memory ability and to brain white matter. Thus, quetiapine appears to be a potentially promising pharmacotherapy for the treatment of alcohol use disorder.

Adverse effects of antenatal glucocorticoids on cerebral myelination in sheep.

OBJECTIVE: To determine in fetal sheep the effect of betamethasone on myelination in relation to stage of myelination, number of treatment courses, dose, and route of administration. METHODS: Fetal expression of myelin basic protein (MBP), a marker of mature oligodendrocytes and myelin, was determined between 0.27 and 0.93 gestation. Short-term betamethasone effects were examined 24 hours after one maternal intramuscular treatment course (weight adjusted to equal the clinical dose of 2 x 8 mg betamethasone to a 70-kg woman) at 0.63, 0.75, and 0.87 gestation or after continuous 48-hour fetal intravenous infusion at 0.75 and 0.87 gestation. Lasting effects were examined 20 days after one and two treatment courses weight-adapted to the clinical dose of 2 x 8 mg or 2 x 12 mg betamethasone at 0.75 gestation. RESULTS: Myelin basic protein immunoreactivity was first detected in the internal capsule at 0.53 gestation, followed by the centrum semiovale, the superficial white matter, and corpus callosum at 0.63 gestation. Within 24 hours after treatment, betamethasone reduced the number of mature oligodendrocytes and MBP immunoreactivity. The effect decreased with gestational age. Maternal and fetal betamethasone administration had similar effects. Loss of MBP immunoreactivity was not reversed 20 days after two treatment courses, independent of dose. CONCLUSION: Betamethasone-induced delayed cerebral myelination is dependent on the stage of brain development in sheep. Betamethasone-related disturbances in myelination and any potential contribution to childhood behavior deficits need to be confirmed in clinical studies.

alpha-Phenyl-n-tert-butyl-nitrone attenuates lipopolysaccharide-induced brain injury and improves neurological reflexes and early sensorimotor behavioral performance in juvenile rats.

Our previous study showed that treatment with alpha-phenyl-n-tert-butyl-nitrone (PBN) after exposure to lipopolysaccharide (LPS) reduced LPS-induced white matter injury in the neonatal rat brain. The object of the current study was to further examine whether PBN has long-lasting protective effects and ameliorates LPS-induced neurological dysfunction. Intracerebral (i.c.) injection of LPS (1 mg/kg) was performed in postnatal day (P) 5 Sprague Dawley rat pups and PBN (100 mg/kg) or saline was administered intraperitoneally 5 min after LPS injection. The control rats were injected (i.c.) with sterile saline. Neurobehavioral tests were carried out from P3 to P21, and brain injury was examined after these tests. LPS exposure resulted in severe brain damage, including enlargement of ventricles bilaterally, loss of mature oligodendrocytes, impaired myelination as indicated by the decrease in myelin basic protein immunostaining, and alterations in dendritic processes in the cortical gray matter of the parietal cortex. Electron microscopic examination showed that LPS exposure caused impaired myelination as indicated by the disintegrated myelin sheaths in the juvenile rat brain. LPS administration also significantly affected neurobehavioral functions such as performance in righting reflex, wire hanging maneuver, cliff avoidance, negative geotaxis, vibrissa-elicited forelimb-placing test, beam walking, and gait test. Treatment with PBN, a free radical scavenger and antioxidant, provided protection against LPS-induced brain injury and associated neurological dysfunction in juvenile rats, suggesting that antioxidation might be an effective approach for therapeutic treatment of neonatal brain injury induced by infection/inflammation.

Steroid protection in the experimental autoimmune encephalomyelitis model of multiple sclerosis.

OBJECTIVES: Based on evidence that pregnant women with multiple sclerosis (MS) show a decline in the relapse rate during the third trimester and an increase during the first 3 months postpartum, the suggestion was made that high levels of circulating sex steroids are responsible for pregnancy-mediated neuroprotection. As both estradiol (E(2)) and progesterone exert neuroprotective and myelinating effects on the nervous system, the effects of sex steroids were studied in the experimental autoimmune encephalomyelitis (EAE) model of MS. METHODS: EAE was induced in female C57BL/6 mice by administration of a myelin oligodendrocyte protein (MOG(40-45)) peptide. Clinical signs of EAE, myelin protein expression and neuronal parameters were determined in mice with or without hormonal treatment. RESULTS: Progesterone given prior to EAE induction attenuated the clinical scores of the disease, slightly delayed disease onset and decreased demyelination foci, according to luxol fast blue staining (LFB), myelin basic protein (MBP) and proteolipid protein (PLP) and mRNA expression. Motoneuron expression of Na,K-ATPase mRNA was also enhanced by progesterone. In turn, combined E(2) plus progesterone therapy more effectively prevented neurological deficits, fully restored LFB staining, MBP and PLP immunoreactivity and avoided inflammatory cell infiltration. On the neuronal side, steroid biotherapy increased brain-derived neurotrophic factor (BDNF) mRNA. CONCLUSION: Early treatment with progesterone alone or more evidently in combination with E(2) showed a clinical benefit and produced myelinating and neuroprotective effects in mice with MOG(40-45)-induced EAE. Therefore, sex steroids should be considered as potential novel therapeutic strategies for MS.

[Exposure to propofol down-regulates myelin basic protein expression in zebrafish embryos: its neurotoxicity on oligodendrocytes and the molecular mechanisms].

OBJECTIVE: To investigate the mechanism underlying propofol- induced down-regulation of myelin basic protein (MBP) in zebrafish embryos. METHODS: Zebrafish embryos (6-48 h post-fertilization [hpf]) were randomized into 4 equal groups for exposure to dimethyl sulfoxide (DMSO), 20 µg/mL propofol, 30 µg/mL propofol, or no particular treatment (control group). The larvae were collected at 48 or 72 hpf for detecting the mRNA levels of MBP, Olig1, Olig2, and Sox10 using qRT-PCR (n=80). The protein expression of MBP was quantitatively detected using Western blotting (n=80), and the apoptosis of the oligodendrocytes was investigated using TUNEL staining (n=6). RESULTS: Exposure to 20 and 30 µg/mL propofol caused significant reductions in the mRNA expressions of Olig1, Olig2, and Sox10 at 48 and 72 hpf (P < 0.05) and also in MBP mRNA and protein levels at 72 hpf (P < 0.05). Exposure to 30 µg/mL propofol induced more obvious reduction in MBP protein expression than 20 µg/mL propofol at 72 hpf (P < 0.05), and the exposures resulted in a significant increase of oligodendrocyte apoptosis at 72 hpf (P < 0.05). CONCLUSIONS: Propofol exposure reduces MBP expression at both the mRNA and protein levels in zebrafish embryos by down-regulating the expressions of Olig1, Olig2 and Sox10 mRNA levels and increasing apoptosis of the oligodendrocytes.

Increased calpain correlates with Th1 cytokine profile in PBMCs from MS patients.

Multiple sclerosis (MS) is a devastating autoimmune demyelinating disease of the central nervous system (CNS). This study investigated whether expression and activity of the calcium-activated protease calpain correlated with Th1/Th2 dysregulation in MS patients during states of relapse and remission. Calpain expression and activity were significantly increased in peripheral blood mononuclear cells (PBMCs) from MS patients, compared to controls, with the highest expression and activity noted during relapse. Th1 cytokines were highest and Th2 cytokines were lowest in MS patients during relapse. Treatment with calpain inhibitor, calpeptin, decreased Th1 cytokines in PBMCs from MS patients. Calpain inhibitor also reduced degradation of myelin basic protein (MBP) by inhibiting the calpain secreted from MBP-specific T cells. Taken together, these results suggested calpain involvement in Th1/Th2 dysregulation in MS patients.

Beneficial effects of r-h-CLU on disease severity in different animal models of peripheral neuropathies.

Clusterin is a protein involved in multiple biological events, including neuronal cytoprotection, membrane recycling and regulation of complement-mediated membrane attack after injury. We investigated the effect of recombinant human clusterin in preclinical models of peripheral neuropathies. Daily treatment with clusterin accelerated the recovery of nerve motor evoked potential parameters after sciatic nerve injury. Prophylactic or therapeutic treatment of experimental autoimmune neuritis rats with clusterin also accelerated the rate of recovery from the disease, associated with remyelination of demyelinated nerve fibers. These data demonstrate that clusterin is capable of ameliorating clinical, neurophysiological and pathological signs in models of peripheral neuropathies.

Activated protein C reduces endotoxin-induced white matter injury in the developing rat brain.

Periventricular leukomalacia (PVL), the dominant form of brain injury in premature infants, is characterized by white matter injury (WMI) and is associated with cerebral palsy. The pathogenesis of PVL is complex and likely involves ischemia/reperfusion, free radical formation, excitotoxicity, impaired regulation of cerebral blood flow, a procoagulant state, and inflammatory mechanisms associated with maternal and/or fetal infection. Using an established animal model of human PVL, we investigated whether activated protein C (APC), an anti-coagulant factor with anti-inflammatory, anti-apoptotic, anti-oxidant, and cytoprotective activities, could reduce endotoxin-induced WMI in the developing rat brain. Intraperitoneal injections of lipopolysaccharide (LPS) (0.5 mg/kg body weight) were given at embryonic days 18 (E18) and 19 (E19) to pregnant Sprague-Dawley rats; control rats were injected with sterile saline. A single intravenous injection of recombinant human (rh) APC (0.2 mg /kg body weight) was given to pregnant rats following the second LPS dose on embryonic day 19 (E19). Reduced cell death in white matter and hypomyelination were shown on TUNEL and myelin basic protein (MBP) staining, respectively, on late postnatal days (P7) in APC-treated groups. There were significantly fewer TUNEL+nuclei in the periventricular WM in the APC+LPS group than in the untreated LPS group. Compared to the APC+LPS and control group, MBP expression was weak in the LPS group on P7, indicating endotoxin-induced hypomyelination in the developing rat brain. APC attenuated the LPS-induced protein expression of inflammatory cytokines, tumor necrosis factor-alpha, and interleukin-6, as evaluated by ELISA in neonatal rat brains. A single intraperitoneal injection of rhAPC (0.2 mg/kg body weight) to neonatal rats on P1 also had similar protective and anti-inflammatory effects against maternally administered LPS. Collectively, these data support the hypothesis that APC may provide protection against an endotoxin-evoked inflammatory response and WMI in the developing rat brain. Moreover, our results suggest that the possible use of APC in treatment of preterm infants and pregnant women with maternal or placental infection may minimize the risk of PVL and cerebral palsy.

Metal-dependent hydrolysis of myelin basic protein by IgGs from the sera of patients with multiple sclerosis.

Homogeneous IgG fractions were obtained by chromatography of the sera of patients with multiple sclerosis (MS) on Protein G-Sepharose under conditions that remove non-specifically bound proteins. These IgGs contained several chelated metals, the relative amount of which decreases in the order: Fe>or=Ca>Cu>or=Zn>or=Mg>or=Mn>or=Pb>or=Co>or=Ni. In contrast to homogeneous IgGs of healthy individuals, Abs of MS patients effectively hydrolyzed human myelin basic protein (MBP). A minor metal-dependent fraction was obtained by chromatography of highly purified IgGs from MS patient on Chelex-100. This IgG fraction did not hydrolyze human MBP in the absence of Me(2+) ions but was activated after addition of Me(2+) ions: Mg(2+)>Mn(2+)>Cu(2+)>Ca(2+). Proteolytic activities of IgGs from other MS patients were also activated by other metal ions (Ni(2+), Fe(2+), Co(2+), Zn(2+), Pb(2+), and Co(2+)) and especially Ni(2+). Ni(2+)-activated IgGs were separated into distinct MBP-hydrolyzing fractions by chromatography on HiTraptrade mark Chelating Sepharose charged with Ni(2+). Detection of Mg(2+)-dependent proteolytic activity in the SDS-PAGE area corresponding only to IgG provided direct evidence that IgG from sera of MS patients possesses metal-dependent human MBP-hydrolyzing activity. Observed properties of MS abzymes distinguish them from other known mammalian metalloproteases and demonstrate their pronounced catalytic diversity. Metal-dependent IgGs from MS patients represent the first example of abzymes with metal-dependent proteolytic activity.

Nerve degeneration is prevented by a single intraneural apotransferrin injection into colchicine-injured sciatic nerves in the rat.

In this work, we have immunohistochemically analyzed the effects of single injections of apotransferrin (aTf) on the expression of myelin (myelin basic proteins [MBPs]) and axonal (protein gene product 9.5 [PGP 9.5] and beta(III)-tubulin [beta(III)-tub]) proteins in colchicine-injected and crushed sciatic nerves of adult rats. A protein redistribution was seen in the distal stump of injured nerves, with the appearance of MBP- and PGP 9.5-immunoreactive (IR) clusters which occurred earlier in crushed nerves (3 days post-injury [PI]) as compared to colchicine-injected nerves (7 days PI). beta(III)-tub-IR clusters appeared at 1 day PI preceding the PGP 9.5- and MBP-IR clusters in colchicine-injected nerves. With image analysis, the peak of clustering formation was found at 14 days PI for MBP and at 3 days PI for beta(III)-tub in colchicine-injected nerves. At 28 days of survival, the protein distribution patterns were almost normal. The intraneural application of aTf, at different concentrations (0.0005 mg/ml, 0.005 mg/ml, 0.05 mg/ml, 0.5 mg/ml), prevented nerve degeneration produced by colchicine, with the appearance of only a small number of MBP- and beta(III)-tub-IR clusters. However, aTf was not able to prevent clustering formation when the nerve was crushed, a kind of injury that also involves necrosis and blood flow alterations. The results suggest that aTf could prevent the colchicine effects by stabilizing the cytoskeleton proteins of the nerve fibers, avoiding the disruption of the axonal transport and thus the myelin degeneration. Transferrin is proposed as a complementary therapeutic avenue for treatment of cytotoxic nerve injuries.

Growth inhibition of astrocytoma cells by farnesyl transferase inhibitors is mediated by a combination of anti-proliferative, pro-apoptotic and anti-angiogenic effects.

While 25% of human cancers harbor oncogenic Ras mutations, such mutations are not found in astrocytomas. We have previously demonstrated that the activation of receptor tyrosine kinases expressed by malignant human astrocytoma cells and specimens results in functional upregulation of the Ras signalling pathway and increased levels of activated Ras*GTP. Farnesyl transferase inhibitors (FTIs) are promising anti-cancer agents in early clinical trials, which may exert their effect through pharmacological inhibition of the Ras signalling pathway. In this study we establish the anti-tumorigenic properties of the FTI L-744,832 against a panel of malignant human astrocytoma cell lines. Furthermore, we demonstrate the multiple mechanisms by which L-744,832 exerts its effect. L-744,832 demonstrates both cytostatic and cytotoxic effects on astrocytoma cells, and cells expressing a truncated constitutively phosphorylated Epidermal Growth Factor Receptor common in high-grade astrocytomas (EGFRvIII/p140EGF-R) demonstrate increased sensitivity to the agent. L-744,832 is capable of inducing apoptosis in astrocytoma cells under anchorage-dependent conditions; this process occurs in a p53-independent manner and is associated with increased expression of Bax and Bak. L-744,832 also induces cell cycle arrest at both the G1/M and G2/S checkpoints; this process is also independent of p53 mutational status. Cell cycle arrest in drug-treated cells can be accompanied by induction of p21WAF1/CIP1, but this induction is not necessary for the cell cycle inhibitory effects, nor is it dependent on functional p53. Finally, angiogenesis in astrocytomas has been shown to be dependent on secretion of Vascular Endothelial Growth Factor (VEGF) by tumour cells, particularly under hypoxic conditions. L-744,832 potently inhibits the secretion of VEGF under hypoxic conditions. These combinations of mechanisms suggest that these tumours, despite the absence of oncogenic Ras mutations, will be amenable to growth inhibition by FTIs, through a combination of anti-proliferative, pro-apoptotic, and anti-angiogenic effects.

The receptor for advanced glycation end-products (RAGE) directly binds to ERK by a D-domain-like docking site.

The receptor for advanced glycation end-products (RAGE)-mediated cellular activation through the mitogen-activated protein kinase (MAPK) cascade, activation of NF-kappaB and Rho family small G-proteins, cdc42/Rac, is implicated in the pathogenesis of inflammatory disorders and tumor growth/metastasis. However, the precise molecular mechanisms for the initiation of cell signaling by RAGE remain to be elucidated. In this study, proteins which directly bind to the cytoplasmic C-terminus of RAGE were purified from rat lung extracts using an affinity chromatography technique and identified to be extracellular signal-regulated protein kinase-1 and -2 (ERK-1/2). Their interactions were confirmed by immunoprecipitation of ERK-1/2 from RAGE-expressing HT1080 cell extracts with anti-RAGE antibody. Furthermore, the augmentation of kinase activity of RAGE-bound ERK upon the stimulation of cells with amphoterin was demonstrated by determining the phosphorylation level of myelin basic protein, an ERK substrate. In vitro binding studies using a series of C-terminal deletion mutants of human RAGE revealed the importance of the membrane-proximal cytoplasmic region of RAGE for the direct ERK-RAGE interaction. This region contained a sequence similar to the D-domain, a ERK docking site which is conserved in some ERK substrates including MAPK-interacting kinase-1/2, mitogen- and stress-activated protein kinase-1, and ribosomal S6 kinase. These data suggest that ERK may play a role in RAGE signaling through direct interaction with RAGE.

Effect of interferon beta on human myelin basic protein-specific T-cell lines: comparison of IFNbeta-1a and IFNbeta-1b.

The effect of interferon (IFN)beta-1a and IFNbeta-1b on human myelin basic protein-reactive T-cell lines was investigated. Both drugs inhibited proliferation and production of lymphotoxin (LT), whereas the production of interleukin-4 was not altered and interleukin-10 was induced. Comparing equal numbers of units IFNbeta-1a and -1b showed almost identical results. These in vitro data indicate that the immunomodulatory capacity of both interferons with respect to T cells paralleled their antiviral effect.

Lead alters the immunogenicity of two neural proteins: a potential mechanism for the progression of lead-induced neurotoxicity.

Some heavy metals have been suspected of playing a role in the pathogenesis of nervous system diseases such as multiple sclerosis, amyotrophic lateral sclerosis, and Alzheimer's disease. In these disorders, autoantibodies against neural proteins are evident at some stage of the disease. Lead is known to affect both the immune and nervous systems. Work in our laboratory has shown that lead exposure leads to the production of autoantibodies against neural proteins, including myelin basic protein (MBP) and glial fibrillary acidic protein (GFAP). We hypothesize that lead aggravates neurological disease by enhancing the immunogenicity of nervous system proteins, including MBP and GFAP. To test this hypothesis, lead-altered protein was prepared by incubating MBP or GFAP with lead acetate for 24 hr. On days 0, 14, and 28, mice received inoculations with either saline, native protein, or lead-altered protein. Anti-MBP and anti-GFAP, isotypes IgM and IgG, were measured in sera by ELISA on day 38. Sera of mice treated with lead-altered MBP had statistically higher anti-MBP IgG titers than both control and native MBP-immunized mice. An analogous response was seen in mice immunized with lead-altered GFAP. Supernatants from lectin-stimulated splenocytes were also examined for antibody titers and for interleukin 2 (IL-2) and interleukin 6 (IL-6) levels. A significant increase in IL-6 production was seen in mice immunized with lead-altered MBP but not with lead-altered GFAP. No changes were observed in the IL-2 levels of mice immunized with either lead-altered protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Transforming growth factor-beta enhances the ultraviolet-mediated stress response in p53-/- keratinocytes.

Skin cancer is the most common tumor type in Caucasians, with an incidence that approaches the lifetime risk for all other cancer subtypes combined. The most common predisposing factor in the development of non-melanoma skin cancer is exposure to ultraviolet (UV) radiation in sun-light. UV radiation activates c-Jun amino-terminal kinases (JNK); this kinase pathway is involved in UV-mediated apoptosis and phosphorylation of c-Jun, all of which are part of the cellular stress response. Transforming growth factor-beta1 (TGF-beta1) is an important negative regulator of keratinocyte proliferation and has other pleiotropic effects in these cells. The purpose of these investigations was to decide whether TGF-beta1 activated c-Jun amino-terminal kinases in a spontaneously immortalized human keratinocyte cell line, HaCaT, and if TGF-beta1 modulated the activation of JNK in keratinocytes exposed to ultraviolet C (UVC) radiation. Results from these investigations showed that TGF-beta1 (10 ng/ml) activated JNK within 5 min. Pretreatment with TGF-beta1 enhanced UV-mediated JNK activation and was time- and UV-dose-dependent. Pretreatment with TGF-beta1 also enhanced activity of the c-Jun promoter-reporter construct, TRE(x5)-CAT. These results suggested that TGF-beta1 modulates the response of keratinocytes to ultraviolet radiation and implicates TGF-beta1 as a potential mediator the cellular of stress response in keratinocytes.