RESUMEN
Multidrug resistance (MDR) contributes to failure of chemotherapy. We here show that biodegradable polymeric nanogels are able to overcome MDR via folic acid targeting. The nanogels are based on hydroxyethyl methacrylamide-oligoglycolates-derivatized poly(hydroxyethyl methacrylamide-co-N-(2-azidoethyl)methacrylamide) (p(HEMAm-co-AzEMAm)-Gly-HEMAm), covalently loaded with the chemotherapeutic drug doxorubicin (DOX) and subsequently decorated with a folic acid-PEG conjugate via copper-free click chemistry. pH-Responsive drug release is achieved via the acid-labile hydrazone bond between DOX and the methacrylamide polymeric network. Cellular uptake and cytotoxicity analyses in folate receptor-positive B16F10 melanoma versus folate receptor-negative A549 lung carcinoma cells confirmed specific uptake of the targeted nanogels. Confocal microscopy demonstrated efficient internalization, lysosomal trafficking, drug release and nuclear localization of DOX. We also show that DOX resistance in 4T1 breast cancer cells results in upregulation of the folate receptor, and that folic acid targeted nanogels can be employed to bypass drug efflux pumps, resulting in highly efficient killing of resistant cancer cells. In conclusion, folic acid functionalized nanogels with pH-controlled drug release seem to hold significant potential for treating multidrug resistant malignancies.
Asunto(s)
Doxorrubicina/administración & dosificación , Sistemas de Liberación de Medicamentos , Resistencia a Múltiples Medicamentos , Resistencia a Antineoplásicos , Receptores de Folato Anclados a GPI/metabolismo , Nanopartículas , Células A549 , Línea Celular Tumoral , Humanos , Concentración de Iones de Hidrógeno , Melanoma Experimental , Terapia Molecular DirigidaRESUMEN
During the last decades, a plethora of nanoparticles have been developed and evaluated and a real hype has been created around their potential application as diagnostic and therapeutic agents. Despite their suggestion as potential diagnostic agents, only a single diagnostic nanoparticle formulation, namely iron oxide nanoparticles, has found its way into clinical routine so far. This fact is primarily due to difficulties in achieving appropriate pharmacokinetic properties and a reproducible synthesis of monodispersed nanoparticles. Furthermore, concerns exist about their biodegradation, elimination and toxicity. The majority of nanoparticle formulations that are currently routinely used in the clinic are used for therapeutic purposes. These therapeutic nanoparticles aim to more efficiently deliver a (chemo-) therapeutic drug to the pathological site, while avoiding its accumulation in healthy organs and tissues, and are predominantly based on the "enhanced permeability and retention" (EPR) effect. Furthermore, based on their ability to integrate diagnostic and therapeutic entities within a single nanoparticle formulation, nanoparticles hold great promise for theranostic purposes and are considered to be highly useful for personalizing nanomedicine-based treatments. In this review article, we present applications of diagnostic and therapeutic nanoparticles, summarize frequently used non-invasive imaging techniques and describe the role of EPR in the accumulation of nanotheranostic formulations. In this context, the clinical potential of nanotheranostics and image-guided drug delivery for individualized and improved (chemo-) therapeutic interventions is addressed.
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Antineoplásicos/uso terapéutico , Diagnóstico por Imagen/métodos , Sistemas de Liberación de Medicamentos/métodos , Nanopartículas/uso terapéutico , Neoplasias/diagnóstico , Neoplasias/terapia , Antineoplásicos/administración & dosificación , Humanos , Nanomedicina/métodos , Nanopartículas/administración & dosificaciónRESUMEN
The aim of this study was the development of imatinib-loaded poly(d,l-lactide-co-glycolide) (PLGA) microspheres with high loading efficiency which can afford continuous release of imatinib over a prolonged period of time. Imatinib mesylate loaded PLGA microspheres with a size of 6-20 µm were prepared by a double emulsion (W1/O/W2) method using dichloromethane as volatile solvent. It was found that the microspheres were spherical with a non-porous surface; imatinib loading efficiency (LE) was highly dependent on the pH of the external water phase (W2). By increasing the pH of W2 phase above the highest pKa of imatinib (pKa 8.1), at which imatinib is mainly uncharged, the LE increased from 10% to 90% (pH 5.0 versus pH 9.0). Conversely, only 4% of its counter ion, mesylate, was retained in the microspheres at the same condition (pH 9.0). Since mesylate is highly water soluble, it is unlikely that it partitions into the organic phase. We demonstrated, using differential scanning calorimetry (DSC), that imatinib was molecularly dispersed in the polymeric matrix at loadings up to 8.0%. At higher drug loading, imatinib partially crystallized in the matrix. Imatinib microspheres released their cargo during three months by a combination of diffusion through the polymer matrix and polymer erosion. In conclusion, we have formulated imatinib microspheres with high LE and LC. Although we started with a double emulsion of imatinib mesylate, the obtained microspheres contained imatinib base which was mainly molecularly dispersed in the polymer matrix. These microspheres release imatinib over a 3-month period which is of interest for local treatment of cancer.
Asunto(s)
Preparaciones de Acción Retardada/química , Liberación de Fármacos , Mesilato de Imatinib , Microesferas , Portadores de Fármacos/química , Ácido Láctico/química , Tamaño de la Partícula , Ácido Poliglicólico/química , Copolímero de Ácido Poliláctico-Ácido Poliglicólico , Solubilidad , Factores de TiempoRESUMEN
Sunitinib is a multi-targeted receptor tyrosine kinase (RTK) inhibitor that blocks several angiogenesis related pathways. The aim of this study was to develop sunitinib-loaded polymeric microspheres that can be used as intravitreal formulation for the treatment of ocular diseases. A series of novel multi-block copolymers composed of amorphous blocks of poly-(D,L-lactide) (PDLLA) and polyethylene glycol (PEG) and of semi-crystalline poly-(L-lactide) (PLLA) blocks were synthesized. Sunitinib-loaded microspheres were prepared by a single emulsion method using dichloromethane as volatile solvent and DMSO as co-solvent. SEM images showed that the prepared microspheres (â¼ 30 µm) were spherical with a non-porous surface. Sunitinib-loaded microspheres were studied for their degradation and in-vitro release behavior. It was found that increasing the percentage of amorphous soft blocks from 10% to 30% accelerated the degradation of the multi-block copolymers. Sunitinib microspheres released their cargo for a period of at least 210 days by a combination of diffusion and polymer erosion. The initial burst (release in 24h) and release rate could be tailored by controlling the PEG-content of the multi-block copolymers. Sunitinib-loaded microspheres suppressed angiogenesis in a chicken chorioallantoic membrane (CAM) assay. These microspheres therefore hold promise for long-term suppression of ocular neovascularization.
Asunto(s)
Sistemas de Liberación de Medicamentos , Indoles/administración & dosificación , Microesferas , Neovascularización Patológica/tratamiento farmacológico , Pirroles/administración & dosificación , Administración Oftálmica , Inhibidores de la Angiogénesis/administración & dosificación , Inhibidores de la Angiogénesis/farmacología , Animales , Embrión de Pollo , Membrana Corioalantoides/irrigación sanguínea , Membrana Corioalantoides/efectos de los fármacos , Preparaciones de Acción Retardada , Portadores de Fármacos/química , Liberación de Fármacos , Indoles/farmacología , Inyecciones Intravítreas , Ácido Láctico/química , Microscopía Electrónica de Rastreo , Neovascularización Patológica/patología , Poliésteres , Polietilenglicoles/química , Polímeros/química , Pirroles/farmacología , Solventes/química , Sunitinib , Factores de TiempoRESUMEN
Anticancer drug research based on natural compounds enabled the discovery of many drugs currently used in cancer therapy. Here, we report the in vitro, in vivo and in silico anticancer and estrogen-like activity of Psidium guajava L. (guava) extracts and enriched mixture containing the meroterpenes guajadial, psidial A and psiguadial A and B. All samples were evaluated in vitro for anticancer activity against nine human cancer lines: K562 (leukemia), MCF7 (breast), NCI/ADR-RES (resistant ovarian cancer), NCI-H460 (lung), UACC-62 (melanoma), PC-3 (prostate), HT-29 (colon), OVCAR-3 (ovarian) and 786-0 (kidney). Psidium guajava's active compounds displayed similar physicochemical properties to estradiol and tamoxifen, as in silico molecular docking studies demonstrated that they fit into the estrogen receptors (ERs). The meroterpene-enriched fraction was also evaluated in vivo in a Solid Ehrlich murine breast adenocarcinoma model, and showed to be highly effective in inhibiting tumor growth, also demonstrating uterus increase in comparison to negative controls. The ability of guajadial, psidial A and psiguadials A and B to reduce tumor growth and stimulate uterus proliferation, as well as their in silico docking similarity to tamoxifen, suggest that these compounds may act as Selective Estrogen Receptors Modulators (SERMs), therefore holding significant potential for anticancer therapy.
Asunto(s)
Antineoplásicos Fitogénicos/farmacología , Proliferación Celular/efectos de los fármacos , Neoplasias/patología , Extractos Vegetales/farmacología , Psidium/química , Moduladores Selectivos de los Receptores de Estrógeno/farmacología , Animales , Línea Celular Tumoral , Biología Computacional/métodos , Estrógenos , Femenino , Humanos , Ratones Endogámicos BALB C , Neoplasias/metabolismo , Extractos Vegetales/química , Hojas de la Planta/química , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Doxorubicin is an anthracycline anticancer agent that is commonly used in the treatment of a variety of cancers, but its application is associated with severe side effects. Biodegradable and thermosensitive polymeric micelles based on poly(ethylene glycol)-b-poly[N-(2-hydroxypropyl) methacrylamide-lactate] (mPEG-b-p(HPMAmLac(n))) have been studied as drug delivery systems for therapeutic and imaging agents and have shown promising in vitro and in vivo results. The purpose of this study was to investigate the covalent coupling of a doxorubicin-glucuronide prodrug (DOX-propGA3) to the core of mPEG-b-p(HPMAmLac(2)) micelles. This prodrug is specifically activated by human ß-glucuronidase, an enzyme that is overexpressed in necrotic tumor areas. To this end, an azide modified block copolymer (mPEG(5000)-b-p(HPMAmLac(2)-r-AzEMA)) was synthesized and characterized, and DOX-propGA3 was coupled to the polymer via click chemistry with a high (95%) coupling efficiency. Micelles formed by this DOX containing polymer were small (50 nm) and monodisperse and released 40% of the drug payload after 5 days incubation at 37 °C in the presence of ß-glucuronidase, but less than 5% in the absence of the enzyme. In vitro cytotoxicity experiments demonstrated that DOX micelles incubated with 14C cells showed the same cytotoxicity as free DOX only in the presence of ß-glucuronidase, indicating full conversion of the polymer-bound DOX into the parent drug. Overall, this novel system is very promising for enzymatically responsive anticancer therapy.
Asunto(s)
Antibióticos Antineoplásicos/administración & dosificación , Doxorrubicina/administración & dosificación , Metacrilatos/química , Micelas , Polietilenglicoles/química , Profármacos/administración & dosificación , Antibióticos Antineoplásicos/química , Antibióticos Antineoplásicos/farmacología , Línea Celular Tumoral , Química Clic , Doxorrubicina/química , Doxorrubicina/farmacología , Humanos , Neoplasias/tratamiento farmacológico , Profármacos/química , Profármacos/farmacología , TemperaturaRESUMEN
The adsorption of blood proteins onto liposomes and other colloidal particles is an important process influencing the circulation time. Proteins adsorbed to the surface of liposomes can mediate recognition of the liposomes by macrophages of the reticuloendothelial system (RES) facilitating their clearance from the circulation. Coating liposomes with poly(ethylene glycol) (PEG) decreases the blood clearance considerably, most likely due to reduced protein adsorption and/or liposome aggregation. By using the relation between clearance and protein binding, the present study introduces an in vitro assay measuring interactions of liposomes with proteins to predict their blood clearance in vivo. Such assay is valuable since it limits time and costs, and importantly reduces the number of animals required for pharmacokinetic investigations of new formulations. In the current study, Surface Plasmon Resonance (SPR) and fluorescence Single Particle Tracking (fSPT) were used to study liposome-protein interactions and blood induced liposome aggregation in vitro. By means of SPR the interactions between proteins and liposomes coated with PEG of different molecular weights and at different densities (PEG(2000) in 2.5%, 5% and 7%; PEG(5000) in 0.5%, 1.5% and 2.5%), were measured for several plasma proteins: human serum albumin (HSA), apolipoprotein E (ApoE), α2-macroglobulin (α2-M), ß2-glycoprotein (ß2-G) and fibronectin (Fn). Liposomes coated with PEG interacted less with all proteins, an effect which increased with the PEG surface density. In parallel, fSPT analysis showed that the exposure of liposomes to full blood did not change the liposome size, indicating that aggregation is not a strong attributive factor in the clearance of these liposomes. In addition, the SPR measurements of the interactions between liposomes and proteins were correlated with the blood clearance of the liposomes. For each protein, the degree of protein-liposome interaction as determined by SPR showed a moderate to strong positive correlation with the clearance of the liposome type.
Asunto(s)
Proteínas Sanguíneas/metabolismo , Liposomas/sangre , Liposomas/metabolismo , Animales , Fluorescencia , Humanos , Proteínas Inmovilizadas/metabolismo , Liposomas/química , Ratones , Unión Proteica , Resonancia por Plasmón de SuperficieRESUMEN
Liposome-encapsulated corticosteroids have shown to exert strong beneficial effects in inflammatory diseases, such as arthritis and cancer. To extend the clinical applicability of these potent nanomedicines, the therapeutic effect of dexamethasone phosphate loaded long-circulating liposomes (LCL-DXP) was evaluated in animal models of multiple sclerosis (MS) and Crohn's disease (CD). In mice with experimental autoimmune encephalitis (EAE), a model for MS, treatment with LCL-DXP, but not free DXP, resulted in a decrease in disease activity when compared to PBS treated mice. In contrast, in mice with chronic DSS-induced colitis, a model for CD, treatment with LCL-DXP did not induce an improvement, but in fact worsened the fecal blood loss after treatment, indicating an aggravation of the disease. It is hypothesized that modulation of macrophage polarization towards a M2 phenotype underlies the efficacy of corticosteroid-based drug delivery systems, which is supported by the presented data. On the one hand, M1 polarized macrophages are part of the pathogenesis of MS; the modulation to M2-polarization by LCL-DXP is therefore beneficial. On the other hand, M1-polarized intestinal macrophages fulfill a protective and inflammation-suppressing role in intestinal homeostasis; changing their phenotype to M2 causes reduced protection to invading microorganisms, leading to a more severe intestinal inflammation. These findings therefore indicate that the interplay between the specific phenotype of macrophages and the specific inflammatory context of the inflammatory disease in question may be an important determining factor in the therapeutic applicability of liposomal corticosteroids in inflammatory disease.
Asunto(s)
Colitis/tratamiento farmacológico , Dexametasona/análogos & derivados , Encefalomielitis Autoinmune Experimental/tratamiento farmacológico , Glucocorticoides/administración & dosificación , Animales , Colitis/fisiopatología , Enfermedad de Crohn/tratamiento farmacológico , Enfermedad de Crohn/fisiopatología , Dexametasona/administración & dosificación , Dexametasona/farmacología , Dexametasona/toxicidad , Modelos Animales de Enfermedad , Encefalomielitis Autoinmune Experimental/fisiopatología , Femenino , Glucocorticoides/farmacología , Glucocorticoides/toxicidad , Inflamación/tratamiento farmacológico , Inflamación/fisiopatología , Liposomas , Macrófagos/metabolismo , Ratones , Esclerosis Múltiple/tratamiento farmacológico , Esclerosis Múltiple/fisiopatologíaRESUMEN
Overwhelming evidence has linked inflammatory disorders to a hypercoagulable state. In fact, thromboembolic complications are among the leading causes of disability and death in many acute and chronic inflammatory diseases. Despite this clinical knowledge, coagulation and immunity were long regarded as separate entities. Recent studies have unveiled molecular underpinnings of the intimate interconnection between both systems. The studies have clearly shown that distinct pro-inflammatory stimuli also activate the clotting cascade and that coagulation in turn modulates inflammatory signaling pathways. In this review, we use evidence from sepsis and inflammatory bowel diseases as a paradigm for acute and chronic inflammatory states in general and rise hypotheses how a systematic molecular understanding of the "inflammation-coagulation" crosstalk may result in novel diagnostic and therapeutic strategies that target the inflammation-induced hypercoagulable state.
Asunto(s)
Inmunidad Innata/fisiología , Mediadores de Inflamación/fisiología , Inflamación/fisiopatología , Transducción de Señal/fisiología , Trombofilia/diagnóstico , Trombofilia/fisiopatología , Animales , Antitrombina III/farmacología , Colitis Ulcerosa/fisiopatología , Enfermedad de Crohn/fisiopatología , Citocinas/sangre , Heparina/farmacología , Humanos , Proteína C/fisiología , Receptores de Reconocimiento de Patrones/fisiología , Sepsis/fisiopatología , Trombofilia/genética , Trombofilia/terapiaRESUMEN
Comparative investigation concerning gelfiltration as well as haemostaseologic analysis of venoms and venom fractions of some snakes (elapidae and viperidae) have shown that in elapidae an inhibition of coagulation is dominant whilst in viperidae the stimulation of coagulation is of importance. Our investigations produce a basis to select substances for activation of coagulation and substances for inhibition of coagulation. Under pharmacological viewpoints the data may produce information to use snake fractions for anticoagulation or for procoagulant therapy in bleeding tendency.
Asunto(s)
Anticoagulantes/farmacología , Coagulación Sanguínea/efectos de los fármacos , Venenos de Serpiente/farmacología , Animales , Tiempo de Sangría , Cromatografía en Gel , Venenos Elapídicos/aislamiento & purificación , Venenos Elapídicos/farmacología , Hemorragia/inducido químicamente , Humanos , Venenos de Serpiente/aislamiento & purificaciónRESUMEN
Drug targeting systems are nanometre-sized carrier materials designed for improving the biodistribution of systemically applied (chemo)therapeutics. Various different tumour-targeted nanomedicines have been evaluated over the years, and clear evidence is currently available for substantial improvement of the therapeutic index of anticancer agents. Here, we briefly summarise the most important targeting systems and strategies, and discuss recent advances and future directions in the development of tumour-targeted nanomedicines.
Asunto(s)
Antineoplásicos/uso terapéutico , Sistemas de Liberación de Medicamentos , Nanomedicina , Neoplasias/tratamiento farmacológico , Humanos , Liposomas , PolímerosRESUMEN
For thrombophilia should be screened, if a first thromboembolic event has occurred at the age of less than 45 years, if family history indicates thrombophilia, or in the case of a special clinical condition. To get valid diagnostic results a few pecularities concerning the physiology of coagulation and laboratory medicine have to be considered. This review gives an overview of pitfalls and options during the preanalytical phase of thrombophilia diagnostics. Furthermore, the laboratory assays to perform a screening are highlighted.
Asunto(s)
Pruebas Hematológicas/métodos , Tamizaje Masivo/métodos , Trombofilia/diagnóstico , Trombofilia/prevención & control , HumanosRESUMEN
Drug targeting systems are nanometer-sized carrier materials designed for improving the biodistribution of systemically applied (chemo-) therapeutics. Reasoning that (I) the temporal and spatial interaction between systemically applied chemotherapy and clinically relevant fractionated radiotherapy is suboptimal, and that (II) drug targeting systems are able to improve the temporal and spatial parameters of this interaction, we have here set out to evaluate the potential of 'carrier-based radiochemotherapy'. N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers were used as a model drug targeting system, doxorubicin and gemcitabine as model drugs, and the syngeneic and radio- and chemoresistant Dunning AT1 rat prostate carcinoma as a model tumour model. Using magnetic resonance imaging and gamma-scintigraphy, the polymeric drug carriers were first shown to circulate for prolonged periods of time, to localise to tumours both effectively and selectively, and to improve the tumour-directed delivery of low molecular weight agents. Subsequently, they were then shown to interact synergistically with radiotherapy, with radiotherapy increasing the tumour accumulation of the copolymers, and with the copolymers increasing the therapeutic index of radiochemotherapy (both for doxorubicin and for gemcitabine). Based on these findings, and on the fact that its principles are likely broadly applicable, we propose carrier-based radiochemotherapy as a novel concept for treating advanced solid malignancies.
Asunto(s)
Acrilamidas/uso terapéutico , Diagnóstico por Imagen , Doxorrubicina/uso terapéutico , Radioisótopos de Yodo/uso terapéutico , Nanomedicina , Neoplasias de la Próstata/tratamiento farmacológico , Neoplasias de la Próstata/radioterapia , Acrilamidas/farmacocinética , Animales , Antimetabolitos Antineoplásicos/farmacocinética , Antimetabolitos Antineoplásicos/uso terapéutico , Terapia Combinada , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacocinética , Desoxicitidina/uso terapéutico , Doxorrubicina/farmacocinética , Sistemas de Liberación de Medicamentos , Resistencia a Antineoplásicos , Gadolinio , Imagen por Resonancia Magnética , Masculino , Dosis Máxima Tolerada , Neoplasias de la Próstata/diagnóstico , Tolerancia a Radiación , Ratas , Ribonucleótido Reductasas/antagonistas & inhibidores , Distribución Tisular , GemcitabinaRESUMEN
Recent surveys of seed coat morphology in Lobelioideae (Campanulaceae) have demonstrated the systematic utility of such data in the subfamily and led to a revision of the supraspecific classification of Lobelia. Expanding upon these studies, we examined via scanning electron microscopy 41 seed accessions, emphasizing lobelioid genera in which only one or no species had been examined. Most conformed to previously described testal patterns. However, five species of the endemic Hawaiian genus Cyanea, comprising the molecularly defined Hardyi Clade, had a unique testal pattern (here termed Type F), characterized by laterally compressed, almost linear, areoles with rounded, knob-like protuberances on the radial walls at opposite ends. This offered a convenient synapomorphy for recognition of a clade originally defined on a molecular basis. A second unique testal pattern was found in the related Hawaiian endemics Brighamia and Delissea, thus supporting their close relationship. In this type (here termed Type G), the seed coat is irregularly wrinkled (rugose), creating broad, rounded ridges that run more-or-less perpendicular to the long axis of the seed and thus to the long axis of the testal cells. Seed coat morphology also supported the monophyly of all 124 species of Hawaiian Lobelioideae and their probable derivation from Asian species of Lobelia subg. Tupa. Additional studies supported close relationships between (1) the neotropical genera Centropogon and Siphocampylus; (2) the western American genera Legenere and Downingia; and (3) Jamaican Hippobroma and Lobelia sect. Tylomium, a group endemic to the West Indies.
RESUMEN
The archaeal transcriptional initiation machinery closely resembles core elements of the eukaryal polymerase II system. However, apart from the established basal archaeal transcription system, little is known about the modulation of gene expression in archaea. At present, no obvious eukaryal-like transcriptional regulators have been identified in archaea. Instead, we have previously isolated an archaeal gene, the Pyrococcus furiosus lrpA, that potentially encodes a bacterial-like transcriptional regulator. In the present study, we have for the first time addressed the actual involvement of an archaeal Lrp homologue in transcription modulation. For that purpose, we have produced LrpA in Escherichia coli. In a cell-free P. furiosus transcription system we used wild-type and mutated lrpA promoter fragments to demonstrate that the purified LrpA negatively regulates its own transcription. In addition, gel retardation analyses revealed a single protein-DNA complex, in which LrpA appeared to be present in (at least) a tetrameric conformation. The location of the LrpA binding site was further identified by DNaseI and hydroxyl radical footprinting, indicating that LrpA binds to a 46-base pair sequence that overlaps the transcriptional start site of its own promoter. The molecular basis of the transcription inhibition by LrpA is discussed.
Asunto(s)
ADN de Archaea/genética , Proteínas de Unión al ADN/genética , Regulación de la Expresión Génica Arqueal , Regiones Promotoras Genéticas , Pyrococcus furiosus/genética , Pyrococcus furiosus/metabolismo , Factores de Transcripción/genética , Transcripción Genética , Secuencia de Aminoácidos , Proteínas Arqueales , Secuencia de Bases , Sitios de Unión , ADN de Archaea/química , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Escherichia coli , Proteína Reguladora de Respuesta a la Leucina , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Factores de Transcripción/química , Factores de Transcripción/metabolismoRESUMEN
Motor evoked responses to focal transcranial magnetic stimulation were investigated over the unaffected hemisphere in 15 patients with hemiparesis after ischaemic stroke and compared with data from normal control subjects. Whereas responses to muscles ipsilateral to the stimulated hemisphere could only be elicited at maximal intensities in two out of 12 normal control subjects, such ipsilateral responses were recorded after stimulation of the unaffected hemisphere in patients with poor recovery after stroke at significantly lower thresholds, but not in patients with good recovery. These responses occurred with a somewhat longer (on average 6 ms) latency than the typical contralateral response. The duration of the silent period ipsilateral to stimulation of the unaffected hemisphere was longer than in control subjects. Also the contralateral threshold for the unaffected hemisphere was elevated in comparison with the control group. In one patient, who developed mirror movements after stroke, the ipsilateral threshold was exceptionally low and the latency of the ipsilateral response identical to that seen contralaterally. It is concluded that the motor outputs in the unaffected hemisphere are significantly changed after stroke, including the unmasking of ipsilateral corticospinal projections. However, these pathways seem to be of little significance for recovery, as the existence of these responses was not correlated with clinical improvement. The unaffected hemisphere after stroke shows plastic changes in motor output organization after a contralateral lesion.
Asunto(s)
Trastornos Cerebrovasculares/fisiopatología , Lateralidad Funcional , Movimiento/fisiología , Plasticidad Neuronal/fisiología , Adulto , Anciano , Umbral Diferencial , Electromiografía , Potenciales Evocados Motores , Femenino , Humanos , Magnetismo , Masculino , Persona de Mediana Edad , Estimulación Física , Tiempo de Reacción , Valores de ReferenciaRESUMEN
It has been postulated that the transduction of mechanical stress signals to bone cells occurs via loading-dependent flow of interstitial fluid through the lacuno-canalicular network of bone. We have shown earlier that chicken osteocytes release enhanced amounts of prostaglandin E2 after 1 h treatment with pulsating fluid flow (PFF, 0.5 +/- 0.02 Pa, 5 Hz). Here we study the acute response to PFF on three cell populations derived from fetal chick calvariae, namely periosteal fibroblasts (PF), an osteoblast and osteocyte containing population (OBmix), and osteocytes (OCY), and the involvement of the actin-cytoskeleton in this process. All three cell populations rapidly (OCY: within 5 min, OBmix, PF: within 10 min) increased their release of prostaglandins E2 and I2 in response to PFF, but the response by OCY was 2-4 times higher than that by OBmix or PF. Disruption of the actin-cytoskeleton by cytochalasin B completely abolished the response. We conclude that osteocytes are more sensitive to fluid shear stress than immature bone cells, and that the actin-cytoskeleton is involved in the response to fluid flow.
Asunto(s)
Osteocitos/fisiología , Prostaglandinas/biosíntesis , 6-Cetoprostaglandina F1 alfa/biosíntesis , Animales , Células Cultivadas , Embrión de Pollo , Medios de Cultivo , Técnicas de Cultivo/métodos , Dinoprostona/biosíntesis , Epoprostenol/biosíntesis , Cinética , Osteocitos/citología , Flujo Pulsátil , Cráneo/citología , Estrés Mecánico , Factores de TiempoRESUMEN
Quartz was injected into a hind food of BALB/c and DBA/2 mice and on days 40, 90, and 180 the progressive response ensuing in the draining popliteal lymph node (PLN) was investigated by histopathology and immunohistopathology. The area of silicotic nodules (ASN) was measured by morphometry, and, by this parameter, strain BALB/c proved to be a high responder to quartz, and strain DBA/2 a low responder, albeit both strains showed a similar degree of reactive lymphoid hyperplasia in the draining PLN. Both strains also showed a similar quartz content in the draining PLN but in BALB/c mice quartz particles were concentrated in the ASN, whereas in DBA/2 mice they were evenly dispersed over the PLN. Because the silicotic response of athymic BALB/c nu/nu mice was even stronger than that of euthymic BALB/c mice, T cells are not required for the development of silicotic nodules. This fits the notion that quartz is not an antigen and that high and low responder strains are MHC-identical. Because quartz-treated BALB/c, but not DBA/2 mice, showed a persistent expression of the macrophage differentiation markers MRP8 and MRP14, phenotypically the observed strain difference in silicotic responsiveness seems to be expressed at the level of macrophages.
Asunto(s)
Ganglios Linfáticos/efectos de los fármacos , Enfermedades Linfáticas/inducido químicamente , Macrófagos/efectos de los fármacos , Cuarzo/toxicidad , Linfocitos T/efectos de los fármacos , Titanio/toxicidad , Animales , Biomarcadores/análisis , Peso Corporal/efectos de los fármacos , Polvo , Femenino , Inmunohistoquímica , Ganglios Linfáticos/patología , Enfermedades Linfáticas/patología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos DBA , Ratones Desnudos , Cuarzo/administración & dosificación , Titanio/administración & dosificaciónRESUMEN
Skeletal tissues contain, apart from cells of the osteogenic and chondrogenic lineage, cells of hemopoietic origin, e.g., macrophages, osteoclasts, and their precursors. In the present study we examined the sensitivity for extracellular ATP4- of the above-mentioned cell types in freshly isolated, bone-derived cell populations and in explanted fetal metatarsal bones. Cells of hemopoietic origin reacted to the presence of ATP4- with an increased permeability for impermeant cytotoxic molecules, e.g., ethidium bromide (EB), thiocyanate (KSCN), and an increased non-ion selective membrane conductance. As a consequence, these cells could be killed by a short treatment with adenosine-5' triphosphate (ATP)+KSCN. On the other hand, cells of nonhemopoietic origin (e.g., osteoblasts, chondrocytes) were found to be insensitive to ATP4- in this respect. These cells survived the treatment without apparent damage to their alkaline phosphatase activities, osteogenic potentials, and osteoclast induction capacities. The elimination of the endogenous cells of hemopoietic origin from bone tissue or cell populations derived therefrom offers the possibility to study the properties and functions of osteogenic or chondrogenic cells without interference by the presence of cells of hemopoietic origin. It also allows the study of interactions between osteogenic cells and selected cell populations of hemopoietic origin in coculture experiments.
Asunto(s)
Huesos/citología , Sistema Hematopoyético/citología , Adenosina Trifosfato/farmacología , Animales , Permeabilidad de la Membrana Celular/efectos de los fármacos , Separación Celular , Feto/citología , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/efectos de los fármacos , Macrófagos/citología , Macrófagos/efectos de los fármacos , Masculino , Potenciales de la Membrana/efectos de los fármacos , Ratones , Rojo Neutro , Osteoclastos/citología , Osteoclastos/efectos de los fármacosRESUMEN
A method is described that permits the removal of hematopoietic cells and macrophages from mouse bone marrow cultures. The method is based on the difference in effect of extracellular ATP4- ions (ATP in the absence of divalent, complexing cations) on cells of hematopoietic origin, including macrophages, and of nonhematopoietic origin, such as fibroblastlike stromal cells. In contrast to fibroblastlike cells, hematopoietic cells and macrophages form under the influence of ATP4- lesions in their plasma membranes, which allows the entrance of molecules such as ethidium bromide (EB) and potassium thiocyanate (KSCN), which normally do not easily cross the membrane. The lesions can be rapidly closed by the addition of Mg2+ to the incubation medium, leaving the EB or KSCN trapped in the cell. This method allows the selective introduction of cell-toxic substances such as KSCN into hematopoietic cells and macrophages. By using this method, fibroblastlike stromal cells can be isolated from mouse bone marrow cultures.