RÉSUMÉ
BACKGROUND: Neural stem cells (NSCs) are a promising source for cell replacement therapies for neurological diseases. Growing evidence suggests an important role of cerebrospinal fluid (CSF) not only on neuroectodermal cells during brain development but also on the survival, proliferation and fate specification of NSCs in the adult brain. Existing in vitro studies focused on embryonic cell lines and embryonic CSF. We therefore studied the effects of adult human leptomeningeal CSF on the behaviour of adult human NSCs (ahNSCs). RESULTS: Adult CSF increased the survival rate of adult human NSCs compared to standard serum free culture media during both stem cell maintenance and differentiation. The presence of CSF promoted differentiation of NSCs leading to a faster loss of their self-renewal capacity as it is measured by the proliferation markers Ki67 and BrdU and stronger cell extension outgrowth with longer and more cell extensions per cell. After differentiation in CSF, we found a larger number of GFAP+ astroglial cells compared to differentiation in standard culture media and a lower number of beta-tubulin III+ neuronal cells. CONCLUSIONS: Our data demonstrate that adult human leptomeningeal CSF creates a beneficial environment for the survival and differentiation of adult human NSCs. Adult CSF is in vitro a strong glial differentiation stimulus and leads to a rapid loss of stem cell potential.
Sujet(s)
Astrocytes/effets des médicaments et des substances chimiques , Différenciation cellulaire/effets des médicaments et des substances chimiques , Protéines du liquide céphalorachidien/pharmacologie , Neurones/effets des médicaments et des substances chimiques , Cellules souches/effets des médicaments et des substances chimiques , Adulte , Astrocytes/cytologie , Astrocytes/métabolisme , Marqueurs biologiques/métabolisme , Broxuridine , Techniques de culture cellulaire , Différenciation cellulaire/physiologie , Prolifération cellulaire/effets des médicaments et des substances chimiques , Survie cellulaire/effets des médicaments et des substances chimiques , Survie cellulaire/physiologie , Cellules cultivées , Liquide cérébrospinal/composition chimique , Liquide cérébrospinal/métabolisme , Protéines du liquide céphalorachidien/métabolisme , Milieux de culture conditionnés/métabolisme , Milieux de culture conditionnés/pharmacologie , Protéine gliofibrillaire acide/métabolisme , Humains , Antigène KI-67/métabolisme , Méninges/métabolisme , Neurogenèse/effets des médicaments et des substances chimiques , Neurogenèse/physiologie , Neurones/cytologie , Neurones/métabolisme , Transplantation de cellules souches/méthodes , Cellules souches/cytologie , Cellules souches/métabolisme , Tubuline/métabolismeRÉSUMÉ
It is widely believed that the potencies of nonsteroid anti-inflammatory drugs (NSAIDs) as inhibitors of cyclooxygenase (COX) are influenced by protein binding in the extracellular fluid, since NSAIDs are bound to circulating albumin by well over 95%. This is an important point because the protein concentrations in synovial fluid and the central nervous system, which are sites of NSAID action, are markedly different from those in plasma. Here we have used a modified whole-blood assay to compare the potencies of aspirin, celecoxib, diclofenac, indomethacin, lumiracoxib, meloxicam, naproxen, rofecoxib, sodium salicylate, and SC560 as inhibitors of COX-1 and COX-2 in the presence of differing concentrations of protein. The potencies of diclofenac, naproxen, rofecoxib, and salicylate, but not aspirin, celecoxib, indomethacin, lumiracoxib, meloxicam, or SC560, against COX-1 (human platelets) increased as protein concentrations were reduced. Varying protein concentrations did not affect the potencies of any of the drugs against COX-2, with the exception of sodium salicylate (A549 cells). Clearly, our findings show that the selectivity of inhibitors for COX-1 and COX-2, which are taken to be linked to their efficacy and side effects, may change in different extracellular fluid conditions. In particular, selectivity in one body compartment does not demonstrate selectivity in another. Thus, whole-body safety or toxicity cannot be linked to one definitive measure of COX selectivity.
Sujet(s)
Protéines du sang/pharmacologie , Cyclooxygenase 1/effets des médicaments et des substances chimiques , Inhibiteurs de la cyclooxygénase 2/pharmacologie , Inhibiteurs des cyclooxygénases/pharmacologie , Acide acétylsalicylique/sang , Acide acétylsalicylique/liquide cérébrospinal , Acide acétylsalicylique/pharmacologie , Plaquettes/effets des médicaments et des substances chimiques , Plaquettes/enzymologie , A-23187/pharmacologie , Calcium/physiologie , Célécoxib , Lignée cellulaire/effets des médicaments et des substances chimiques , Protéines du liquide céphalorachidien/pharmacologie , Inhibiteurs de la cyclooxygénase 2/effets indésirables , Inhibiteurs de la cyclooxygénase 2/sang , Inhibiteurs de la cyclooxygénase 2/liquide cérébrospinal , Inhibiteurs des cyclooxygénases/effets indésirables , Inhibiteurs des cyclooxygénases/sang , Inhibiteurs des cyclooxygénases/liquide cérébrospinal , Diclofenac/sang , Diclofenac/liquide cérébrospinal , Diclofenac/pharmacologie , Dinoprostone/biosynthèse , Dinoprostone/sang , Dinoprostone/liquide cérébrospinal , Humains , Indométacine/sang , Indométacine/liquide cérébrospinal , Indométacine/pharmacologie , Ionophores/pharmacologie , Lactones/sang , Lactones/liquide cérébrospinal , Lactones/pharmacologie , Méloxicam , Naproxène/sang , Naproxène/liquide cérébrospinal , Naproxène/pharmacologie , Spécificité d'organe , Composés chimiques organiques/sang , Composés chimiques organiques/liquide cérébrospinal , Composés chimiques organiques/pharmacologie , Liaison aux protéines , Pyrazoles/sang , Pyrazoles/liquide cérébrospinal , Pyrazoles/pharmacologie , Salicylate de sodium/sang , Salicylate de sodium/liquide cérébrospinal , Salicylate de sodium/pharmacologie , Sulfonamides/sang , Sulfonamides/liquide cérébrospinal , Sulfonamides/pharmacologie , Sulfones/sang , Sulfones/liquide cérébrospinalRÉSUMÉ
Early in development, the behavior of neuroepithelial cells is controlled by several factors acting in a developmentally regulated manner. Recently it has been shown that diffusible factors contained within embryonic cerebrospinal fluid (CSF) promote neuroepithelial cell survival, proliferation, and neurogenesis in mesencephalic explants lacking any known organizing center. In this paper, we show that mesencephalic and mesencephalic+isthmic organizer explants cultured only with basal medium do not express the typically expressed mesencephalic or isthmic organizer genes analyzed (otx2 and fgf8, respectively) and that mesencephalic explants cultured with embryonic CSF-supplemented medium do effect such expression, although they exhibit an altered pattern of gene expression, including ectopic shh expression domains. Other trophic sources that are able to maintain normal neuroepithelial cell behavior, i.e., fibroblast growth factor-2, fail to activate this ectopic shh expression. Conversely, the expression pattern of the analyzed genes in mesencephalic+isthmic organizer explants cultured with embryonic cerebrospinal fluid-supplemented medium mimics the pattern for control embryos developed in ovo. We demonstrate that embryonic CSF collaborates with the isthmic organizer in regulation of the expression pattern of some characteristic neuroectodermal genes during early stages of central nervous system (CNS) development, and we suggest that this collaboration is not restricted to the maintenance of neuroepithelial cell survival. Data reported in this paper corroborate the hypothesis that factors contained within embryonic CSF contribute to the patterning of the CNS during early embryonic development.
Sujet(s)
Protéines du liquide céphalorachidien/métabolisme , Régulation de l'expression des gènes au cours du développement/physiologie , Mésencéphale/embryologie , Facteurs de croissance nerveuse/métabolisme , Neurones/métabolisme , Cellules souches/métabolisme , Animaux , Plan d'organisation du corps/effets des médicaments et des substances chimiques , Plan d'organisation du corps/physiologie , Différenciation cellulaire/effets des médicaments et des substances chimiques , Différenciation cellulaire/physiologie , Prolifération cellulaire/effets des médicaments et des substances chimiques , Survie cellulaire/effets des médicaments et des substances chimiques , Survie cellulaire/physiologie , Protéines du liquide céphalorachidien/pharmacologie , Embryon de poulet , Milieux de culture conditionnés/métabolisme , Milieux de culture conditionnés/pharmacologie , Facteur de croissance fibroblastique de type 2/métabolisme , Facteur de croissance fibroblastique de type 2/pharmacologie , Régulation de l'expression des gènes au cours du développement/effets des médicaments et des substances chimiques , Protéines Hedgehog , Mésencéphale/cytologie , Mésencéphale/effets des médicaments et des substances chimiques , Facteurs de croissance nerveuse/pharmacologie , Neurones/cytologie , Neurones/effets des médicaments et des substances chimiques , Techniques de culture d'organes , Cellules souches/cytologie , Cellules souches/effets des médicaments et des substances chimiques , Transactivateurs/effets des médicaments et des substances chimiques , Transactivateurs/métabolismeRÉSUMÉ
OBJECTIVES: To investigate the effect of cerebrospinal fluid (CSF) from patients with subarachnoid hemorrhage (SAH) on the activation of polymorphonuclear neutrophils (PMN) in response to receptor-dependent stimulation with N-formyl-l-methionyl-l-leucyl-l-phenylalanine and TNFalpha or non-receptor-dependent stimulation with phorbol 12-myristate 13-acetate. METHODS: CSF from 12 patients with SAH due to ruptured cerebral aneurysm was collected. Samples of CSF were drawn at different time points. CSF from 6 healthy subjects receiving spinal anesthesia served as the control group. After stimulation of PMN the generation of reactive oxygen intermediates was analyzed on a flow cytometer. RESULTS: In the presence of CSF, PMN showed a significant suppression of the oxidative burst following stimulation compared to stimulation without CSF. The reduction of the oxidative burst following stimulation was higher in the presence of CSF from patients with SAH. After pretreatment at 56 degrees C, the extent of the suppression observed following receptor-dependent stimulation and CSF from patients with SAH was similar to that seen after stimulation with CSF from healthy individuals. CONCLUSIONS: These data show that the presence of CSF resulted in a suppression of neutrophil oxidative function. A more distinct depression was seen in the presence of CSF from patients with SAH. We suggest a complex physiological inhibitory and protective mechanism against unfavorable activation of PMN by CSF.
Sujet(s)
Chimiotaxie des leucocytes/immunologie , Tolérance immunitaire/immunologie , Granulocytes neutrophiles/métabolisme , Stimulation du métabolisme oxydatif/immunologie , Hémorragie meningée/liquide cérébrospinal , Hémorragie meningée/immunologie , Adulte , Sujet âgé , Protéines du liquide céphalorachidien/immunologie , Protéines du liquide céphalorachidien/métabolisme , Protéines du liquide céphalorachidien/pharmacologie , Chimiotaxie des leucocytes/effets des médicaments et des substances chimiques , Femelle , Radicaux libres/métabolisme , Humains , Tolérance immunitaire/effets des médicaments et des substances chimiques , Mâle , Adulte d'âge moyen , N-Formyl-méthionyl-leucyl-phénylalanine/pharmacologie , Granulocytes neutrophiles/effets des médicaments et des substances chimiques , Granulocytes neutrophiles/immunologie , Valeurs de référence , Stimulation du métabolisme oxydatif/effets des médicaments et des substances chimiques , Hémorragie meningée/physiopathologie , 12-Myristate-13-acétate de phorbol/pharmacologie , Facteur de nécrose tumorale alpha/pharmacologieRÉSUMÉ
Early in development, the behavior of neuroepithelial cells is controlled by several factors, which act in a developmentally regulated manner. Diffusible factors are secreted locally by the neuroepithelium itself, although other nearby structures may also be involved. Evidence suggests a physiological role for the cerebrospinal fluid in the development of the brain. Here, using organotypic cultures of chick embryo neuroepithelial explants from the mesencephalon, we show that the neuroepithelium in vitro is not able to self-induce cell survival, replication, and neurogenesis. We also show that the embryonic cerebrospinal fluid (E-CSF) promotes neuroepithelial stem cell survival and induces proliferation and neurogenesis in mesencephalic explants. These data strongly suggest that E-CSF is involved in the regulation of neuroepithelial cells behavior, supporting the hypothesis that this fluid plays a key role during the early development of the central nervous system.
Sujet(s)
Protéines du liquide céphalorachidien/pharmacologie , Liquide cérébrospinal/physiologie , Développement embryonnaire/effets des médicaments et des substances chimiques , Mésencéphale/effets des médicaments et des substances chimiques , Cellules neuroépithéliales/effets des médicaments et des substances chimiques , Animaux , Différenciation cellulaire/effets des médicaments et des substances chimiques , Prolifération cellulaire/effets des médicaments et des substances chimiques , Survie cellulaire/effets des médicaments et des substances chimiques , Liquide cérébrospinal/composition chimique , Embryon de poulet , Mésencéphale/embryologie , Cellules neuroépithéliales/anatomopathologie , Techniques de culture d'organesRÉSUMÉ
OBJECTIVE: To determine the role of oxidative stress in mediating HIV dementia and to identify novel therapeutic compounds that may block this oxidative stress. METHODS: Brain tissue from patients with HIV encephalitis and macaques with simian immune deficiency virus encephalitis was immunostained for lipid peroxidation. Oxidized proteins in CSF of patients with various stages of HIV dementia were quantitated and we determined whether CSF from these patients could alter mitochondrial function. Several novel compounds with antioxidant effects were screened to determine their relative efficacy in protecting against CSF-induced neurotoxicity. RESULTS: Evidence for oxidative stress was present both in brain and in CSF. The presence of oxidized proteins in the CSF and CSF-induced progressive decrease in mitochondrial activity correlated with the severity of cognitive impairment, but only the group of patients with moderate to severe dementia reached statistical significance. L-deprenyl, didox, imidate, diosgenin, and ebselen blocked the CSF-induced toxicity. No effect of trimidox, ruthenium red, or Quercetin was seen. CONCLUSIONS: Increased oxidative stress is present in brain and CSF of HIV-infected patients. There is also an accumulation of toxic substances in the CSF that are capable of inducing oxidative stress. The authors have identified several novel compounds that are capable of blocking the CSF-induced toxicity, the therapeutic potential of which is worthy of further exploration.
Sujet(s)
Démence associée au SIDA/physiopathologie , Antioxydants/pharmacologie , Stress oxydatif/effets des médicaments et des substances chimiques , Démence associée au SIDA/anatomopathologie , Aldéhydes/analyse , Aldéhydes/métabolisme , Animaux , Apoptose/effets des médicaments et des substances chimiques , Encéphale/cytologie , Encéphale/métabolisme , Encéphale/anatomopathologie , Cellules cultivées , Liquide cérébrospinal/composition chimique , Liquide cérébrospinal/métabolisme , Protéines du liquide céphalorachidien/composition chimique , Protéines du liquide céphalorachidien/métabolisme , Protéines du liquide céphalorachidien/pharmacologie , Cytochromes de type c/métabolisme , Colorants fluorescents , Humains , Cétones/analyse , Métabolisme lipidique , Macaca , Potentiels de membrane/effets des médicaments et des substances chimiques , Mitochondries/effets des médicaments et des substances chimiques , Neurones/cytologie , Neurones/effets des médicaments et des substances chimiques , Neurones/métabolisme , Oxydoréduction/effets des médicaments et des substances chimiques , Virus de l'immunodéficience simienneRÉSUMÉ
Neuronal damage seems to be a major source of disability in multiple sclerosis (MS) patients and at present magnetic resonance imaging (MRI) is a sensitive method to evaluate lesion and disease activity. We studied the potential correlation between changes in MS patients' disability after relapse, the degree of T1 lesion hypointensity on MRI in vivo and neuronal apoptosis induced by cerebrospinal fluid (CSF) on neuron cultures. In this study, we included 24 MS patients with relapsing disease. Clinical recovery from relapse was measured by the Expanded Disability Status Scale (EDSS). T1-weighted MRI studies were done according to established standards and neuronal apoptosis was induced by treatment of neuronal cultures with CSF from patients while relapsing. Recovery after relapse is inversely correlated with neuronal apoptosis (r=-0.725, p<0.0001). A correlation was found between T1 lesion hypointensity and a poor recovery from relapse (r=0.656, p=0.0005) and such hypointensity correlated strongly with neuronal apoptosis (r=-0.779, p<0.0001). CSF from all patients with hypointense T1 lesions caused significantly increased neuronal apoptosis, whereas all CSF that did not induced such effects corresponded to patients without T1 lesions. The recovery from an acute MS relapse is significantly worse in patients with hypointense T1 lesions in MRI and in those whose CSF damaged neurons on cultures in vitro, phenomena that closely correlated each other.
Sujet(s)
Apoptose/effets des médicaments et des substances chimiques , Système nerveux central/métabolisme , Protéines du liquide céphalorachidien/pharmacologie , Sclérose en plaques/liquide cérébrospinal , Dégénérescence nerveuse/liquide cérébrospinal , Dégénérescence nerveuse/induit chimiquement , Neurones/effets des médicaments et des substances chimiques , Adolescent , Adulte , Albumines/liquide cérébrospinal , Apoptose/physiologie , Cellules cultivées/effets des médicaments et des substances chimiques , Cellules cultivées/métabolisme , Cellules cultivées/anatomopathologie , Système nerveux central/anatomopathologie , Système nerveux central/physiopathologie , Protéines du liquide céphalorachidien/métabolisme , Évaluation de l'invalidité , Femelle , Humains , Immunoglobuline G/liquide cérébrospinal , Numération des leucocytes , Imagerie par résonance magnétique , Mâle , Adulte d'âge moyen , Sclérose en plaques/anatomopathologie , Sclérose en plaques/physiopathologie , Dégénérescence nerveuse/physiopathologie , Neurones/métabolisme , Neurones/anatomopathologie , Récupération fonctionnelle/physiologie , RécidiveRÉSUMÉ
To determine the possible contribution of glial cells via oxidative stress/cytokine secretion in the pathogenesis of Parkinson's disease (PD), Alzheimer disease (AD), amyotrophic lateral sclerosis (ALS) or multiple sclerosis (MS) the concentration of nitric oxide (NO) (by the Griess method) and Interleukin-6 (IL-6) (by enzyme-linked immunosorbent assay) were measured in resting rat microglial and astrocytic cell culture supernatants stimulated by cerebrospinal fluid (CSF) (dilution 1:4, 1:10) from patients with the aforementioned diseases. Neither the concentration of NO (optical density at 450 nm: control, 0.036+/-0.006; MS, 0.034+/-0.008; AD, 0.031+/-0.006; PD, 0.02+/-0.01; lipopolysaccharide (LPS), 0.26+/-0.018) nor the amount of IL-6 (ng/ml: control, 0.112+/-0.026; PD, 0.12+/-0.027; MS, 0.123+/-0.008; ALS, 0.137+/-0.01; LPS, 1.81+/-0.11) differed in any disease group from those of unaffected controls. These findings suggest that the stimuli for inflammatory activation of glia are quite localized and not present in sufficient concentrations in the CSF of affected patients.
Sujet(s)
Cytokines/immunologie , Encéphalite/liquide cérébrospinal , Gliose/liquide cérébrospinal , Maladies neurodégénératives/liquide cérébrospinal , Névroglie/immunologie , Monoxyde d'azote/immunologie , Stress oxydatif/immunologie , Astrocytes/effets des médicaments et des substances chimiques , Astrocytes/immunologie , Astrocytes/métabolisme , Communication autocrine/effets des médicaments et des substances chimiques , Communication autocrine/immunologie , Cellules cultivées/effets des médicaments et des substances chimiques , Cellules cultivées/immunologie , Cellules cultivées/métabolisme , Protéines du liquide céphalorachidien/immunologie , Protéines du liquide céphalorachidien/métabolisme , Protéines du liquide céphalorachidien/pharmacologie , Cytokines/métabolisme , Cytokines/pharmacologie , Encéphalite/immunologie , Encéphalite/physiopathologie , Gliose/induit chimiquement , Gliose/immunologie , Humains , Interféron gamma/immunologie , Interféron gamma/métabolisme , Interféron gamma/pharmacologie , Interleukine-1/immunologie , Interleukine-1/métabolisme , Interleukine-1/pharmacologie , Interleukine-6/immunologie , Interleukine-6/métabolisme , Interleukine-6/pharmacologie , Lipopolysaccharides/pharmacologie , Microglie/effets des médicaments et des substances chimiques , Microglie/immunologie , Microglie/métabolisme , Maladies neurodégénératives/immunologie , Maladies neurodégénératives/physiopathologie , Névroglie/effets des médicaments et des substances chimiques , Névroglie/métabolisme , Monoxyde d'azote/métabolisme , Facteur de nécrose tumorale alpha/immunologie , Facteur de nécrose tumorale alpha/métabolisme , Facteur de nécrose tumorale alpha/pharmacologieRÉSUMÉ
Primary cultures of murine cerebellar granule neurones were exposed to cerebrospinal fluid from patients with subtypes of multiple sclerosis or acute polyradiculoneuropathy (Guillain-Barré syndrome) for 2 days. Cells were then stained with Hoechst 33342 or terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL) to detect apoptotic bodies. The results were compared with control cultures exposed to cerebrospinal fluid from patients with no known neurological disease or deficit. There was no significant difference in the level of apoptosis induced between these controls and cultures not exposed to cerebrospinal fluid at all. Cultures exposed to cerebrospinal fluid samples from patients with relapsing-remitting multiple sclerosis did not have higher levels of apoptosis than cells exposed to controls, regardless of whether the sample was taken during relapse or remission. However, a significant increase in apoptosis was observed in cultures exposed to cerebrospinal fluid from patients with primary progressive multiple sclerosis, and apoptosis correlated with disease severity. This supports the existence of biochemical differences between subgroups of multiple sclerosis. A significant increase in apoptosis was also induced by cerebrospinal fluid samples from patients with acute polyradiculoneuropathy, suggesting the presence of neurotoxic factor(s) here also. The relevance to disease pathology is unclear.
Sujet(s)
Apoptose/effets des médicaments et des substances chimiques , Protéines du liquide céphalorachidien/pharmacologie , Syndrome de Guillain-Barré/liquide cérébrospinal , Sclérose en plaques récurrente-rémittente/liquide cérébrospinal , Neurones/cytologie , Adulte , Animaux , Cellules cultivées , Cervelet/cytologie , Femelle , Humains , Méthode TUNEL , Mâle , Souris , Adulte d'âge moyen , Sclérose en plaques chronique progressive/liquide cérébrospinal , Neurones/effets des médicaments et des substances chimiquesRÉSUMÉ
Our earlier studies have shown that cerebrospinal fluid (CSF) of patients with amyotrophic lateral sclerosis (ALS), when intrathecally injected into the neonatal rats, produces an aberrant phosphorylation of neurofilaments (NF) in the ventral horn neurons and reactive astrogliosis in the spinal cord. We wanted to investigate the effect of cyclophosphamide in the spinal cords of neonatal rats exposed to ALS-CSF. A single dose (5 microg in 5 microl saline) of cyclophosphamide was injected, 24 h after the administration of CSF samples from ALS and non-ALS neurological patients into the spinal subarachnoid space of 3-day-old rat pups. Rats were sacrificed after a period of 24 h, and stained with antibodies against the phosphorylated NF (SMI-31 antibody) and glial fibrillary acidic protein (GFAP). Cyclophosphamide treatment resulted in a 50% decrease in the number of SMI-31 stained neuronal soma in ventral horns of spinal cords of ALS-CSF exposed rats. This was accompanied by a decrease in the number of GFAP immunoreactive astrocytes. Furthermore, lactate dehydrogenase (LDH) activity was also decreased significantly, following cyclophosphamide treatment. These results suggest that cyclophosphamide could exert a neuroprotective effect against the neurotoxic action of factor(s) present in the ALS-CSF.
Sujet(s)
Sclérose latérale amyotrophique/liquide cérébrospinal , Cellules de la corne ventrale/effets des médicaments et des substances chimiques , Protéines du liquide céphalorachidien/pharmacologie , Cyclophosphamide/pharmacologie , Immunosuppresseurs/pharmacologie , Dégénérescence nerveuse/prévention et contrôle , Neuroprotecteurs/pharmacologie , Sclérose latérale amyotrophique/traitement médicamenteux , Sclérose latérale amyotrophique/physiopathologie , Animaux , Animaux nouveau-nés/métabolisme , Cellules de la corne ventrale/anatomopathologie , Astrocytes/effets des médicaments et des substances chimiques , Astrocytes/anatomopathologie , Relation dose-effet des médicaments , Calendrier d'administration des médicaments , Protéine gliofibrillaire acide/effets des médicaments et des substances chimiques , Protéine gliofibrillaire acide/métabolisme , Gliose/induit chimiquement , Gliose/physiopathologie , Gliose/prévention et contrôle , Immunohistochimie , L-Lactate dehydrogenase/effets des médicaments et des substances chimiques , L-Lactate dehydrogenase/métabolisme , Dégénérescence nerveuse/induit chimiquement , Dégénérescence nerveuse/physiopathologie , Protéines neurofilamenteuses/effets des médicaments et des substances chimiques , Protéines neurofilamenteuses/métabolisme , Phosphorylation/effets des médicaments et des substances chimiques , Rats , Rat WistarRÉSUMÉ
OBJECTIVE: Meningiomas are benign neoplasms that derive from coverings of the brain. Approximately 10% of benign tumors progress into atypical, malignant tumors, thus constituting a subset of histopathologically benign tumors that are clinically invasive. The aim of this study was to evaluate cathepsins B and L and their inhibitors as new prognostic factors that could distinguish malignant from benign forms of meningiomas. METHODS: Using immunohistochemical analysis and specific monoclonal antibodies, we evaluated the levels of cathepsins B and L and the levels of the endogenous cysteine proteinase inhibitors stefin A and cystatin C in 88 meningiomas. Immunohistochemical scores were determined as the sum of the frequency (0-3) and intensity (0-3) of immunolabeling of the tumor cells. RESULTS: Of the 88 tumors studied, 67 were benign meningiomas and 21 were atypical meningiomas. Among the benign group, nine tumors had certain features of malignancy. These tumors were classified as border benign meningiomas, and the rest were classified as clear benign meningiomas. A high immunohistochemical score (4-6) for cathepsin B was more frequent in atypical tumors than in clear benign tumors (P < 0.001). Compared with clear benign tumors, higher cathepsin B immunohistochemical scores were found in atypical tumors (P < 0.001) and border benign tumors (P < 0.03). No statistical difference in immunohistochemical staining of cathepsin B was found between atypical meningiomas and border benign meningiomas. Higher expression of cathepsin L was found in atypical tumors as compared with clear benign tumors (P < 0.03), but it was not observed in border benign as compared with clear benign meningiomas. No immunostaining for stefin A and cystatin C was detected in any of the tumors. CONCLUSION: We show that the levels of cathepsin B and cathepsin L antigens are significantly higher in invasive types of benign meningioma. Specifically, cathepsin B may be used as a diagnostic marker to distinguish histomorphologically benign but invasive meningiomas from histomorphologically clear benign tumors.
Sujet(s)
Marqueurs biologiques tumoraux/analyse , Cathepsine B/analyse , Cathepsines/analyse , Tumeurs des méninges/composition chimique , Tumeurs des méninges/anatomopathologie , Méningiome/composition chimique , Méningiome/anatomopathologie , Adolescent , Adulte , Sujet âgé , Cathepsine B/antagonistes et inhibiteurs , Cathepsine L , Cathepsines/antagonistes et inhibiteurs , Protéines du liquide céphalorachidien/pharmacologie , Cystatine A , Cystatine C , Cystatines/pharmacologie , Cysteine endopeptidases , Inhibiteurs de la cystéine protéinase/pharmacologie , Femelle , Humains , Mâle , Adulte d'âge moyen , Invasion tumorale , PronosticRÉSUMÉ
An earlier study reported that human cerebrospinal fluid (CSF) has fungistatic activity for Cryptococcus neoformans. The present study reports that molecular sieve fractionation of concentrated CSF yielded three protein peaks, one of which (p2) had anticryptococcal activity. On a DEAE-Sephacel anion-exchange column the active molecular sieve peak (p2) gave two peaks that contained anticryptococcal activity. The first (DEAE-1) eluted with 0.1 M NaCl and the second (DEAE-2) eluted with 0.2 M NaCl in buffer. Fungistatic activity of DEAE-1 was reversed by FeCl3. Moreover, FeCl3 reversed inhibition of C. neoformans growth by CSF. In contrast, activity of DEAE-2 was not reversed by FeCl3, indicating that inhibition was produced by an iron-independent mechanism. Immunoblot assays showed that transferrin was present in DEAE-1 but not in DEAE-2, whereas albumin was present in DEAE-2 but not in DEAE-1. On NuPAGE, DEAE-1 protein migrated as a single band corresponding to transferrin and DEAE-2 protein gave a single band corresponding to albumin. In control experiments, human serum albumin subjected to the same isolation protocol acquired anticryptococcal activity similar to that of DEAE-2. Therefore, CSF albumin (DEAE-2) activity was associated with the isolation protocol. These data indicate that transferrin, present in or isolated from CSF, sequesters trace amounts of ferric iron, inhibits growth of C. neoformans and acts as an innate defence mechanism.
Sujet(s)
Protéines du liquide céphalorachidien/isolement et purification , Protéines du liquide céphalorachidien/pharmacologie , Cryptococcus neoformans/effets des médicaments et des substances chimiques , Transferrine/liquide cérébrospinal , Transferrine/pharmacologie , Liquide cérébrospinal/composition chimique , Protéines du liquide céphalorachidien/composition chimique , Chromatographie sur gel , Chromatographie d'échange d'ions/méthodes , Cryptococcus neoformans/croissance et développement , Électrophorèse sur gel de polyacrylamide/méthodes , Composés du fer III/pharmacologie , Humains , Immunotransfert/méthodes , Focalisation isoélectrique/méthodes , Sérumalbumine/liquide cérébrospinal , Sérumalbumine/composition chimique , Sérumalbumine/pharmacologie , Transferrine/composition chimique , Transferrine/isolement et purificationRÉSUMÉ
The close relationship between neurodegeneration and gliosis could play a relevant role in propagating the degenerative event in the brain. Although there is evidence of the neurotoxicity of activated glia, the ability of damaged neurons to modulate glial response remains unexplored. Exposure of primary glial cells to damaged or dead hippocampal neurons was followed by glial release of tumor necrosis factor-alpha (TNF-alpha). This release was reduced by a partial prevention of neural death. By contrast, no TNF-alpha was released when glial cells were exposed to damaged murine fibroblasts. Exposure of glial cells to the cerebrospinal fluid (CSF) of patients with Alzheimer's disease was also followed by TNF-alpha release, while the CSF of subjects with nondegenerative brain disorders evoked no response. These data suggest that damaged neurons both in vitro and in vivo release factor(s) that activate glial response. Heat treatment of sonicated neurons or use of a mixture of protease inhibitors, among them the caspase inhibitors Z-DEVD-FMK and Z-YVAD-FMK, prevented TNF-alpha release from glial cells. We conclude that a primary neurodegenerative event may induce glial response by releasing a neurospecific protein factor via activation of a caspase.
Sujet(s)
Endopeptidases/métabolisme , Gliose/enzymologie , Gliose/physiopathologie , Dégénérescence nerveuse/enzymologie , Dégénérescence nerveuse/physiopathologie , Névroglie/enzymologie , Maladie d'Alzheimer/enzymologie , Maladie d'Alzheimer/anatomopathologie , Maladie d'Alzheimer/physiopathologie , Animaux , Animaux nouveau-nés , Marqueurs biologiques/analyse , Inhibiteurs des caspases , Caspases/effets des médicaments et des substances chimiques , Caspases/métabolisme , Extrait cellulaire/pharmacologie , Cellules cultivées , Protéines du liquide céphalorachidien/métabolisme , Protéines du liquide céphalorachidien/pharmacologie , Endopeptidases/effets des médicaments et des substances chimiques , Gliose/anatomopathologie , Hippocampe/cytologie , Hippocampe/enzymologie , Lipopolysaccharides/pharmacologie , Dégénérescence nerveuse/anatomopathologie , Névroglie/effets des médicaments et des substances chimiques , Neurones/effets des médicaments et des substances chimiques , Neurones/enzymologie , Neurones/anatomopathologie , Inhibiteurs de protéases/pharmacologie , Rats , Rat Sprague-Dawley , Facteur de nécrose tumorale alpha/métabolismeRÉSUMÉ
The Hydra bioassay system utilizes a tentacle ball formation (TBF), a component of the feeding response of hydra, elicited by S-methyl-glutathione. TBF is modulated by many biologically active peptides in a specific way to individual peptides, and is useful in investigating biologically active peptides in a complex biological sample. We applied the hydra bioassay to explore a possible biologically active substance responsible for the decrease in the motor activity of the mice. The suppression of the CSF obtained from rats after exhaustive exercise was marked lower than that of sedentary rats. Addition of transforming growth factor-beta (TGF-beta), which is the only substance known to nullify TBF, to CSF of the sedentary rat reproduced this change in the suppression of the TBF. This system is useful to screen active peptides in small amounts of biological samples containing very low concentrations of peptides.
Sujet(s)
Dosage biologique/méthodes , Protéines du liquide céphalorachidien/analyse , Fatigue/liquide cérébrospinal , Hydra/effets des médicaments et des substances chimiques , Neuropeptides/liquide cérébrospinal , Animaux , Comportement animal/effets des médicaments et des substances chimiques , Protéines du liquide céphalorachidien/pharmacologie , Comportement alimentaire/effets des médicaments et des substances chimiques , Hydra/croissance et développement , Mâle , Neuropeptides/pharmacologie , Effort physique , Rats , Rat Sprague-Dawley , Repos/physiologie , TrypsineRÉSUMÉ
The environment presented by host tissue may influence cellular transplants in the CNS depending on injury or disease. Here we examined whether chronic pain alters cerebrospinal fluid (CSF), thereby enhancing the analgesic effect of transplanted adrenal cells. CSF samples were taken intracisternally from rats with neuropathic pain induced by chronic constriction injury of the sciatic nerve. The samples were applied to cultured bovine chromaffin-cell clusters while catecholamine release was measured by fast cyclic voltammetry. This caused marked and sustained elevations in catecholamine levels, compared to CSF from sham-operated controls, which were reversible by the nicotinic antagonist mecamylamine. These results suggest that chronic neuropathic pain produces increased CSF levels of secretogogues for chromaffin cells, and illustrates the importance of host microenvironmental factors in determining graft function.
Sujet(s)
Médulla surrénale/effets des médicaments et des substances chimiques , Médulla surrénale/transplantation , Catécholamines/métabolisme , Protéines du liquide céphalorachidien/pharmacologie , Cellules chromaffines/effets des médicaments et des substances chimiques , Cellules chromaffines/transplantation , Douleur/physiopathologie , Neuropathies périphériques/physiopathologie , Moelle spinale/physiopathologie , Médulla surrénale/métabolisme , Animaux , Bovins , Cellules cultivées , Liquide cérébrospinal/composition chimique , Liquide cérébrospinal/métabolisme , Cellules chromaffines/métabolisme , Maladie chronique/traitement médicamenteux , Électrophysiologie , Mâle , Mécamylamine/pharmacologie , Microélectrodes , Antagonistes nicotiniques/pharmacologie , Douleur/liquide cérébrospinal , Douleur/traitement médicamenteux , Neuropathies périphériques/liquide cérébrospinal , Neuropathies périphériques/traitement médicamenteux , Rats , Rat Sprague-Dawley , Récepteurs nicotiniques/effets des médicaments et des substances chimiques , Récepteurs nicotiniques/métabolisme , Moelle spinale/composition chimique , Moelle spinale/métabolismeRÉSUMÉ
Malignant progression is accompanied by degradation of extracellular matrix proteins. Here we describe a novel confocal assay in which we can observe proteolysis by living human breast cancer cells (BT20 and BT549) through the use of quenched-fluorescent protein substrates. Degradation thus was imaged, by confocal optical sectioning, as an accumulation of fluorescent products. With the BT20 cells, fluorescence was localized to pericellular focal areas that coincide with pits in the underlying matrix. In contrast, fluorescence was localized to intracellular vesicles in the BT549 cells, vesicles that also label for lysosomal markers. Neither intracellular nor pericellular fluorescence was observed in the BT549 cells in the presence of cytochalasin B, suggesting that degradation occurred intracellularly and was dependent on endocytic uptake of substrate. In the presence of a cathepsin B-selective cysteine protease inhibitor, intracellular fluorescence was decreased approximately 90% and pericellular fluorescence decreased 67% to 96%, depending on the protein substrate. Matrix metallo protease inhibitors reduced pericellular fluorescence approximately 50%, i.e., comparably to a serine and a broad spectrum cysteine protease inhibitor. Our results suggest that: 1) a proteolytic cascade participates in pericellular digestion of matrix proteins by living human breast cancer cells, and 2) the cysteine protease cathepsin B participates in both pericellular and intracellular digestion of matrix proteins by living human breast cancer cells.
Sujet(s)
Tumeurs du sein/enzymologie , Peptide hydrolases/métabolisme , Cellules cancéreuses en culture/cytologie , Tumeurs du sein/métabolisme , Tumeurs du sein/anatomopathologie , Cathepsine B/antagonistes et inhibiteurs , Cathepsine B/métabolisme , Protéines du liquide céphalorachidien/pharmacologie , Cystatine C , Cystatines/pharmacologie , Inhibiteurs de la cystéine protéinase/pharmacologie , Dipeptides/pharmacologie , Matrice extracellulaire/métabolisme , Colorants fluorescents , Humains , Traitement d'image par ordinateur , Techniques immunoenzymatiques , Foie/enzymologie , Lysosomes/enzymologie , Lysosomes/métabolisme , Microscopie confocale , Sérumalbumine bovine/métabolisme , Cellules cancéreuses en culture/métabolismeRÉSUMÉ
The effect of human cerebrospinal fluid (CSF) on the growth of Cryptococcus neoformans and Candida species was tested in RPMI-1640. CSF alone was highly fungistatic for both yeasts and inhibited growth in a concentration-dependent manner. Unlike human serum, CSF did not collaborate with fluconazole for killing C. neoformans. Molecular sieve fractionation of CSF on a G-200 Sephadex column yielded a highly antifungal fraction with a molecular weight around 66 kDa. On SDS-PAGE this fraction migrated as a major and a minor band corresponding to the mobility of bovine serum albumin. These novel findings suggest that CSF contains a factor(s) that provides resistance to the growth of C. neoformans or Candida species.
Sujet(s)
Antifongiques/pharmacologie , Candida/croissance et développement , Protéines du liquide céphalorachidien/pharmacologie , Liquide cérébrospinal/physiologie , Cryptococcus neoformans/croissance et développement , Animaux , Candida/effets des médicaments et des substances chimiques , Bovins , Liquide cérébrospinal/composition chimique , Protéines du liquide céphalorachidien/isolement et purification , Numération de colonies microbiennes , Cryptococcus neoformans/effets des médicaments et des substances chimiques , Électrophorèse sur gel de polyacrylamide , Fluconazole/pharmacologie , HumainsRÉSUMÉ
Cerebrospinal fluid from L-dopa-treated Parkinson's disease patients and subjects without neurodegenerative diseases (controls) was explored in its trophic properties as culture medium on a variety of cells from neural origin. Primary cultures of regional brain dissociates from rat and Cebus apella monkey fetuses, immature rat adrenal chromaffin cells, phaeochromocytoma (PC12), and neuroblastoma (NB69) cell lines as well as subcultured fetal rat astroglia were used as target cells for 24- to 48-h culture periods. Most cerebrospinal fluid samples from L-dopa-treated patients had a general dystrophic effect. This phenomenon was more apparent on striatum and ventral mesencephalon than on cerebral cortex cell dissociates. The deleterious effect of these samples was abolished by previous exposure to fetal astroglial cells. Neuroblastoma cells showed no differential response when exposed to samples from control and L-dopa-treated patients. Phaeochromocytoma cells did not grow processes under any of the samples assayed in the time interval explored, but neither showed evidence of dystrophy. The relevance of these findings to the transplantation of different cell types as one of the possible therapies for Parkinson's disease is discussed. The suggestion is made that CSF testing prior to transplantation may aid in anticipating its possible outcome. Cotransplantation of neuronal cells with subcultured astroglia may foster survival and growth of the former cells.
Sujet(s)
Antiparkinsoniens/administration et posologie , Astrocytes/effets des médicaments et des substances chimiques , Protéines du liquide céphalorachidien/pharmacologie , Lévodopa/administration et posologie , Neurones/effets des médicaments et des substances chimiques , Maladie de Parkinson/liquide cérébrospinal , Adulte , Sujet âgé , Sujet âgé de 80 ans ou plus , Animaux , Astrocytes/cytologie , Astrocytes/métabolisme , Cebus , Cortex cérébral/cytologie , Cellules chromaffines/cytologie , Cellules chromaffines/effets des médicaments et des substances chimiques , Humains , Mésencéphale/cytologie , Adulte d'âge moyen , Néostriatum/cytologie , Neurones/cytologie , Cellules PC12 , Maladie de Parkinson/traitement médicamenteux , Rats , Rat Sprague-DawleyRÉSUMÉ
Interleukin (IL) 8 was measured in CSF of 14 patients with severe traumatic brain injury. IL-8 levels were significantly higher in CSF (up to 8,000 pg/ml) than serum (up to 2,400 pg/ml) (p < 0.05), suggesting intrathecal production. Maximal IL-8 values in CSF correlated with a severe dysfunction of the blood-brain barrier. Nerve growth factor (NGF) was detected in CSF of 7 of 14 patients (range of maximal NGF: 62-12,130 pg/ml). IL-8 concentrations were significantly higher in these patients than in those without NGF (p < 0.01). CSF containing high IL-8 (3,800-7,900 pg/ml) induced greater NGF production in cultured astrocytes (202-434 pg/ml) than samples with low IL-8 (600-1,000 pg/ml), which showed a smaller NGF increase (0-165 pg/ml). Anti-IL-8 antibodies strongly reduced (52-100%) the release of NGF in the group of high IL-8, whereas in the group with low IL-8, this effect was lower (0-52%). The inability of anti-IL-8 antibodies to inhibit the synthesis of NGF completely may depend on cytokines like tumor necrosis factor alpha and IL-6 found in these CSF samples, which may act in association with IL-8. Thus, IL-8 may represent a pivotal cytokine in the pathology of brain injury.
Sujet(s)
Astrocytes/effets des médicaments et des substances chimiques , Barrière hémato-encéphalique , Lésions encéphaliques/liquide cérébrospinal , Protéines du liquide céphalorachidien/métabolisme , Interleukine-8/liquide cérébrospinal , Facteurs de croissance nerveuse/biosynthèse , Adolescent , Adulte , Sujet âgé , Animaux , Astrocytes/métabolisme , Lésions encéphaliques/sang , Lésions encéphaliques/physiopathologie , Cellules cultivées , Protéines du liquide céphalorachidien/pharmacologie , Femelle , Régulation de l'expression des gènes/effets des médicaments et des substances chimiques , Humains , Interleukine-8/sang , Interleukine-8/métabolisme , Interleukine-8/pharmacologie , Mâle , Souris , Adulte d'âge moyen , Facteurs de croissance nerveuse/génétique , Protéines recombinantes/pharmacologieRÉSUMÉ
Cerebrospinal fluid from patients with aneurysmal subarachnoid hemorrhage induces the elevation of cytosolic free calcium [Ca2+]i in cultured vascular smooth muscle cells. We have purified a [Ca2+]i elevating factor from cerebrospinal fluid of a patient with subarachnoid hemorrhage due to aneurysm rupture. The calcium-elevating protein factor was purified to homogeneity by ammonium sulfate precipitation and a combination of Mono Q, Superose 12, and Mono S columns using liquid chromatography. Fifteen microgram of the purified protein was obtained from 340 mg of cerebrospinal fluid proteins and the molecular mass of the protein was estimated to be 81 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis. It was also shown that the purified protein was cross-reactive with anti-human transferrin antibody. These results suggested that transferrin may be involved with the cerebral vasospasm after subarachnoid hemorrhage.