RESUMO
Background: Negative neurocognitive bias is a core feature of depression that is reversed by antidepressant drug treatment. However, it is unclear whether modulation of neurocognitive bias is a common mechanism of distinct biological treatments. This randomized controlled functional magnetic resonance imaging study explored the effects of a single electroconvulsive therapy session on self-referent emotional processing. Methods: Twenty-nine patients with treatment-resistant major depressive disorder were randomized to one active or sham electroconvulsive therapy session at the beginning of their electroconvulsive therapy course in a double-blind, between-groups design. The following day, patients were given a self-referential emotional word categorization test and a free recall test. This was followed by an incidental word recognition task during whole-brain functional magnetic resonance imaging at 3T. Mood was assessed at baseline, on the functional magnetic resonance imaging day, and after 6 electroconvulsive therapy sessions. Data were complete and analyzed for 25 patients (electroconvulsive therapy: n = 14, sham: n = 11). The functional magnetic resonance imaging data were analyzed using the FMRIB Software Library randomize algorithm, and the Threshold-Free Cluster Enhancement method was used to identify significant clusters (corrected at P < .05). Results: A single electroconvulsive therapy session had no effect on hippocampal activity during retrieval of emotional words. However, electroconvulsive therapy reduced the retrieval-specific neural response for positive words in the left frontopolar cortex. This effect occurred in the absence of differences between groups in behavioral performance or mood symptoms. Conclusions: The observed effect of electroconvulsive therapy on prefrontal response may reflect early facilitation of memory for positive self-referent information, which could contribute to improvements in depressive symptoms including feelings of self-worth with repeated treatments.
Assuntos
Encéfalo/fisiopatologia , Transtorno Depressivo Maior/fisiopatologia , Transtorno Depressivo Maior/terapia , Eletroconvulsoterapia , Emoções/fisiologia , Memória/fisiologia , Adulto , Antidepressivos/uso terapêutico , Encéfalo/diagnóstico por imagem , Mapeamento Encefálico , Transtorno Depressivo Maior/diagnóstico por imagem , Transtorno Depressivo Maior/psicologia , Transtorno Depressivo Resistente a Tratamento/diagnóstico por imagem , Transtorno Depressivo Resistente a Tratamento/fisiopatologia , Transtorno Depressivo Resistente a Tratamento/psicologia , Transtorno Depressivo Resistente a Tratamento/terapia , Método Duplo-Cego , Feminino , Humanos , Idioma , Imageamento por Ressonância Magnética , Masculino , Testes Neuropsicológicos , AutoimagemRESUMO
BACKGROUND: The British Society of Audiology has produced clear guidelines as to how otoscopy should be undertaken; however, no nationally recognised guidelines exist for the wider clinical community. Images of otoscopy appear in many books, journals, magazines and websites. OBJECTIVE: This study aimed to determine the rate of non-compliance with good practice in images of otoscopy, the seriousness of the breach, and whether this is more common in sites for professionals or the general public. METHOD: Google Images was searched using the terms 'otoscopy' and 'ear examination'. A total of 200 images were identified and collated. The images were reviewed for compliance with good practice standards. RESULTS: Only 12.75 per cent of the images were graded as having no breach of good practice standards. CONCLUSION: Professional websites have a responsibility to show best practice. When choosing an image, the source of the image needs to be carefully considered.
Assuntos
Recursos Audiovisuais/normas , Fidelidade a Diretrizes/estatística & dados numéricos , Otoscopia/normas , Guias de Prática Clínica como Assunto/normas , Audiologia/normas , Inglaterra , Humanos , Internet , Sociedades Médicas/normasRESUMO
Non-uniform sampling combined with maximum entropy reconstruction is a powerful technique used in multi-dimensional NMR spectroscopy to reduce sample measurement time. We adapted this technique to the pulse EPR experiment hyperfine sublevel correlation (HYSCORE) and show that experimental times can be shortened by approximately an order of magnitude as compared to conventional linear sampling with negligible loss of information.
RESUMO
BACKGROUND AND PURPOSE: Multiple antibiotic resistant strains of plague are emerging, driving a need for the development of novel antibiotics effective against Yersinia pestis. DNA adenine methylation regulates numerous fundamental processes in bacteria and alteration of DNA adenine methlytransferase (Dam) expression is attenuating for several pathogens, including Y. pestis. The lack of a functionally similar enzyme in humans makes Dam a suitable target for development of novel therapeutics for plague. EXPERIMENTAL APPROACH: Compounds were evaluated for their ability to inhibit Dam activity in a high-throughput screening assay. DNA was isolated from Yersinia grown in the presence of lead compounds and restricted to determine the effect of inhibitors on DNA methylation. Transcriptional analysis was undertaken to determine the effect of an active inhibitor on virulence-associated phenotypes. KEY RESULTS: We have identified a series of aryl stibonic acids which inhibit Dam in vitro. The most active, 4-stibonobenzenesulfonic acid, exhibited a competitive mode of inhibition with respect to DNA and a K(i) of 6.46 nM. One compound was found to inhibit DNA methylation in cultured Y. pestis. The effects of this inhibition on the physiology of the cell were widespread, and included altered expression of known virulence traits, including iron acquisition and Type III secretion. CONCLUSIONS AND IMPLICATIONS: We have identified a novel class of potent Dam inhibitors. Treatment of bacterial cell cultures with these inhibitors resulted in a decrease in DNA methylation. Expression of virulence factors was affected, suggesting these inhibitors may attenuate bacterial infectivity and function as antibiotics.
Assuntos
Antibacterianos/farmacologia , Vacina contra a Peste/farmacologia , DNA Metiltransferases Sítio Específica (Adenina-Específica)/antagonistas & inibidores , Yersinia pestis/efeitos dos fármacos , Yersinia pestis/enzimologia , Antibacterianos/química , Metilação de DNA/efeitos dos fármacos , Perfilação da Expressão Gênica , Humanos , Testes de Sensibilidade Microbiana , Vacina contra a Peste/química , Vacina contra a Peste/genética , Relação Estrutura-Atividade , Virulência/efeitos dos fármacos , Virulência/genética , Fatores de Virulência/metabolismo , Yersinia pestis/patogenicidadeRESUMO
Over approximately the last 15 years the electron paramagnetic resonance (EPR) technique of double electron electron resonance (DEER) has attracted considerable attention since it allows for the precise measurement of the dipole-dipole coupling between radicals and thus can lead to distance information between pairs of radicals separated by up to ca. 8 nm. The "deadtime free" 4-pulse DEER sequence is widely used but can suffer from poor sensitivity if the electron spin-echo decays too quickly to allow collection of a sufficiently long time trace. In this paper we present a method which takes advantage of the much greater sensitivity that the 3-pulse sequence offers over the 4-pulse sequence since the measured electron spin-echo intensity (for equal sequence lengths) is greater. By combining 3- and 4-pulse DEER time traces using a method coined DEER-Stitch (DEERS) accurate dipole-dipole coupling measurements can be made which combine the sensitivity of the 3-pulse DEER sequence with the deadtime free advantage of the 4-pulse DEER sequence. To develop the DEER-Stitch method three systems were measured: a semi-rigid bis-nitroxide labeled nanowire, the bis-nitroxide labeled protein CD55 with a distance between labels of almost 8 nm and a dimeric copper amine oxidase from Arthrobacter globiformis (AGAO).
Assuntos
Espectroscopia de Ressonância de Spin Eletrônica/métodos , Algoritmos , Amina Oxidase (contendo Cobre)/química , Arthrobacter/química , Antígenos CD55/química , Antígenos CD55/genética , Cristalização , Cisteína/química , DNA/química , DNA/genética , Humanos , Nanofios , Óxidos de Nitrogênio/química , Distribuição Normal , Proteínas/química , Marcadores de SpinRESUMO
The increased blood oxygenation level-dependent contrast-to-noise ratio at ultrahigh field (7 T) has been exploited in a comparison of the spatial location and strength of activation in high-resolution (1.5 mm isotropic) gradient echo (GE) and spin echo (SE), echo planar imaging data acquired during the execution of a simple motor task in five subjects. SE data were acquired at six echo times from 30 to 55 ms. Excellent fat suppression was achieved in the SE echo planar images using slice-selective gradient reversal. Threshold-free cluster enhancement was used to define regions of interest (ROIs) containing voxels showing significant stimulus-locked signal changes from the GE and average SE data. These were used to compare the signal changes and spatial locations of activated regions in SE and GE data. T(2) and T(2)* values were measured, with means of 48.3 ± 1.1 ms and 36.5 ± 3.4 ms in the SE ROI. In addition, we identified a dark band in SE images of the motor cortex corresponding to a region in which T(2) and T(2)* were significantly lower than in the surrounding grey matter. The fractional SE signal change in the ROI was found to vary linearly as a function of TE, with a slope that was dependent on the particular ROI assessed: the mean ΔR(2) value was found to be 0.85 ± 0.11 s(-1) for the SE ROI and -0.37 ± 0.05 s(-1) for the GE ROI. The fractional signal change relative to the shortest TE revealed that the largest signal change occurred at a TE of 45 ms outside of the dark band. At this TE, the ratio of the fractional signal change in GE and SE data was found to be 0.48 ± 0.05. Phase maps produced from high-resolution GE images spanning the right motor cortex were used to identify veins. The GE ROI was found to contain 18% more voxels overlying the venous mask than the SE ROI.
Assuntos
Imagem Ecoplanar/métodos , Imageamento por Ressonância Magnética/métodos , Córtex Motor/anatomia & histologia , Adulto , Mapeamento Encefálico/métodos , Feminino , Humanos , Processamento de Imagem Assistida por Computador/métodos , Imageamento por Ressonância Magnética/instrumentação , Masculino , Oxigênio/sangue , Sensibilidade e Especificidade , Adulto JovemRESUMO
A range of sol-gel synthesis conditions were used to prepare high surface area N-doped TiO(2) in the anatase phase. The N dopant was derived either from NH(3) in solution or from NH(3) gas bubbled through solution. Bulk N doping levels were determined by an inert gas fusion method and were compared with surface N doping levels determined by X-ray photoelectron spectroscopy. Comparison was also made with the concentration of paramagnetic species measured by electron spin resonance spectroscopy. It was found that both surface and bulk doping levels were typically around 3 orders of magnitude higher than the concentration of paramagnetic N-containing species. All N-doped samples showed higher visible region (lambda > 395 nm) photocatalytic activity than undoped anatase itself. It is argued that catalytic activity is associated with the presence of nitrogen bound to lattice oxygen to give (NO)'O which can be photoexcited to give (NO)(O)(X).
Assuntos
Nitrogênio/química , Titânio/química , Catálise , Espectroscopia de Ressonância de Spin Eletrônica , Oxigênio/química , Processos Fotoquímicos , Espectroscopia FotoeletrônicaRESUMO
Double electron-electron resonance (DEER) spectroscopy can determine, from measurement of the dipolar interaction, the distance and orientation between two paramagnetic centres in systems lacking long-range order such as powders or frozen solution samples. In spin systems with considerable anisotropy, the microwave pulses excite only a fraction of the electron paramagnetic resonance (EPR) spectrum and the resulting orientation selection needs to be explicitly taken into account if a meaningful distance and orientation is to be determined. Here, a general method is presented to analyze the dipolar interaction between two paramagnetic spin centres from a series of DEER traces recorded so that different orientations of the spin-spin vector are sampled. Delocalised spin density distributions and spin projection factors (as for example in iron-sulfur clusters), are explicitly included. Application of the analysis to a spin-labelled flavoprotein reductase/reduced iron-sulfur ferredoxin protein complex and a bi-radical with two Cu(ii) ions provides distance and orientation information between the radical centres. In the protein complex this enables the protein-protein binding geometry to be defined. Experimentally, orientationally selective DEER measurements are possible on paramagnetic systems where the resonator bandwidth allows the frequencies of pump and detection pulses to be separated sufficiently to excite enough orientations to define adequately the spin-spin vector.
Assuntos
Espectroscopia de Ressonância de Spin Eletrônica/métodos , Conformação Molecular , Algoritmos , Cobre/química , Ferredoxinas/química , Hidrogenase/química , Metaloporfirinas/química , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Rodopseudomonas/químicaRESUMO
AIMS: The mechanisms by which obesity confers increased cardiovascular risk and the effects of moderate weight loss on cardiovascular health are incompletely understood. We sought to characterize the preclinical changes in cardiac and vascular health that accompany obesity and the influence of lifestyle modification on these parameters. METHODS: Preclinical markers of vasculopathy in resistance vessels and conduit arteries and left ventricular structure and function were assessed in 39 obese subjects (BMI > 30 kg/m(2)) and 11 healthy weight controls. The influence of serum on cellular adhesion molecule (CAM) expression on human endothelial cells was studied ex vivo in a subgroup of 13 obese and nine healthy weight subjects. These analyses were repeated in all 17 of the obese subjects who complied with 4-9 months of lifestyle modification treatment (six with weight loss >5% and 11 with weight loss <5%). RESULTS: Compared with healthy weight controls, obese subjects had decreased peak hyperaemic forearm blood flow (p = 0.015), increased carotid intima-media thickness (p = 0.009), increased left ventricular wall thickness and volume and evidence of systolic and diastolic dysfunction as assessed using tissue Doppler imaging (S', p = 0.09; E'/A', p = 0.02), and serum from obese subjects increased the intercellular CAM-1 expression on human endothelial cells (p = 0.009). However, arterial endothelial function assessed by flow-mediated dilatation was not altered (p = 0.99). Lifestyle modification treatment resulted in potentially beneficial changes in fibrinogen (p = 0.003), HDL cholesterol (p = 0.05) and soluble vascular CAM-1 (p = 0.06). In subjects with weight loss greater than 5% of body weight, there was also a decrease in low-level inflammation (high-sensitivity C-reactive protein, p = 0.05), lipid peroxidation (thiobarbituric acid-reactive substances, p = 0.05) and triglycerides (p = 0.07). CONCLUSIONS: Obesity is associated with widespread alterations in cardiac and vascular structure and function. Moderate short-term weight loss by lifestyle modification results in some beneficial changes in serum profile; however, these are not accompanied by significant alterations to either cardiac or vascular structure and function.
Assuntos
Exercício Físico/fisiologia , Obesidade/fisiopatologia , Redução de Peso/fisiologia , Adulto , Biomarcadores/sangue , Artérias Carótidas/diagnóstico por imagem , Estudos de Casos e Controles , Distribuição de Qui-Quadrado , Selectina E/sangue , Ecocardiografia , Feminino , Humanos , Molécula 1 de Adesão Intercelular/sangue , Masculino , Obesidade/sangue , Obesidade/terapia , Estudos Prospectivos , Fluxo Sanguíneo Regional/fisiologia , Túnica Íntima/diagnóstico por imagem , Molécula 1 de Adesão de Célula Vascular/sangue , Disfunção Ventricular Esquerda/sangue , Disfunção Ventricular Esquerda/diagnóstico por imagemRESUMO
1. Ischaemia-reperfusion (IR) injury is an important contributor to tissue damage and has been shown to be attenuated by preconditioning (PC) in some animal models. A recent report has suggested that the forearm can be used for the study of this phenomenon in humans. We aimed to reproduce and further characterize this model. 2. Healthy young adult volunteers (mean (+/-SEM) age 32+/-6 years) were studied on two occasions. During one visit, IR alone was induced by 10 min of upper arm cuff occlusion, whereas on another occasion a PC stimulus (three 3 min cuff inflations) preceded IR. Endothelial function in the ischaemic arm was assessed by measuring arterial flow-mediated dilatation (FMD) and by calculation of forearm blood flow at baseline and 15 and 60 min after IR. Systemic venous blood was sampled from the non-ischaemic arm at baseline, after PC and at 2, 15 and 30 min after IR to assess neutrophil/leucocyte (CD11b) and platelet (bound glycoprotein IIb/IIIa and fibrinogen) activation, as well as numbers of platelet-leucocyte complexes, which were determined by flow cytometry. Because of a lack of measurable effects, the IR experiment was repeated with 20 min ischaemia in six subjects. 3. Five females and eight males completed the study. Flow-mediated dilatation was significantly impaired 30 min after IR (4.1 vs 6.2% at baseline; P<0.05);however, this was not significantly attenuated by ischaemic PC (FMD reduction at 30 min compared with baseline was 2.1+/-0.5% with IR alone and 2.6+/-1.4% with IR after PC; NS). No significant effect was seen on the number of platelet-leucocyte aggregates or on white cell or platelet activation after IR alone or after IR with PC (P>0.6 for all comparisons). Similar results were obtained in six subjects studied subjected to 20 min ischaemia. 4. In conclusion, in healthy young adults, brief periods of skeletal muscle ischaemia lead to arterial endothelial dysfunction, but no significant platelet or white cell activation. Preconditioning does not attenuate this effect on the endothelium. Further experiments with longer ischaemia times and varying PC stimuli may be necessary to produce measurable effects; however, this may prove difficult in conscious human subjects.
Assuntos
Antebraço/irrigação sanguínea , Antebraço/fisiopatologia , Precondicionamento Isquêmico , Traumatismo por Reperfusão/prevenção & controle , Traumatismo por Reperfusão/fisiopatologia , Adulto , Endotélio Vascular/fisiopatologia , Feminino , Citometria de Fluxo , Humanos , Leucócitos/fisiologia , Ativação de Macrófagos/fisiologia , Masculino , Neutrófilos/fisiologia , Fluxo Sanguíneo Regional/fisiologia , Vasodilatação/fisiologiaRESUMO
The interaction of Cu(II) with the ligand tdci (1,3,5-trideoxy-1,3,5-tris(dimethylamino)-cis-inositol) was studied both in the solid state and in solution. The complexes that were formed were also tested for phosphoesterase activity. The pentanuclear complex [Cu(5)(tdciH(-2))(tdci)(2)(OH)(2)(NO(3))(2)](NO(3))(4).6H(2)O consists of two dinuclear units and one trinuclear unit, having two shared copper(II) ions. The metal centers within the pentanuclear structure have three distinct coordination environments. All five copper(II) ions are linked by hydroxo/alkoxo bridges forming a Cu(5)O(6) cage. The Cu-Cu separations of the bridged centers are between 2.916 and 3.782 A, while those of the nonbridged metal ions are 5.455-5.712 A. The solution equilibria in the Cu(II)-tdci system proved to be extremely complicated. Depending on the pH and metal-to-ligand ratio, several differently deprotonated mono-, di-, and trinuclear complexes are formed. Their presence in solution was supported by mass, CW, and pulse EPR spectroscopic study, too. In these complexes, the metal ions are presumed to occupy tridentate [O(ax),N(eq),O(ax)] coordination sites and the O-donors of tdci may serve as bridging units between two metal ions. Additionally, deprotonation of the metal-bound water molecules may occur. The dinuclear Cu(2)LH(-3) species, formed around pH 8.5, provides outstanding rate acceleration for the hydrolysis of the activated phosphodiester bis(4-nitrophenyl)phosphate (BNPP). The second-order rate constant of BNPP hydrolysis promoted by the dinuclear complex (T = 298 K) is 0.95 M(-1) s(-1), which is ca. 47600-fold higher than that of the hydroxide ion catalyzed hydrolysis (k(OH)). Its activity is selective for the phosphodiester, and the hydrolysis was proved to be catalytic. The proposed bifunctional mechanism of the hydrolysis includes double Lewis acid activation and intramolecular nucleophilic catalysis.
Assuntos
Cobre , Inositol/química , Compostos Organofosforados/química , Diester Fosfórico Hidrolases/metabolismo , Catálise , Cristalografia por Raios X , Hidrólise , Indicadores e Reagentes , Inositol/análogos & derivados , Cinética , Ligantes , Modelos Químicos , Modelos Moleculares , Conformação Molecular , Potenciometria , EspectrofotometriaRESUMO
BACKGROUND AND AIMS: With increasing resources being spent on nutritional supplements, this study sought to evaluate the effect of introducing guidelines on prescribing of supplements, by auditing practice, prior to, and after the implementation of guidelines. METHODS: Prescribing practice was evaluated from patient interviews, and knowledge of health professionals examined from questionnaires from 50 GP practices. Training on the use of guidelines on prescribing supplements was implemented, incorporating a Nutritional Screening Tool and practical application of high-energy dietary advice, targeting GPs and Community Nurses. RESULTS: Education to GPs and Community Nurses significantly reduced total prescribing by 15% and reduced the levels of inappropriate prescribing from 77% to 59% due to an improvement in monitoring of patients prescribed supplements. Although knowledge regarding high-energy dietary advice for nutritionally 'at risk' patients did improve as a result of the training, this was not demonstrated in practice. This lack of relevant dietary advice remained the main reason that inappropriate supplement prescriptions remained high. CONCLUSION: Education on guidelines incorporating a Nutritional Screening Tool has proved to be an effective method of achieving more appropriate prescribing of supplements, suggesting the need for ongoing training of health professionals in Primary Care.
Assuntos
Enfermagem em Saúde Comunitária/educação , Suplementos Nutricionais/estatística & dados numéricos , Ciências da Nutrição/educação , Médicos de Família/educação , Atenção Primária à Saúde/normas , Idoso , Competência Clínica , Prescrições de Medicamentos/estatística & dados numéricos , Inglaterra , Feminino , Humanos , Masculino , Programas de Rastreamento , Avaliação Nutricional , Educação de Pacientes como Assunto , Guias de Prática Clínica como Assunto , Padrões de Prática Médica , Qualidade da Assistência à Saúde , Inquéritos e QuestionáriosRESUMO
Antiphospholipid (aPL) syndrome (APS) is characterized by thromboembolic events, thrombocytopenia, or recurrent miscarriage associated with aPL Abs with specificity for beta2-glycoprotein-I (beta2GPI). We recently reported that at least 44% of patients with the APS possess circulating type 1 (Th1) CD4+ T cells that proliferate and secrete IFN-gamma when stimulated with beta2GPI in vitro. In this study, we show that stimulation of PBMCs from 20 APS patients with beta2GPI induced substantial monocyte tissue factor (TF) (80 +/- 11 TF stimulation index (TF-SI)), whereas no induction was observed using PBMCs from 13 patients with aPL Abs without APS (6 +/- 1 TF-SI) or 7 normal and 7 autoimmune controls (5 +/- 1 and 3 +/- 1 TF-SI, respectively) (p < 0.0001). TF induction on monocytes by beta2GPI was dose dependent and required CD4+ T lymphocytes and class II MHC molecules. Because monocyte TF induction by beta2GPI was observed in all patients with APS, but not in any patient with aPL Abs without APS, this response is a potentially useful predictor for APS in patients with aPL Abs, as well as providing mechanistic insight into thrombosis and fetal loss in these patients.
Assuntos
Anticorpos Antifosfolipídeos/sangue , Morte Fetal/imunologia , Glicoproteínas/imunologia , Ativação Linfocitária , Monócitos/imunologia , Subpopulações de Linfócitos T/imunologia , Tromboplastina/biossíntese , Trombose/imunologia , Anticoagulantes/imunologia , Testes de Coagulação Sanguínea , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Relação Dose-Resposta Imunológica , Epitopos de Linfócito T/sangue , Epitopos de Linfócito T/imunologia , Feminino , Morte Fetal/sangue , Humanos , Masculino , Monócitos/metabolismo , Subpopulações de Linfócitos T/metabolismo , Tromboplastina/fisiologia , Trombose/sangue , beta 2-Glicoproteína IRESUMO
We have used immunoelectron microscopy to investigate the subcellular location of Pfs16 in Plasmodium falciparum. It was detected in the outer membrane region of gametocytes and more specifically on the parasitophorous vacuole membrane (pvm), since, during gametogenesis when the pvm disintegrates, the majority of the antigen was detected on the remains of this membrane in multilaminated whorls and not on the gamete plasma membrane. The antigen was also present on other gametocyte cellular structures, including those which we believe to be Garnham bodies, present in the host cell cytoplasm of some gametocytes. The antigen was present too on the membrane surrounding cytosomes and the resulting food vacuoles in the parasite cytoplasm.
Assuntos
Antígenos de Protozoários/análise , Proteínas de Membrana/análise , Plasmodium falciparum/imunologia , Animais , Membrana Celular/imunologia , Eritrócitos/imunologia , Eritrócitos/parasitologia , Eritrócitos/ultraestrutura , Feminino , Humanos , Imuno-Histoquímica , Membranas Intracelulares/imunologia , Masculino , Microscopia Imunoeletrônica , Plasmodium falciparum/ultraestrutura , Vacúolos/imunologiaRESUMO
Frozen sections and cell cultures of 50 human astrocytomas, fetal and adult human brain were examined for immunofluorescence reactivity with antisera to glial fibrillary acidic protein (GFAP), actin, fibronectin and factor VIII antigen. In frozen tissue sections GFAP expression was restricted to normal and neoplastic astrocytes while fibronectin and factor VIII antigen were localized to blood vessels. In primary cell culture, 80-100% neoplastic astrocytes expressed GFAP but not fibronectin or factor VIII antigen while actin was present as diffuse cytoplasmic staining of the cell body and cell processes. By the 5th-6th passage in vitro, GFAP immunoreactivity was lost while fibronectin and actin cables were prominently expressed. Factor VIII antigen remained negative throughout serial subculture. In double fluorochrome experiments, GFAP positive cells did not express fibronectin or actin cables, while GFAP negative cells expressed fibronectin and had prominent actin cables. Our results suggest a change in population of astrocytoma cells with increasing passage in vitro, reflecting either an overgrowth of tumour glioblasts or dedifferentiation of tumour astrocytes.
Assuntos
Actinas/análise , Antígenos/análise , Astrocitoma/análise , Química Encefálica , Neoplasias Encefálicas/análise , Fator VIII/imunologia , Fibronectinas/análise , Glioblastoma/análise , Proteínas de Filamentos Intermediários/análise , Astrocitoma/patologia , Encéfalo , Neoplasias Encefálicas/patologia , Linhagem Celular , Células Cultivadas , Fator VIII/análise , Feto/análise , Imunofluorescência , Secções Congeladas , Proteína Glial Fibrilar Ácida , Glioblastoma/patologia , Humanos , Fator de von WillebrandRESUMO
We investigated the development of astrocytes in mechanically dissociated primary cultures of foetal and neonatal rat brain grown in different culture media using immunofluorescence and antiserum to glial fibrillary acidic protein (GFAP). GFAP-positive cells developed at a time corresponding to the 16th day of embryonic development and initially grew more slowly in Dulbecco's modified Eagle's Medium (DMEM) than in Medium 199 (M199) or Modified Eagle's Medium (MEM), but as the cultures matured a greater proportion of GFAP-positive cells was obtained in DMEM, resulting in relatively pure populations of GFAP-positive cells after 1-2 months in vitro. GFAP-positive cells comprised process-bearing and fibroblastoid cell types and cells with intermediate morphology. Cultures in DMEM were also characterized by the appearance after 8-18 days in vitro of thick long bands of glial processes surrounding cyst-like spaces. The morphological change may represent a gliotic reaction to necrosis in vitro.