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1.
Sci Transl Med ; 11(476)2019 01 23.
Artigo em Inglês | MEDLINE | ID: mdl-30674657

RESUMO

Retinoblastoma is a pediatric solid tumor of the retina activated upon homozygous inactivation of the tumor suppressor RB1 VCN-01 is an oncolytic adenovirus designed to replicate selectively in tumor cells with high abundance of free E2F-1, a consequence of a dysfunctional RB1 pathway. Thus, we reasoned that VCN-01 could provide targeted therapeutic activity against even chemoresistant retinoblastoma. In vitro, VCN-01 effectively killed patient-derived retinoblastoma models. In mice, intravitreous administration of VCN-01 in retinoblastoma xenografts induced tumor necrosis, improved ocular survival compared with standard-of-care chemotherapy, and prevented micrometastatic dissemination into the brain. In juvenile immunocompetent rabbits, VCN-01 did not replicate in retinas, induced minor local side effects, and only leaked slightly and for a short time into the blood. Initial phase 1 data in patients showed the feasibility of the administration of intravitreous VCN-01 and resulted in antitumor activity in retinoblastoma vitreous seeds and evidence of viral replication markers in tumor cells. The treatment caused local vitreous inflammation but no systemic complications. Thus, oncolytic adenoviruses targeting RB1 might provide a tumor-selective and chemotherapy-independent treatment option for retinoblastoma.

3.
Front Oncol ; 8: 127, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29755954

RESUMO

Background and objective: Diffuse intrinsic pontine glioma (DIPG) is a lethal brainstem tumor in children. Dendritic cells (DCs) have T-cell stimulatory capacity and, therefore, potential antitumor activity for disease control. DCs vaccines have been shown to reactivate tumor-specific T cells in both clinical and preclinical settings. We designed a phase Ib immunotherapy (IT) clinical trial with the use of autologous dendritic cells (ADCs) pulsed with an allogeneic tumors cell-lines lysate in patients with newly diagnosed DIPG after irradiation (radiation therapy). Methods: Nine patients with newly diagnosed DIPG met enrollment criteria. Autologous dendritic cell vaccines (ADCV) were prepared from monocytes obtained by leukapheresis. Five ADCV doses were administered intradermally during induction phase. In the absence of tumor progression, patients received three boosts of tumor lysate every 3 months during the maintenance phase. Results: Vaccine fabrication was feasible in all patients included in the study. Non-specific KLH (9/9 patients) and specific (8/9 patients) antitumor response was identified by immunologic studies in peripheral blood mononuclear cells (PBMC). Immunological responses were also confirmed in the T lymphocytes isolated from the cerebrospinal fluid (CSF) of two patients. Vaccine administration resulted safe in all patients treated with this schema. Conclusion: These preliminary results demonstrate that ADCV preparation is feasible, safe, and generate a DIPG-specific immune response detected in PBMC and CSF. This strategy shows a promising backbone for future schemas of combination IT.

4.
J Control Release ; 264: 34-44, 2017 Oct 28.
Artigo em Inglês | MEDLINE | ID: mdl-28830790

RESUMO

Treatment of retinoblastoma -a pediatric cancer of the developing retina- might benefit from strategies to inhibit the blood-retinal barrier (BRB). The potent anticancer agent topotecan is a substrate of efflux transporters BCRP and P-gp, which are expressed at the BRB to restrict vitreous and retinal distribution of xenobiotics. In this work we have studied vitreous and retinal distribution, tumor accumulation and antitumor activity of topotecan, using pantoprazole as inhibitor of BCRP and P-gp. We used rabbit and mouse eyes as BRB models and patient-derived xenografts as retinoblastoma models. To validate the rabbit BRB model we stained BCRP and P-gp in the retinal vessels. Using intravitreous microdialysis we showed that the penetration of the rabbit vitreous by lactone topotecan increased significantly upon concomitant administration of pantoprazole (P=0.0285). Pantoprazole also increased topotecan penetration of the mouse vitreous, measured as the vitreous-to-plasma topotecan concentration ratio at the steady state (P=0.0246). Pantoprazole increased topotecan antitumor efficacy and intracellular penetration in retinoblastoma in vitro, but did not enhance intratumor drug distribution and survival in mice bearing the intraocular human tumor HSJD-RBT-2. Anatomical differences with the clinical setting likely limited our in vivo study, since xenografts were poorly vascularized masses that loaded most of the vitreous compartment. We conclude that pharmacological modulation of the BRB is feasible, enhances anticancer drug distribution into the vitreous and might have clinical implications in retinoblastoma. CHEMICAL COMPOUNDS INCLUDED IN THIS MANUSCRIPT: Topotecan (PubChem CID: 60700) Pantoprazole sodium (PubChem CID: 15008962).


Assuntos
2-Piridinilmetilsulfinilbenzimidazóis/farmacologia , Barreira Hematorretiniana/efeitos dos fármacos , Neoplasias da Retina/tratamento farmacológico , Retinoblastoma/tratamento farmacológico , Inibidores da Topoisomerase I/uso terapêutico , Topotecan/uso terapêutico , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/antagonistas & inibidores , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/antagonistas & inibidores , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/genética , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Barreira Hematorretiniana/metabolismo , Humanos , Camundongos Nus , Pantoprazol , Coelhos , Neoplasias da Retina/genética , Neoplasias da Retina/metabolismo , Retinoblastoma/genética , Retinoblastoma/metabolismo , Inibidores da Topoisomerase I/farmacocinética , Topotecan/farmacocinética , Corpo Vítreo/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
5.
J Control Release ; 255: 108-119, 2017 06 10.
Artigo em Inglês | MEDLINE | ID: mdl-28412222

RESUMO

Neuroblastoma is a pediatric solid tumor with high expression of the tumor associated antigen disialoganglioside GD2. Despite initial response to induction therapy, nearly 50% of high-risk neuroblastomas recur because of chemoresistance. Here we encapsulated the topoisomerase-I inhibitor SN-38 in polymeric nanoparticles (NPs) surface-decorated with the anti-GD2 mouse mAb 3F8 at a mean density of seven antibody molecules per NP. The accumulation of drug-loaded NPs targeted with 3F8 versus with control antibody was monitored by microdialysis in patient-derived GD2-expressing neuroblastoma xenografts. We showed that the extent of tumor penetration by SN-38 was significantly higher in mice receiving the targeted nano-drug delivery system when compared to non-targeted system or free drug. This selective penetration of the tumor extracellular fluid translated into a strong anti-tumor effect prolonging survival of mice bearing GD2-high neuroblastomas in vivo.


Assuntos
Anticorpos Monoclonais/administração & dosagem , Antineoplásicos Fitogênicos/administração & dosagem , Camptotecina/análogos & derivados , Líquido Extracelular/metabolismo , Imunoglobulina G/administração & dosagem , N-Acetilgalactosaminiltransferases/antagonistas & inibidores , Nanopartículas/administração & dosagem , Neuroblastoma/metabolismo , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais Murinos , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/metabolismo , Antineoplásicos Fitogênicos/química , Antineoplásicos Fitogênicos/farmacocinética , Camptotecina/administração & dosagem , Camptotecina/química , Camptotecina/farmacocinética , Linhagem Celular Tumoral , Pré-Escolar , Liberação Controlada de Fármacos , Regulação Neoplásica da Expressão Gênica , Humanos , Imunoglobulina G/química , Irinotecano , Masculino , Camundongos Nus , N-Acetilgalactosaminiltransferases/genética , N-Acetilgalactosaminiltransferases/imunologia , N-Acetilgalactosaminiltransferases/metabolismo , Nanopartículas/química , Neuroblastoma/tratamento farmacológico , Distribuição Tecidual , Ensaios Antitumorais Modelo de Xenoenxerto
6.
Cancer Lett ; 380(1): 10-9, 2016 09 28.
Artigo em Inglês | MEDLINE | ID: mdl-27319373

RESUMO

Translational research in retinoblastoma - a pediatric tumor that originates during the development of the retina - would be improved by the creation of new patient-derived models. Using tumor samples from enucleated eyes we established a new battery of preclinical models that grow in vitro in serum-free medium and in vivo in immunodeficient mice. To examine whether the new xenografts recapitulate human disease and disseminate from the retina to the central nervous system, we evaluated their histology and the presence of molecular markers of dissemination that are used in the clinical setting to detect extraocular metastases. We evaluated GD2 synthase and CRX as such markers and generated a Taqman real-time quantitative PCR method to measure CRX mRNA for rapid, sensitive and specific quantification of local and metastatic tumor burden. This approach was able to detect 1 human retinoblastoma cell in 100.000 mouse brain cells. Our research adds novel preclinical tools for the discovery of new retinoblastoma treatments for clinical translation.


Assuntos
Biomarcadores Tumorais/metabolismo , Neoplasias Encefálicas/enzimologia , Movimento Celular , Proteínas de Homeodomínio/metabolismo , N-Acetilgalactosaminiltransferases/metabolismo , Neoplasias Experimentais/enzimologia , Neoplasias da Retina/enzimologia , Retinoblastoma/enzimologia , Transativadores/metabolismo , Animais , Biomarcadores Tumorais/genética , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/secundário , Linhagem Celular Tumoral , Pré-Escolar , Feminino , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Xenoenxertos , Proteínas de Homeodomínio/genética , Humanos , Lactente , Camundongos Nus , N-Acetilgalactosaminiltransferases/genética , Micrometástase de Neoplasia , Transplante de Neoplasias , Neoplasias Experimentais/genética , Neoplasias Experimentais/patologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Neoplasias da Retina/genética , Neoplasias da Retina/patologia , Retinoblastoma/genética , Retinoblastoma/secundário , Transdução de Sinais , Transativadores/genética , Células Tumorais Cultivadas
7.
BMC Genomics ; 17: 304, 2016 Apr 23.
Artigo em Inglês | MEDLINE | ID: mdl-27108081

RESUMO

BACKGROUND: cAMP signaling produces dramatic changes in astrocyte morphology and physiology. However, its involvement in phenotype acquisition and the transcriptionally mediated mechanisms of action are largely unknown. RESULTS: Here we analyzed the global transcriptome of cultured astroglial cells incubated with activators of cAMP pathways. A bulk of astroglial transcripts, 6221 annotated genes, were differentially regulated by cAMP signaling. cAMP analogs strongly upregulated genes involved in typical functions of mature astrocytes, such as homeostatic control, metabolic and structural support to neurons, antioxidant defense and communication, whereas they downregulated a considerable number of proliferating and immaturity-related transcripts. Moreover, numerous genes typically activated in reactive cells, such as scar components and immunological mediators, were repressed by cAMP. GSEA analysis contrasting gene expression profiles with transcriptome signatures of acutely isolated astrocytes and in situ evaluation of protein levels in these cells showed that cAMP signaling conferred mature and in vivo-like transcriptional features to cultured astrocytes. CONCLUSIONS: These results indicate that cAMP signaling is a key pathway promoting astrocyte maturation and restricting their developmental and activation features. Therefore, a positive modulation of cAMP signaling may promote the normal state of differentiated astrocytes and favor the protection and function of neuronal networks.


Assuntos
Astrócitos/metabolismo , AMP Cíclico/metabolismo , Transdução de Sinais , Transcriptoma , Animais , Animais Recém-Nascidos , Antioxidantes/metabolismo , Diferenciação Celular , Células Cultivadas , Regulação da Expressão Gênica no Desenvolvimento , Camundongos , Análise de Sequência com Séries de Oligonucleotídeos , Regulação para Cima
8.
PLoS One ; 10(12): e0145107, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26670220

RESUMO

BACKGROUND: Collagen VI related myopathies encompass a range of phenotypes with involvement of skeletal muscle, skin and other connective tissues. They represent a severe and relatively common form of congenital disease for which there is no treatment. Collagen VI in skeletal muscle and skin is produced by fibroblasts. AIMS & METHODS: In order to gain insight into the consequences of collagen VI mutations and identify key disease pathways we performed global gene expression analysis of dermal fibroblasts from patients with Ullrich Congenital Muscular Dystrophy with and without vitamin C treatment. The expression data were integrated using a range of systems biology tools. Results were validated by real-time PCR, western blotting and functional assays. FINDINGS: We found significant changes in the expression levels of almost 600 genes between collagen VI deficient and control fibroblasts. Highly regulated genes included extracellular matrix components and surface receptors, including integrins, indicating a shift in the interaction between the cell and its environment. This was accompanied by a significant increase in fibroblasts adhesion to laminin. The observed changes in gene expression profiling may be under the control of two miRNAs, miR-30c and miR-181a, which we found elevated in tissue and serum from patients and which could represent novel biomarkers for muscular dystrophy. Finally, the response to vitamin C of collagen VI mutated fibroblasts significantly differed from healthy fibroblasts. Vitamin C treatment was able to revert the expression of some key genes to levels found in control cells raising the possibility of a beneficial effect of vitamin C as a modulator of some of the pathological aspects of collagen VI related diseases.


Assuntos
Matriz Extracelular/metabolismo , Fibroblastos/metabolismo , Perfilação da Expressão Gênica , Distrofias Musculares/genética , Esclerose/genética , Ácido Ascórbico/farmacologia , Adesão Celular/efeitos dos fármacos , Adesão Celular/genética , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/genética , Matriz Extracelular/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Fibroblastos/patologia , Redes Reguladoras de Genes/efeitos dos fármacos , Humanos , Integrina alfa3/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Distrofias Musculares/patologia , Esclerose/patologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/genética , Cicatrização/efeitos dos fármacos , Cicatrização/genética
10.
Pharm Res ; 32(9): 2889-900, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-25773723

RESUMO

PURPOSE: To develop a reproducible microdialysis-tumor homogenate method for the study of the intratumor distribution of a highly hydrophobic anticancer drug (SN-38; 7-ethyl-10-hydroxycamptothecin) in neuroblastoma patient-derived xenografts. METHODS: We studied the nonspecific binding of SN-38 to the microdialysis tubing in the presence of 2-hydroxypropyl-beta-cyclodextrin (HPBCD) in the perfusate. We calibrated the microdialysis probes by the zero flow rate (ZFR) method and calculated the enhancement factor (f = extrapolated SN-38 concentration at the ZFR / SN-38 concentration in the dialysed solution) of HPBCD. We characterized the extravasation of HPBCD to tumors engrafted in mice. In vivo microdialysis and terminal homogenate data at the steady state (subcutaneous pump infusions) were used to calculate the volume of distribution of unbound SN-38 (Vu,tumor) in neuroblastoma. RESULTS: HPBCD (10% w/v) in the perfusate prevented the nonspecific binding of SN-38 to the microdialysis probe and enhanced SN-38 recovery (f = 1.86). The extravasation of HPBCD in the tumor during microdialysis was lower than 1%. Vu,tumor values were above 3 mL/g tumor for both neuroblastoma models and suggested efficient cellular penetration of SN-38. CONCLUSIONS: The method contributes to overcome the limitations of the microdialysis technique in hydrophobic drugs and provides a powerful tool to characterize compartmental anticancer drug distribution in xenografts.


Assuntos
Antineoplásicos/metabolismo , Xenoenxertos/metabolismo , Neuroblastoma/metabolismo , 2-Hidroxipropil-beta-Ciclodextrina , Animais , Antineoplásicos/farmacologia , Camptotecina/análogos & derivados , Camptotecina/metabolismo , Camptotecina/farmacologia , Humanos , Interações Hidrofóbicas e Hidrofílicas , Irinotecano , Camundongos , Camundongos Nus , Microdiálise/métodos , Neuroblastoma/tratamento farmacológico , beta-Ciclodextrinas/metabolismo , beta-Ciclodextrinas/farmacologia
11.
BMC Genomics ; 15: 91, 2014 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-24484525

RESUMO

BACKGROUND: Mutations in the gene encoding thymidine kinase 2 (TK2) result in the myopathic form of mitochondrial DNA depletion syndrome which is a mitochondrial encephalomyopathy presenting in children. In order to unveil some of the mechanisms involved in this pathology and to identify potential biomarkers and therapeutic targets we have investigated the gene expression profile of human skeletal muscle deficient for TK2 using cDNA microarrays. RESULTS: We have analysed the whole transcriptome of skeletal muscle from patients with TK2 mutations and compared it to normal muscle and to muscle from patients with other mitochondrial myopathies. We have identified a set of over 700 genes which are differentially expressed in TK2 deficient muscle. Bioinformatics analysis reveals important changes in muscle metabolism, in particular, in glucose and glycogen utilisation, and activation of the starvation response which affects aminoacid and lipid metabolism. We have identified those transcriptional regulators which are likely to be responsible for the observed changes in gene expression. CONCLUSION: Our data point towards the tumor suppressor p53 as the regulator at the centre of a network of genes which are responsible for a coordinated response to TK2 mutations which involves inflammation, activation of muscle cell death by apoptosis and induction of growth and differentiation factor 15 (GDF-15) in muscle and serum. We propose that GDF-15 may represent a potential novel biomarker for mitochondrial dysfunction although further studies are required.


Assuntos
Perfilação da Expressão Gênica , Fator 15 de Diferenciação de Crescimento/genética , Miopatias Mitocondriais/genética , Timidina Quinase/genética , Proteína Supressora de Tumor p53/metabolismo , Adolescente , Adulto , Biomarcadores/metabolismo , Caspase 3/metabolismo , Criança , Pré-Escolar , Biologia Computacional , DNA Mitocondrial/genética , DNA Mitocondrial/metabolismo , Fator 15 de Diferenciação de Crescimento/sangue , Fator 15 de Diferenciação de Crescimento/metabolismo , Humanos , Lactente , Miopatias Mitocondriais/patologia , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Transdução de Sinais , Timidina Quinase/metabolismo
12.
PLoS One ; 8(10): e77430, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24223098

RESUMO

Ullrich congenital muscular dystrophy (UCMD), caused by collagen VI deficiency, is a common congenital muscular dystrophy. At present, the role of collagen VI in muscle and the mechanism of disease are not fully understood. To address this we have applied microarrays to analyse the transcriptome of UCMD muscle and compare it to healthy muscle and other muscular dystrophies. We identified 389 genes which are differentially regulated in UCMD relative to controls. In addition, there were 718 genes differentially expressed between UCMD and dystrophin deficient muscle. In contrast, only 29 genes were altered relative to other congenital muscular dystrophies. Changes in gene expression were confirmed by real-time PCR. The set of regulated genes was analysed by Gene Ontology, KEGG pathways and Ingenuity Pathway analysis to reveal the molecular functions and gene networks associated with collagen VI defects. The most significantly regulated pathways were those involved in muscle regeneration, extracellular matrix remodelling and inflammation. We characterised the immune response in UCMD biopsies as being mainly mediated via M2 macrophages and the complement pathway indicating that anti-inflammatory treatment may be beneficial to UCMD as for other dystrophies. We studied the immunolocalisation of ECM components and found that biglycan, a collagen VI interacting proteoglycan, was reduced in the basal lamina of UCMD patients. We propose that biglycan reduction is secondary to collagen VI loss and that it may be contributing towards UCMD pathophysiology. Consequently, strategies aimed at over-expressing biglycan and restore the link between the muscle cell surface and the extracellular matrix should be considered.


Assuntos
Colágeno Tipo VI/deficiência , Distrofias Musculares/metabolismo , Esclerose/metabolismo , Transcriptoma , Estudos de Casos e Controles , Criança , Retículo Endoplasmático/genética , Retículo Endoplasmático/metabolismo , Matriz Extracelular/metabolismo , Perfilação da Expressão Gênica , Genes MHC da Classe II , Humanos , Mitocôndrias/genética , Mitocôndrias/metabolismo , Anotação de Sequência Molecular , Terapia de Alvo Molecular , Músculo Esquelético/metabolismo , Distrofias Musculares/genética , Distrofias Musculares/terapia , Análise de Sequência com Séries de Oligonucleotídeos , Proteólise , Esclerose/genética , Esclerose/terapia
13.
Brain Pathol ; 23(3): 274-84, 2013 May.
Artigo em Inglês | MEDLINE | ID: mdl-22998035

RESUMO

The secretory sorting receptors carboxypeptidase E (CPE) and secretogranin III (SgIII) critically activate peptidic messengers and targeting them at the regulated secretory pathway. In Alzheimer's disease (AD), the wide range of changes includes impaired function of key secretory peptidic cargos such as brain-derived neurotrophic factor (BDNF) and neuropeptides. Here, we analyzed CPE and SgIII in the cerebral cortex of AD patients and transgenic mice. In the normal human cortex, a preferential location in dendrites and perikarya was observed for CPE, whereas SgIII was mainly associated with axons and terminal-like buttons. Interestingly, SgIII and CPE were consistently detected in astroglial cell bodies and thin processes. In AD cortices, a strong wide accumulation of both sorting receptors was detected in dystrophic neurites surrounding amyloid plaques. Occasionally, increased levels of SgIII were also observed in plaque associate-reactive astrocytes. Of note, the main alterations detected for CPE and SgIII in AD patients were faithfully recapitulated by APPswe/PS1dE9 mice. These results implicate for the first time the sorting receptors for regulated secretion in amyloid ß-associated neural degeneration. Because CPE and SgIII are essential in the process and targeting of neuropeptides and neurotrophins, their participation in the pathological progression of AD may be suggested.


Assuntos
Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/genética , Peptídeos beta-Amiloides/metabolismo , Carboxipeptidase H/genética , Carboxipeptidase H/metabolismo , Cromograninas/genética , Cromograninas/metabolismo , Degeneração Neural/metabolismo , Degeneração Neural/patologia , Idoso , Idoso de 80 Anos ou mais , Animais , Astrócitos/metabolismo , Astrócitos/patologia , Western Blotting , Córtex Cerebral/metabolismo , Córtex Cerebral/patologia , Feminino , Proteína Glial Fibrilar Ácida/metabolismo , Humanos , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Transgênicos , Microscopia Eletrônica , Pessoa de Meia-Idade , Placa Amiloide/metabolismo , Placa Amiloide/patologia
14.
J Neuropathol Exp Neurol ; 71(10): 894-906, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-22975586

RESUMO

Ullrich congenital muscular dystrophy (UCMD) is a common form of muscular dystrophy associated with defects in collagen VI. It is characterized by loss of individual muscle fibers and muscle mass and proliferation of connective and adipose tissues. We sought to investigate the mechanisms by which collagen VI regulates muscle cell survival, size, and regeneration and, in particular, the potential role of the ubiquitin-proteasome and calpain-proteolytic systems. We studied muscle biopsies of UCMD (n = 6), other myopathy (n = 12), and control patients (n = 10) and found reduced expression of atrogin-1, MURF1, and calpain-3 mRNAs in UCMD cases. Downregulation of calpain-3 was associated with changes in the nuclear immunolocalization of nuclear factor-κB. We also observed increased expression versus controls of regeneration markers at the protein and RNA levels. Satellite cell numbers did not differ in collagen VI-deficient muscle versus normal nonregenerating muscle, indicating that collagen VI does not play a key role in the maintenance of the satellite cell pool. Our results indicate that alterations in calpain-3 and nuclear factor-κB signaling pathways may contribute to muscle mass loss in UCMD muscle, whereas atrogin-1 and MURF1 are not likely to play a major role.


Assuntos
Calpaína/fisiologia , Colágeno Tipo VI/deficiência , Fibras Musculares Esqueléticas/fisiologia , Proteínas Musculares/fisiologia , Atrofia Muscular/metabolismo , NF-kappa B/fisiologia , Regeneração/fisiologia , Transdução de Sinais/fisiologia , Criança , Pré-Escolar , Feminino , Humanos , Masculino , Fibras Musculares Esqueléticas/patologia , Músculo Esquelético/patologia , Músculo Esquelético/fisiologia , Atrofia Muscular/patologia , Distrofias Musculares/metabolismo , Distrofias Musculares/patologia , Adulto Jovem
15.
Hippocampus ; 21(2): 185-97, 2011 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-20082296

RESUMO

Taurine is one of the most abundant free amino acids in the mammalian central nervous system, where it is crucial to proper development. Moreover, taurine acts as a neuroprotectant in various diseases; in epilepsy, for example, it has the capacity to reduce or abolish seizures. In the present study, taurine levels has been determine in mice treated with Kainic Acid (KA) and results showed an increase of this amino acid in hippocampus but not in whole brain after 3 and 7 days of KA treatment. This increase occurs when gliosis was observed. Moreover, taurine transporter (TAUT) was found in astrocytes 3 and 7 days after KA treatment, together with an increase in cysteine sulfinic acid decarboxylase (csd) mRNA, that codifies for the rate-limiting enzyme of taurine synthesis, in the hippocampus at the same times after KA treatment. Glial cultures enriched in astrocytes were developed to demonstrate that these cells are responsible for changes in taurine levels after an injury to the brain. The cultures were treated with proinflammatory cytokines to reproduce gliosis. In this experimental model, an increase in the immunoreactivity of GFAP was observed, together with an increase in CSD and taurine levels. Moreover, an alteration in the taurine uptake-release kinetics was detected in glial cells treated with cytokine. All data obtained indicate that astrocytes could play a key role in taurine level changes induced by neuronal damage. More studies are, therefore, needed to clarify the role taurine has in relation to neuronal death and repair.


Assuntos
Astrócitos/metabolismo , Hipocampo/metabolismo , Taurina/metabolismo , Animais , Astrócitos/efeitos dos fármacos , Sequência de Bases , Transporte Biológico Ativo/efeitos dos fármacos , Carboxiliases/genética , Carboxiliases/metabolismo , Células Cultivadas , Citocinas/farmacologia , Primers do DNA/genética , Proteína Glial Fibrilar Ácida , Gliose/induzido quimicamente , Gliose/metabolismo , Hipocampo/citologia , Hipocampo/efeitos dos fármacos , Mediadores da Inflamação/farmacologia , Ácido Caínico/toxicidade , Masculino , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Camundongos , Proteínas do Tecido Nervoso/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
16.
Cereb Cortex ; 20(6): 1386-97, 2010 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-19892786

RESUMO

Astrocytes release peptide and nonpeptide transmitters that influence neuronal development, function, and plasticity. However, the molecular components of the astroglial secretory pathways in vivo are largely unknown. Here, we analyze in astrocytes the production, expression regulation, trafficking, and release of secretogranin III (SgIII), a member of the multifunctional granin family. We show that astroglial cells in culture synthesize and release a nonprocessed form of SgIII. In vivo studies show that many neuronal populations produce and transport SgIII. In particular, the highest SgIII expression in the cerebral cortex in vivo is present in astroglial cells. Both SgIII protein and mRNA are abundantly detected in cortical astrocytes and in Bergmann glial cells. Moreover, the levels of SgIII mRNA and protein in reactive astrocytes, induced by perforating injury increase dramatically. These results implicate SgIII in the astrocyte secretory pathway in vivo and show that its expression is finely regulated during glial activation. The robust expression of SgIII in astrocytes and its regulation in the injured brain suggest both intracellular and extracellular roles for this glial granin in the physiology and repair/damage of neuronal circuits.


Assuntos
Astrócitos/metabolismo , Cromograninas/biossíntese , Cromograninas/genética , Regulação da Expressão Gênica/fisiologia , Gliose/metabolismo , Animais , Astrócitos/patologia , Lesões Encefálicas/genética , Lesões Encefálicas/metabolismo , Lesões Encefálicas/patologia , Células COS , Células Cultivadas , Cromograninas/metabolismo , Modelos Animais de Doenças , Espaço Extracelular/genética , Espaço Extracelular/metabolismo , Espaço Extracelular/fisiologia , Gliose/genética , Gliose/patologia , Líquido Intracelular/metabolismo , Líquido Intracelular/fisiologia , Camundongos , Regeneração Nervosa/fisiologia , Plasticidade Neuronal/fisiologia , Ratos , Ratos Sprague-Dawley , Regulação para Cima/genética , Regulação para Cima/fisiologia
17.
J Neurochem ; 110(1): 143-56, 2009 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-19594665

RESUMO

Vesicular transmitter release from astrocytes influences neuronal development, function and plasticity. However, secretory pathways and the involved molecular mechanisms in astroglial cells are poorly known. In this study, we show that a variety of SNARE and Munc18 isoforms are expressed by cultured astrocytes, with syntaxin-4, Munc18c, SNAP-23 and VAMP-3 being the most abundant variants. Exocytotic protein expression was differentially regulated by activating and differentiating agents. Specifically, proteins controlling Ca(2+)-dependent secretion in neuroendocrine cells were up-regulated after long-term 8Br-cAMP administration in astrocytes, but not by proinflammatory cytokines. Moreover, 8Br-cAMP treatment greatly increased the cellular content of the peptidic vesicle marker secretogranin-2. Release assays performed on cAMP-treated astrocytes showed that basal and stimulated secretogranin-2 secretion are dependent on [Ca(2+)](i). As shown release of the chimeric hormone ANP.emd from transfected cells, cAMP-induced differentiation in astrocytes enhances Ca(2+)-regulated peptide secretion. We conclude that astroglial cells display distinctive molecular components for exocytosis. Moreover, the regulation of both exocytotic protein expression and Ca(2+)-dependent peptide secretion in astrocytes by differentiating and activating agents suggest that glial secretory pathways are adjusted in different physiological states.


Assuntos
Astrócitos/metabolismo , Sinalização do Cálcio/fisiologia , Exocitose/fisiologia , Proteínas do Tecido Nervoso/metabolismo , Neurossecreção/fisiologia , Peptídeos/metabolismo , 8-Bromo Monofosfato de Adenosina Cíclica/farmacologia , Animais , Astrócitos/efeitos dos fármacos , Encéfalo/citologia , Encéfalo/metabolismo , Cálcio/metabolismo , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/fisiologia , Linhagem Celular , Células Cultivadas , Cromograninas/efeitos dos fármacos , Cromograninas/metabolismo , Cães , Exocitose/efeitos dos fármacos , Camundongos , Proteínas Munc18/química , Proteínas Munc18/metabolismo , Proteínas do Tecido Nervoso/química , Neurossecreção/efeitos dos fármacos , Ratos , Proteínas SNARE/química , Proteínas SNARE/metabolismo , Vesículas Secretórias/efeitos dos fármacos , Vesículas Secretórias/metabolismo , Regulação para Cima/fisiologia
18.
Brain Res ; 1239: 85-91, 2008 Nov 06.
Artigo em Inglês | MEDLINE | ID: mdl-18789906

RESUMO

Cajal-Retzius (CR) cells are transient neurons of the developing cerebral cortex that play a pivotal role in the lamination and construction of neural circuits. One physiological feature of CR cells is the failure to switch GABAergic transmission from excitation to inhibition. To examine the mechanisms underlying the persistence of the depolarizing action of GABA we analyzed the mRNA expression of the K+/Cl- co-transporter type 2 (KCC2) in mouse CR by in situ hybridization. During the second postnatal week, the developmentally regulated expression of KCC2 reached adult levels in most neurons of the cerebral cortex. Double labeling with the CR-cell marker calretinin and KCC2 in situ hybridization showed that CR cells were consistently devoid of KCC2 expression in several cortical areas such as neocortex and hippocampus. Since most cortical calretinin- and calbindin-containing non-CR neurons did express KCC2 mRNA, we conclude that CR cells specifically fail to trigger the developmental expression of the K+/Cl- co-transporter KCC2. These results suggest that absence of KCC2 preserves the depolarizing action of GABA in CR cells and support the notion that KCC2 is a key factor controlling Cl- homeostasis and preventing hyperexcitability.


Assuntos
Encéfalo/crescimento & desenvolvimento , Encéfalo/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Neurônios/fisiologia , Simportadores/genética , Simportadores/metabolismo , Animais , Animais Recém-Nascidos , Northern Blotting , Encéfalo/embriologia , Calbindina 2 , Hipocampo/embriologia , Hipocampo/crescimento & desenvolvimento , Hipocampo/fisiologia , Imuno-Histoquímica , Hibridização In Situ , Camundongos , Camundongos Endogâmicos , Neocórtex/embriologia , Neocórtex/crescimento & desenvolvimento , Neocórtex/fisiologia , Fotomicrografia , Células Piramidais/embriologia , Células Piramidais/crescimento & desenvolvimento , Células Piramidais/fisiologia , RNA Mensageiro , Proteína G de Ligação ao Cálcio S100/metabolismo , Ácido gama-Aminobutírico/metabolismo
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