RÉSUMÉ
BACKGROUND: As surgical education has evolved, most curricula have favoured a competency-based approach over traditional apprenticeship models. Surgical simulation can be a useful aide in the training of both oncological and reconstructive breast surgery trainees. This review investigates the extent to which simulation of breast surgery procedures has been validated as a training tool. METHODS: A comprehensive literature search for studies evaluating the objective validity of breast surgery simulators was performed, using MEDLINE, EMBASE and the Cochrane Library databases. Studies assessing construct, concurrent or predictive validity were included, as well as those demonstrating skill acquisition. FINDINGS: The initial literature search returned 1,625 hits, with only five articles meeting the inclusion criteria. Simulators were designed to train procedures such as breast augmentation, lesion biopsy and excision. Of these, breast biopsy was the most simulated procedure (three studies). Two studies evaluated animal models, two evaluated synthetic models and one study assessed both a synthetic and animal model. Construct validity was confirmed in two studies, concurrent validity in one study and a learning curve demonstrated in another study. No association between experience and performance was seen in the remaining study. The quality of the evidence presented in each article was low due to numerous limitations. Despite the abundance of breast surgery simulators created for trainees, few have been objectively validated and they only cover a narrow range of breast procedures. Although early results are promising, further studies are required before routine use of simulators is considered in breast surgery curricula.
Sujet(s)
Tumeurs du sein/chirurgie , Région mammaire/chirurgie , Formation par simulation , Chirurgiens/enseignement et éducation , Procédures de chirurgie opératoire/enseignement et éducation , Animaux , Femelle , Humains , Mammoplastie/enseignement et éducation , Mastectomie/enseignement et éducationRÉSUMÉ
Spontaneous urinary bladder perforation is a very rare disease. The main cause of urinary perforation, indeed, is a damage to the urinary bladder wall by blunt or penetrating trauma. There are only few idiopathic spontaneous rupture of urinary bladder (ISRUB) cases reported in the literature. Pre-operative diagnosis is very difficult due to similar symptoms, laboratory and imaging findings of a gastrointestinal perforation that is usually excluded intraoperatively. Herein we report a case of a 91-year-old man presented to the emergency department with a spontaneous bladder perforation mimicking an ileal perforation.
Sujet(s)
Erreurs de diagnostic , Maladies de l'iléon/diagnostic , Perforation intestinale/diagnostic , Maladies du jéjunum/diagnostic , Maladies de la vessie/diagnostic , Abdomen aigu/étiologie , Sujet âgé de 80 ans ou plus , Urgences , Humains , Laparotomie , Mâle , Péritonite/étiologie , Rupture spontanée , Techniques de suture , Tomodensitométrie , Maladies de la vessie/complications , Maladies de la vessie/imagerie diagnostique , Maladies de la vessie/chirurgieRÉSUMÉ
A prevalent T helper type 1 (Th1) subset of lymphocytes has been described in Hashimoto's thyroiditis (HT), but whether a similar polarization may characterize HT when associated with non-endocrine autoimmune disorders (NEAD) is not known. The aim of the present study was to analyse the intracellular Th1 and Th2 distinctive cytokines in patients with isolated HT or associated with non-endocrine autoimmune disorders. Intracellular cytokine expression was assessed in peripheral blood lymphocytes (PBL) of 68 out-patients (females = 55; males = 13; median age = 6 years) with HT : 33 had isolated HT and 35 had a concurrent NEAD. The percentage of interferon (IFN)-γ and interleukin (IL)-2 Th1- and IL-4 Th2-positive cells was measured by flow cytometric analysis. We found an increased percentage of IL-2-positive cells in all patients, without differences between patients with isolated HT or associated with NEAD. IFN-γ(+) cells were also increased in both groups, but the median percentage of those with isolated HT was lower than in patients with HT+NEAD (19·0 versus 29·9%; P = 0·0082). An increased number of IL-4-positive cells was observed in three of 33 (9·1%) patients with isolated HT and in 25 of 35 patients with NEAD [71%; P < 0·0001; relative risk (RR) = 3·18]. The median values of IL-4(+) cells (HT = 5·0% versus HT + NEAD = 16·8%) confirmed this large difference (P < 0·0001). A clear-cut increase of IL-4(+) lymphocytes characterizes patients with autoimmune thyroiditis who have associated non-endocrine autoimmune disorders. These findings may represent an initial tool to detect patients with autoimmune thyroiditis in which additional non-endocrine autoimmune disorders may be awaited.
Sujet(s)
Maladies auto-immunes/complications , Maladies auto-immunes/diagnostic , Maladie de Hashimoto/diagnostic , Maladie de Hashimoto/immunologie , Interleukine-4/analyse , Lymphocytes auxiliaires Th2/immunologie , Adulte , Cytokines/sang , Diagnostic différentiel , Femelle , Cytométrie en flux , Maladie de Hashimoto/complications , Humains , Interféron gamma/biosynthèse , Interleukine-2/analyse , Numération des lymphocytes , Mâle , Adulte d'âge moyen , Polyendocrinopathies auto-immunes/complications , Polyendocrinopathies auto-immunes/diagnostic , Lymphocytes auxiliaires Th1/immunologieRÉSUMÉ
The molecular mechanism by which the lipido-sterolic extract of Serenoa repens (LSESr, Permixon) affects prostate cells remains to be fully elucidated. In androgen-independent PC3 prostate cancer cells, the LSESr-induced effects on proliferation and apoptosis were evaluated by counting cells and using a FACScan cytofluorimeter. PC3 cells were stained with JC-1 dye to detect mitochondrial membrane potential. Cell membrane lipid composition was evaluated by thin layer chromatography and gas chromatographic analysis. Akt phosphorylation was analyzed by Western blotting and cellular ultrastructure through electron microscopy. LSESr (12.5 and 25 microg/ml) administration exerted a biphasic action by both inhibiting proliferation and stimulating apoptosis. After 1 h, it caused a marked reduction in the mitochondrial potential, decreased cholesterol content and modified phospholipid composition. A decrease in phosphatidylinositol-4,5-bisphosphate (PIP2) level was coupled with reduced Akt phosphorylation. After 24 h, all of these effects were restored to pre-treatment conditions; however, the saturated (SFA)/unsaturated fatty acid (UFA) ratio increased, mainly due to a significant decrease in omega 6 content. The reduction in cholesterol content could be responsible for both membrane raft disruption and redistribution of signaling complexes, allowing for a decrease of PIP2 levels, reduction of Akt phosphorylation and apoptosis induction. The decrease in omega 6 content appears to be responsible for the prolonged and more consistent increase in the apoptosis rate and inhibition of proliferation observed after 2-3 days of LSESr treatment. In conclusion, LSESr administration results in complex changes in cell membrane organization and fluidity of prostate cancer cells that have progressed to hormone-independent status.
Sujet(s)
Antagonistes des androgènes/pharmacologie , Lignée cellulaire tumorale , Membrane cellulaire/effets des médicaments et des substances chimiques , Préparations à base de plantes/pharmacologie , Tumeurs de la prostate , Serenoa/composition chimique , Apoptose/effets des médicaments et des substances chimiques , Lignée cellulaire tumorale/cytologie , Lignée cellulaire tumorale/effets des médicaments et des substances chimiques , Membrane cellulaire/composition chimique , Prolifération cellulaire/effets des médicaments et des substances chimiques , Humains , Mâle , Lipides membranaires/composition chimique , Lipides membranaires/métabolisme , Potentiel de membrane mitochondriale/effets des médicaments et des substances chimiques , Potentiel de membrane mitochondriale/physiologie , Phytothérapie , Préparations à base de plantes/composition chimique , Tumeurs de la prostate/métabolisme , Tumeurs de la prostate/anatomopathologie , Protéines proto-oncogènes c-akt/métabolismeRÉSUMÉ
Stimulus reduction is an effective way to study visual performance. Cues such as surface characteristics, colour and inner lines can be removed from stimuli, revealing how the change affects recognition and neural processing. An extreme reduction is the removal of the very stimulus, defining it with illusory lines. Perceived boundaries without physical differences between shape and background are called illusory (or subjective) contours. Illusory and real contours activate early stages of the macaque visual pathway in similar ways. However, data relating to the processing of illusory contours in higher visual areas are scarce. We recently reported how illusory contours based on abutting-line gratings affect neurones in the monkey inferotemporal cortex, an area essential for object and shape vision. We now present data on how inferotemporal cortical neurones of monkeys react to another type of shapes, the Kanizsa figures. A set of line drawings, silhouettes, their illusory contour-based counterparts, and control shapes have been presented to awake, fixating rhesus monkeys while single-cell activity was recorded in the anterior part of the inferotemporal cortex. Most of the recorded neurones were responsive and selective to shapes presented as illusory contours. Shape selectivity was proved to be different for line drawings and illusory contours, and also for silhouettes and illusory contours. Neuronal response latencies for Kanizsa figures were significantly longer than those for line drawings and silhouettes. These results reveal differences in processing for Kanizsa figures and shapes having real contours in the monkey inferotemporal cortex.
Sujet(s)
Illusions/physiologie , Reconnaissance visuelle des formes/physiologie , Lobe temporal/physiologie , Cortex visuel/physiologie , Voies optiques/physiologie , Potentiels d'action/physiologie , Animaux , Cartographie cérébrale , Sensibilité au contraste/physiologie , Électrophysiologie , Macaca , Imagerie par résonance magnétique , Neurones/physiologie , Tests neuropsychologiques , Stimulation lumineuse , Temps de réaction/physiologie , Traitement du signal assisté par ordinateur , Lobe temporal/anatomie et histologie , Cortex visuel/anatomie et histologie , Voies optiques/anatomie et histologieRÉSUMÉ
OBJECTIVES: To investigate whether the results of optical platelet aggregometry indicate the risk of recurrent ischemic events. MATERIALS AND METHODS: Cerebro- and cardiovascular patients taking aspirin for at least 30 days were studied retrospectively. Ischemic vascular events occurring prior to testing and the presence of vascular risk factors were recorded. RESULTS: 241 subjects were included. Among the 78 patients (32.4%) who displayed recurrent vascular episodes, the age (62.5 +/- 10.6 vs. 58.4 +/- 11.6, P = 0.009) and the proportion of hypertensives (80.8% vs. 68.1%, P = 0.040) were significantly higher when compared with the participants who exhibited single events. The degree of platelet aggregation did not differ significantly between the patients with and those without recurrent episodes. Logistic regression analysis identified only age (OR 1.033, 95% CI 1.008-1.058, P = 0.010), and not aggregation values, as a risk condition for recurrent vascular episodes. CONCLUSIONS: Results of optical platelet aggregometry were not indicative of the risk of recurrent vascular events. The role of conventional risk factors appeared to be more important.
Sujet(s)
Acide acétylsalicylique/pharmacologie , Encéphalopathie ischémique/diagnostic , Encéphalopathie ischémique/traitement médicamenteux , Agrégation plaquettaire/effets des médicaments et des substances chimiques , Accident vasculaire cérébral/diagnostic , Accident vasculaire cérébral/traitement médicamenteux , Sujet âgé , Acide acétylsalicylique/usage thérapeutique , Encéphalopathie ischémique/physiopathologie , Résistance aux substances/effets des médicaments et des substances chimiques , Résistance aux substances/physiologie , Traitement automatique des données/méthodes , Traitement automatique des données/statistiques et données numériques , Femelle , Humains , Accident ischémique transitoire/diagnostic , Accident ischémique transitoire/traitement médicamenteux , Accident ischémique transitoire/physiopathologie , Mâle , Adulte d'âge moyen , Infarctus du myocarde/diagnostic , Infarctus du myocarde/traitement médicamenteux , Infarctus du myocarde/physiopathologie , Agrégation plaquettaire/physiologie , Antiagrégants plaquettaires/pharmacologie , Antiagrégants plaquettaires/usage thérapeutique , Valeur prédictive des tests , Récidive , Études rétrospectives , Facteurs de risque , Accident vasculaire cérébral/physiopathologieRÉSUMÉ
OBJECTIVES: We speculated that in patients with hypercholesterolemia CD40L overexpression could depend on low-density lipoprotein (LDL)-induced enhanced intraplatelet formation of O(2)*(-) and statin could reduce platelet CD40L via interference with platelet O(2)*(-) production. BACKGROUND: CD40L is a protein with inflammatory and thrombotic properties. CD40L is upregulated in platelets from hypercholesterolemic (HC) patients but the underlying mechanism is unclear. METHODS: Collagen-induced platelet CD40L and platelet O(2)*(-) expression were investigated in 40 HC patients and 40 healthy subjects. HC patients were then randomized to either a diet (n = 20) (group A) or atorvastatin 10 mg day (n = 20) (group B); the above variables were measured at baseline and after 3 and 30 days of treatment. O(2)*(-) and CD40L were also measured in vitro in LDL-treated platelets with or without nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor or atorvastatin added. RESULTS: Compared with controls, HC patients showed higher values of platelet CD40L (P < 0.001) and O(2)*(-) (P < 0.001). Platelet CD40L was significantly correlated with O(2)*(-) (P < 0.001). The interventional trial showed no changes in group A and a significant and parallel decrease in platelet CD40L (P < 0.001) and O(2)*(-) (P < 0.001) in group B. In vitro studies demonstrated that LDL-induced platelet CD40L and GP IIb/IIIa (PAC1 binding) activation via the NADPH oxidase pathway. CD40L upregulation was counteracted by atorvastatin in a dose-dependent fashion. CONCLUSIONS: This study suggests that in patients with hypercholesterolemia platelet CD40L is upregulated via NADPH oxidase-dependent O(2)*(-) generation. Atorvastatin downregulated CD40L with an oxidative stress-mediated mechanism likely involving platelet NADPH oxidase, an effect that seemed to be independent of its cholesterol-lowering action.
Sujet(s)
Plaquettes/immunologie , Plaquettes/métabolisme , Ligand de CD40/sang , Acides heptanoïques/usage thérapeutique , Inhibiteurs de l'hydroxyméthylglutaryl-CoA réductase/usage thérapeutique , Hypercholestérolémie/sang , Hypercholestérolémie/traitement médicamenteux , Pyrroles/usage thérapeutique , Atorvastatine , Autoanticorps/sang , Plaquettes/effets des médicaments et des substances chimiques , Études cas-témoins , Antienzymes/usage thérapeutique , Femelle , Humains , Hypercholestérolémie/immunologie , Techniques in vitro , Mâle , Adulte d'âge moyen , NADPH oxidase/antagonistes et inhibiteurs , NADPH oxidase/sang , Stress oxydatif , Complexe glycoprotéique IIb-IIIa de la membrane plaquettaire/immunologie , Superoxydes/sang , Régulation positiveRÉSUMÉ
We describe a 10-year-old boy with glycogen storage disease type Ib (GSD Ib) with neutropenia and neutrophil dysfunction who never suffered from severe recurrent infections. Lymphocyte subpopulations and assay of intracellular cytokines (IL-2, IL-4 and IFN-gamma) showed a pattern of lymphocyte activation suggesting a shift of T(H)1/T(H)2 balance towards a T(H)1 response. This is the first report of GSD Ib without severe recurrent infections in spite of neutropenia and neutrophil dysfunction.
Sujet(s)
Glycogénose de type I/complications , Glycogénose de type I/anatomopathologie , Infections/diagnostic , Neutropénie/anatomopathologie , Granulocytes neutrophiles/anatomopathologie , Enfant , Humains , Interféron gamma/biosynthèse , Interleukine-2/biosynthèse , Interleukine-4/biosynthèse , Activation des lymphocytes , Mâle , Récidive , Lymphocytes auxiliaires Th1/métabolisme , Lymphocytes auxiliaires Th2/métabolismeRÉSUMÉ
Oxidative stress-mediated LDL modification has a key role in initiation of the atherosclerotic process. Platelets produce reactive oxidant species (ROS) upon stimulation with agonist, but it is uncertain whether they are able to oxidatively modify LDL. Human platelets taken from healthy subjects were incubated with LDL, then stimulated with collagen. Compared with unstimulated platelets, collagen-stimulated platelets induced LDL modification as shown by enhanced conjugated dienes and lysophosphatidylcholine formation, electrophoretic mobility, Apo B-100 degradation, and monocyte LDL uptake. Activated platelets also induced a marked reduction of vitamin E contained in LDL. A significant inhibition of LDL oxidation was observed in platelets treated with arachidonyl trifluomethyl ketone (AACOCF3), an inhibitor of phospholipase A2. The experiments reported above were also conducted in patients with hereditary deficiency of gp91phox, the central core of NADPH oxidase, and in patients with hypercholesterolemia. Platelets from gp91 phox-deficient patients produced a small amount of ROS and weakly modified LDL. Conversely, platelets from hypercholesterolemic patients showed enhanced ROS formation and oxidized LDL more than platelets from healthy subjects. This study provides evidence that platelets modify LDL via NADPH oxidase-mediated oxidative stress, a phenomenon that could be dependent on arachidonic acid activation. This finding suggests a role for platelets in favoring LDL accumulation within atherosclerotic plaque.
Sujet(s)
Plaquettes/métabolisme , Lipoprotéines LDL/métabolisme , Glycoprotéines membranaires/métabolisme , Monocytes/métabolisme , NADPH oxidase/métabolisme , Électrophorèse sur gel de polyacrylamide , Humains , Hypercholestérolémie/métabolisme , Hypercholestérolémie/anatomopathologie , Lysolécithine/isolement et purification , NADPH Oxidase 2 , Oxydoréduction , Espèces réactives de l'oxygène/métabolisme , Vitamine E/métabolismeRÉSUMÉ
AIM: The liver as a solid graft has a known immunological privilege. Its tolerogenic property has been demonstrated in rodents. In humans the onset of chronic rejection and the severity of such complication is less frequent after liver transplantation compared to other organs. The underlying events whose effect is graft acceptance instead of rejection should be further investigated. Their control could open new ways to decrease the need for long-term immunosuppression after transplantation of other organs. Aim of this study is to evaluate a model of liver transplantation in swine as a preliminary step for immunological studies. METHODS: Ten outbred Landrace/Large White mismatched swine underwent to liver transplantation with a simple passive portocaval jugular bypass. The onset of rejection was monitored daily by liver function test. After death or sacrifice the liver parenchyma was studied to evaluate tissue damage and inflammatory infiltrate. RESULTS: The postoperative liver function showed a critical period for organ rejection about postoperative day 5. The animals that survived longer were sacrificed with a normal biochemical hepatic function. However, histology consistently showed a pattern of mild rejection in a still preserved architecture. CONCLUSIONS: The evidence of a prolonged liver function in a rejecting model of liver transplantation makes this model suitable for studies of tolerance induction.
Sujet(s)
Rejet du greffon/physiopathologie , Transplantation hépatique , Tolérance à la transplantation , Animaux , Test d'histocompatibilité , Tests de la fonction hépatique , Modèles animaux , Anastomose portocave chirurgicale , Suidae , Transplantation homologueRÉSUMÉ
AIM: Organ transplantation is the most effective treatment for several degenerative end-stage diseases. While the mainstream immunosuppression can achieve satisfactory results, the therapy has either side effects and flaws. The golden target to reach should be a stable tolerance with the transplanted organ accepted without a long term drug administration. Recent studies demonstrated a tolerogenic effect of spleen cells. Aim of this study is to evaluate a model of combined spleen and whole organ transplantation in a significant preclinical setting in swine. METHODS: Twenty-five outbred Landrace/Large-White swine underwent combined spleen/kidney transplantation (SKTx). The experiments were stratified into 3 groups per randomization. Group 1 (N=7) received kidney transplantation (KTx) alone with no immunosuppressive treatment. Group 2 (N=9) had a combined KTx and whole graft spleen Tx. Group 3 (N=9) had KTx and spleen cells (DST), injected through the portal vein. Renal lab tests were collected to evaluate the onset of rejection. Survivals were evaluated as well. The end-point of the study was at onset of kidney failure or at the limit of 60 postoperative day (POD) in non-rejecting animals. Tissue samples were collected to evaluate grade and severity of rejection. RESULTS: Controls died from kidney failure within 10(th) POD. Group 2 and 3, had a delayed renal graft rejection and an overall prolonged graft survival. Whole graft and spleen cells injection share this effect, while spleen administration through the portal route proved a superior effect, which is significant compared to controls (Kaplan Meier survival analysis P<0.05). CONCLUSIONS: These results, from a non immunosuppressed setting, suggest that spleen plays a key role as an immunomodulatory organ.
Sujet(s)
Immunosuppression thérapeutique/méthodes , Transplantation rénale , Rate/transplantation , Animaux , Femelle , Transplantation rénale/anatomopathologie , Mâle , Rate/anatomopathologie , SuidaeRÉSUMÉ
Aim of this study was to analyse the relationship between the plasma levels of polyphenols and the antioxidant activity of red and white wine. Twenty healthy subjects (HS) were randomly allocated to drink 300 ml of red (n = 10) or white n = 10 wine for 15 days. Ten HS who refrained from any alcohol beverage for 15 days were used as control. Urinary PGF-2alpha-III, a marker of oxidative stress and plasma levels of polyphenols were measured. Urinary PGF-2alpha-III significantly fell in subjects taking wine with a higher percentage decrease in subjects given red wine (-38.5 +/- 6%, p < 0.001) than in those given white wine (-23.1 +/- 6%). Subjects taking red wine had higher plasma polyphenols than those taking white wine (1.9 +/- 0.6 microM versus 1.5 +/- 0.33 microM, p < 0.001). Plasma polyphenols were inversely correlated with urinary PGF2alpha (r = 0.77, p < 0.001). No changes of urinary isoprostanes were observed in subjects who refrained from wine intake. In vitro study demonstrated that only a mixture of polyphenols, all in a range corresponding to that found in human circulation, inhibited LDL oxidation and PKC-mediated NADPH oxidase activation. Such inhibitory effects were more marked using the concentrations of polyphenols detected in human circulation after red wine intake. This study shows that red wine is more antioxidant than white wine in virtue of its higher content of polyphenols, an effect that may be dependent upon a synergism among polyphenols.
Sujet(s)
Flavonoïdes/sang , Flavonoïdes/pharmacologie , Stress oxydatif/effets des médicaments et des substances chimiques , Phénols/sang , Phénols/pharmacologie , Vin , Dinoprost/urine , Flavonoïdes/analyse , Humains , Lipoprotéines LDL/métabolisme , Oxydoréduction , Phénols/analyse , Polyphénols , Protéine kinase C/effets des médicaments et des substances chimiques , Vin/analyseRÉSUMÉ
BACKGROUND: CD40 ligand (CD40L) expression on platelets is mediated by agonists, but the underlying mechanism is still unclear. METHODS AND RESULTS: CD40L expression was measured in platelets from healthy subjects both with and without the addition of antioxidants or a phospholipase A2 (PLA2) inhibitor and in platelets from 2 patients with an inherited deficiency of gp91phox. Immunoprecipitation analysis was also performed to determine whether normal platelets showed gp91phox expression. Unlike catalase and mannitol, superoxide dismutase inhibited agonist-induced platelet CD40L expression in healthy subjects. Immunoprecipitation analysis also showed that platelets from healthy subjects expressed gp91phox. In 2 male patients with inherited gp91phox deficiency, collagen-, thrombin-, and arachidonic acid-stimulated platelets showed an almost complete absence of superoxide anion (O(2)(-)) and CD40L expression. Incubation of platelets from healthy subjects with a PLA2 inhibitor almost completely prevented agonist-induced O(2)(-) and CD40L expression. CONCLUSIONS: These data provide the first evidence that platelet CD40L expression occurs via arachidonic acid-mediated gp91phox activation.
Sujet(s)
Acide arachidonique/métabolisme , Plaquettes/métabolisme , Ligand de CD40/biosynthèse , Glycoprotéines membranaires/physiologie , NADPH oxidase/physiologie , Espèces réactives de l'oxygène/métabolisme , Adulte , Antioxydants/pharmacologie , Acides arachidoniques/pharmacologie , Acide acétylsalicylique/pharmacologie , Plaquettes/effets des médicaments et des substances chimiques , Ligand de CD40/sang , Ligand de CD40/génétique , Catalase/pharmacologie , Collagène/pharmacologie , Granulomatose septique chronique/sang , Granulomatose septique chronique/enzymologie , Humains , Mâle , Mannitol/pharmacologie , Glycoprotéines membranaires/déficit , Glycoprotéines membranaires/génétique , NADPH Oxidase 2 , NADPH oxidase/déficit , NADPH oxidase/génétique , Phospholipases A/antagonistes et inhibiteurs , Phospholipases A2 , Activation plaquettaire/effets des médicaments et des substances chimiques , Antiagrégants plaquettaires/pharmacologie , Superoxide dismutase/pharmacologie , Superoxydes/métabolisme , Thrombine/pharmacologieRÉSUMÉ
Apoptosis was induced in cultured cerebellar granule cells by lowering extracellular K+ concentrations (usually from 25 to 10 mM). The apoptotic phenotype was preceded by an early and transient increase in the intracellular levels of the disialoganglioside, GD3, which behaves as a putative pro-apoptotic factor. We examined whether activation of Fas receptor mediates the increase in GD3 formation in granule cells committed to die. Degenerating granule cells showed increased expression of both Fas receptor and its ligand (Fas-L), at times that coincided with the increase in GD3 levels and the induction of GD3 synthase mRNA. Addition of neutralizing anti-Fas-L antibodies reduced the extent of 'low-K+'-induced apoptosis and abolished the increase in GD3 levels and GD3 synthase mRNA. Similar reductions were observed in cultures prepared from gld or lpr mice, which harbor loss-of-function mutations of Fas-L and Fas receptor, respectively. In addition, exogenous application of soluble Fas-L further enhanced both the increase in GD3 formation and cell death in cultured granule cells switched from 25 into 10 mM K+. We conclude that activation of Fas receptor is entirely responsible for the increase in GD3 levels and contributes to the development of apoptosis by trophic deprivation in cultured cerebellar granule cells.
Sujet(s)
Apoptose/physiologie , Cervelet/cytologie , Gangliosides/métabolisme , Neurones/métabolisme , Antigènes CD95/métabolisme , Animaux , Animaux nouveau-nés , Anticorps/pharmacologie , Apoptose/effets des médicaments et des substances chimiques , Technique de Western/méthodes , Caspase-3 , Caspases/métabolisme , Cellules cultivées , Céramides/analyse , Chromatine/métabolisme , Relation dose-effet des médicaments , Ligand de Fas , Technique d'immunofluorescence/méthodes , Galactosyltransferases/génétique , Galactosyltransferases/métabolisme , Humains , Glycoprotéines membranaires/déficit , Glycoprotéines membranaires/immunologie , Souris , Souches mutantes de souris , Neurones/effets des médicaments et des substances chimiques , Potassium/métabolisme , ARN messager/biosynthèse , Rats , Rat Sprague-Dawley , RT-PCR/méthodes , Facteurs temps , Facteur de nécrose tumorale alpha/immunologie , Antigènes CD95/génétiqueRÉSUMÉ
Carnitine is a physiological cellular constituent that favors intracellular fatty acid transport, whose role on platelet function and O(2) free radicals has not been fully investigated. The aim of this study was to seek whether carnitine interferes with arachidonic acid metabolism and platelet function. Carnitine (10-50 microM) was able to dose dependently inhibit arachidonic acid incorporation into platelet phospholipids and agonist-induced arachidonic acid release. Incubation of platelets with carnitine dose dependently inhibited collagen-induced platelet aggregation, thromboxane A(2) formation, and Ca(2+) mobilization, without affecting phospholipase A(2) activation. Furthermore, carnitine inhibited platelet superoxide anion (O(2)(-)) formation elicited by arachidonic acid and collagen. To explore the underlying mechanism, arachidonic acid-stimulated platelets were incubated with NADPH. This study showed an enhanced platelet O(2)(-) formation, suggesting a role for NADPH oxidase in arachidonic acid-mediated platelet O(2)(-) production. Incubation of platelets with carnitine significantly reduced arachidonic acid-mediated NADPH oxidase activation. Moreover, the activation of protein kinase C was inhibited by 50 microM carnitine. This study shows that carnitine inhibits arachidonic acid accumulation into platelet phospholipids and in turn platelet function and arachidonic acid release elicited by platelet agonists.
Sujet(s)
Acide arachidonique/métabolisme , Plaquettes/physiologie , Carnitine/pharmacologie , Stress oxydatif/physiologie , Plaquettes/métabolisme , Protéines du sang/métabolisme , Calcium/métabolisme , Humains , Membranes intracellulaires/métabolisme , Concentration osmolaire , Phospholipides/métabolisme , Phosphorylation , Agrégation plaquettaire/physiologie , Espèces réactives de l'oxygène/métabolisme , Thromboxane A2/biosynthèseRÉSUMÉ
Gangliosides are sialic acid-containing glycosphingolipids present in the outer leaflet of the plasma membrane of many cell types where they modulate adhesive processes. The main population of glycolipids in resting platelets is represented by ganglioside M3 (GM3). It has been demonstrated that following platelet activation ganglioside D3 (GD3) is rapidly formed from the GM3 pool. The present study was designed to evaluate the link between platelet activation and GD3 expression and to verify whether this ganglioside might play a role in modulating signal transduction events. Our results suggest that following platelet activation, GD3 is rapidly expressed on the platelet surface and internalised to the cytoskeleton where it transiently associates first with the Src family tyrosine kinase Lyn then with the Fc receptor gamma chain. This sequence of events ultimately leads to an enhanced CD32 (the Fc receptor isoform present in platelets) expression on the platelet membrane. These data drive us to speculate that GD3 might act as second messenger in the activatory cascade, which leads to CD32 expression and triggers platelet adhesion and spreading to the subendothelial matrix.
Sujet(s)
Plaquettes/métabolisme , Gangliosides/métabolisme , Activation plaquettaire , Récepteurs du fragment Fc des IgG/métabolisme , ADP/pharmacologie , Acide arachidonique/métabolisme , Acide arachidonique/pharmacologie , Acides arachidoniques/pharmacologie , Plaquettes/effets des médicaments et des substances chimiques , Technique de Western , Cellules cultivées , Collagène/pharmacologie , Cytosquelette/effets des médicaments et des substances chimiques , Cytosquelette/métabolisme , Antienzymes/pharmacologie , Gangliosides/immunologie , Gangliosides/pharmacologie , Humains , Phospholipases A/antagonistes et inhibiteurs , Phospholipases A/métabolisme , Récepteurs du fragment Fc des IgG/effets des médicaments et des substances chimiques , src-Family kinases/effets des médicaments et des substances chimiques , src-Family kinases/métabolismeRÉSUMÉ
We induced apoptosis in primary cultures of cerebellar granule neurons by switching the growing medium into a medium containing lower concentrations of K(+) (5 or 10 mM instead of 25 mM) or, alternatively, by addition of staurosporine. The apoptotic phenotype was always preceded by an early increase in the intracellular levels of the disialoganglioside GD3, which peaked at 2-6 h and returned back to normal at 12 h. GD3 synthase, the enzyme that forms GD3 from the monosialoganglioside GM3, was also induced at early times after the induction of apoptosis in granule cells. Immunofluorescent staining showed that GD3 increased in neuronal cell bodies and neurites, but was never localized in cell nuclei. In cultures switched into a low K(+)-containing medium, exogenously applied GD3, but not the disialoganglioside GD1a, accelerated the development of neuronal apoptosis. In contrast, the antisense-induced knock-down of GD3 synthase was protective against granule cell death induced by lowering extracellular K(+) from 25 to 10 - but not 5 - mM. These results demonstrate that an early and transient increase in GD3 synthesis is one of the factors that contribute to the induction of neuronal apoptosis in culture.
Sujet(s)
Apoptose/physiologie , Gangliosides/physiologie , Neurones/physiologie , Animaux , Cellules cultivées , Technique d'immunofluorescence , Rats , Rat Sprague-DawleyRÉSUMÉ
The presence of nuclear magnetic resonance (NMR)-visible mobile lipid (ML) domains in apoptotic lymphoblasts suggests alterations in neutral lipid metabolism and compartmentation during programmed cell death. The detection of similar ML signals in activated lymphocytes raises questions about common mechanisms of ML formation during apoptosis and upon lymphoblast stimulation. Structure and subcellular localization of ML domains were therefore investigated by NMR, fluorescence and electron microscopy in Jurkat T-lymphoblasts either induced to apoptosis (by anthracyclines or dexamethasone or by serum deprivation) or activated by phorbol myristate acetate (PMA) plus ionomycin. ML contents in drug-treated cells correlated linearly with apoptosis, irrespective of the specific inducer and cell cycle arrest phase (r = 0.993, P < 0.001). Similar ML levels were measured in drug-induced apoptotic cells (A approximately 30-40%) and in non-apoptotic PMA/ionomycin-treated lymphoblasts (72 h). Lower ML contents were instead formed in serum-deprived apoptotic cells, with respect to controls. Increases in ML signals were associated, in either apoptotic or activated cells, with the accumulation of cytoplasmic, osmophilic lipid bodies (diameter < or = 1.0 microm), surrounded by own membrane, possessing intramembrane particles. The results support the hypothesis that ML are formed in the cytoplasm of drug-induced apoptotic cells during an early, 'biochemically active' phase of programmed cell death.
Sujet(s)
Cytoplasme/métabolisme , Lipides/analyse , Lymphocytes T/métabolisme , Apoptose , Colorants fluorescents , Cryofracture , Humains , Ionomycine , Cellules Jurkat , Activation des lymphocytes , Spectroscopie par résonance magnétique , Microscopie électronique , Oxazines , Lymphocytes T/ultrastructure , 12-Myristate-13-acétate de phorbolRÉSUMÉ
BACKGROUND: Epidemiologic studies have shown an inverse relation between moderate consumption of red wine and cardiovascular disease. Studies have shown that red wine and its component flavonoids inhibit in vivo platelet activation, but the underlying mechanism has not yet been identified. OBJECTIVE: Because we showed previously that collagen-induced platelet aggregation is associated with a burst of hydrogen peroxide, which in turn contributes to stimulating the phospholipase C pathway, the aim of this study was to investigate whether flavonoids synergize in inhibiting platelet function and interfere with platelet function by virtue of their antioxidant effect. DESIGN: We tested the effect of 2 flavonoids, quercetin and catechin, on collagen-induced platelet aggregation and hydrogen peroxide and on platelet adhesion to collagen. RESULTS: Catechin (50-100 micromol/L) and quercetin (10-20 micromol/L) inhibited collagen-induced platelet aggregation and platelet adhesion to collagen. The combination of 25 micromol catechin/L and 5 micromol quercetin/L, neither of which had any effect on platelet function when used alone, significantly inhibited collagen-induced platelet aggregation and platelet adhesion to collagen. Such a combination strongly inhibited collagen-induced hydrogen peroxide production, calcium mobilization, and 1,3,4-inositol triphosphate formation. CONCLUSIONS: These data indicate that flavonoids inhibit platelet function by blunting hydrogen peroxide production and, in turn, phospholipase C activation and suggest that the synergism among flavonoids could contribute to an understanding of the relation between the moderate consumption of red wine and the decreased risk of cardiovascular disease.