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1.
Nature ; 547(7662): 222-226, 2017 07 13.
Artigo em Inglês | MEDLINE | ID: mdl-28678784

RESUMO

T cells directed against mutant neo-epitopes drive cancer immunity. However, spontaneous immune recognition of mutations is inefficient. We recently introduced the concept of individualized mutanome vaccines and implemented an RNA-based poly-neo-epitope approach to mobilize immunity against a spectrum of cancer mutations. Here we report the first-in-human application of this concept in melanoma. We set up a process comprising comprehensive identification of individual mutations, computational prediction of neo-epitopes, and design and manufacturing of a vaccine unique for each patient. All patients developed T cell responses against multiple vaccine neo-epitopes at up to high single-digit percentages. Vaccine-induced T cell infiltration and neo-epitope-specific killing of autologous tumour cells were shown in post-vaccination resected metastases from two patients. The cumulative rate of metastatic events was highly significantly reduced after the start of vaccination, resulting in a sustained progression-free survival. Two of the five patients with metastatic disease experienced vaccine-related objective responses. One of these patients had a late relapse owing to outgrowth of ß2-microglobulin-deficient melanoma cells as an acquired resistance mechanism. A third patient developed a complete response to vaccination in combination with PD-1 blockade therapy. Our study demonstrates that individual mutations can be exploited, thereby opening a path to personalized immunotherapy for patients with cancer.


Assuntos
Vacinas Anticâncer/genética , Vacinas Anticâncer/imunologia , Melanoma/imunologia , Melanoma/terapia , Mutação/genética , Medicina de Precisão/métodos , RNA/genética , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais/uso terapêutico , Antígeno B7-H1/imunologia , Antígenos CD8/imunologia , Vacinas Anticâncer/uso terapêutico , Epitopos/genética , Epitopos/imunologia , Humanos , Imunoterapia/métodos , Melanoma/genética , Metástase Neoplásica , Recidiva Local de Neoplasia/prevenção & controle , Nivolumabe , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Linfócitos T/imunologia , Vacinação , Microglobulina beta-2/deficiência
2.
SLAS Technol ; 29(1): 100103, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-37595636

RESUMO

Automation of diagnostic assays generally aims to increase reproducibility and throughput while decreasing human errors and hands-on time. Here, we introduce a protocol for the automated chemical conjugation of glycans to color-coded magnetic beads using the KingFisher Flex magnetic particle processor. The resulting glycan-coupled magnetic beads allow the detection of anti-glycan antibodies of different isotypes from various species. By generating anti-glycan antibody profiles, monoclonal antibodies can be screened for their specificity and cross-reactivity, while anti-glycan antibody profiles from different human body fluids can aid in predicting response to treatment or outcome of disease. This efficient, scalable protocol can also be adapted to attach proteins and other biomolecules to beads, making it useful for a wider range of applications that require bead-based laboratory methods.


Assuntos
Anticorpos Monoclonais , Magnetismo , Humanos , Reprodutibilidade dos Testes , Automação , Polissacarídeos/análise
3.
BMC Bioinformatics ; 12: 324, 2011 Aug 04.
Artigo em Inglês | MEDLINE | ID: mdl-21816082

RESUMO

BACKGROUND: Peptide microarrays offer an enormous potential as a screening tool for peptidomics experiments and have recently seen an increased field of application ranging from immunological studies to systems biology. By allowing the parallel analysis of thousands of peptides in a single run they are suitable for high-throughput settings. Since data characteristics of peptide microarrays differ from DNA oligonucleotide microarrays, computational methods need to be tailored to these specifications to allow a robust and automated data analysis. While follow-up experiments can ensure the specificity of results, sensitivity cannot be recovered in later steps. Providing sensitivity is thus a primary goal of data analysis procedures. To this end we created rapmad (Robust Alignment of Peptide MicroArray Data), a novel computational tool implemented in R. RESULTS: We evaluated rapmad in antibody reactivity experiments for several thousand peptide spots and compared it to two existing algorithms for the analysis of peptide microarrays. rapmad displays competitive and superior behavior to existing software solutions. Particularly, it shows substantially improved sensitivity for low intensity settings without sacrificing specificity. It thereby contributes to increasing the effectiveness of high throughput screening experiments. CONCLUSIONS: rapmad allows the robust and sensitive, automated analysis of high-throughput peptide array data. The rapmad R-package as well as the data sets are available from http://www.tron-mz.de/compmed.


Assuntos
Algoritmos , Biomarcadores Tumorais , Neoplasias/metabolismo , Análise Serial de Proteínas/métodos , Software , Humanos , Sensibilidade e Especificidade
4.
Differentiation ; 77(1): 60-9, 2009 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-19281765

RESUMO

UNLABELLED: We describe the use of rotary cultures (72 rpm) as an excellent method for generating spheroids from dispersed bovine granulosa cells (GC). The GC spheroids were symmetrical (diameter between 100 and 200 microm), easily accessible, and could be obtained at high yields. On day one, the spheroids showed a two-layered outer zone of cells that stained lighter than the inner zone in semi-thin sections. Bromodeoxyuridine (BrdU) uptake was frequent and randomly distributed. By day two, a striking decrease in BrdU uptake was noted. Apoptotic bodies appeared up to day four, as did TUNEL and propidium iodide labelled dead cells. At that time, the inner zone contained cells with large-sized vacuoles and the core was amorphous. The large-sized vacuoles were identified at the ultrastructural level and represented autophagosomes and autophagolysosomes that were in different stages of development. Surprisingly, conspicuous signs of cell death were accompanied by an increase in spontaneous luteinization compared to conventional stationary cultures. We detected high levels of progesterone (immunoassay) accompanied by high levels of the proteins and enzymes relevant for steroidogenesis (StAR, P450scc, 3beta-HSD by immunoblot and immunohistochemistry, respectively). CONCLUSIONS: Concomitant to cell death, GC spheroids augment progesterone synthesis. The GC spheroids provide an ideal model for studying steroidogenesis coupled to programmed cell death at the level of the mitochondria.


Assuntos
Apoptose , Células da Granulosa/metabolismo , Progesterona/biossíntese , Animais , Bovinos , Técnicas de Cultura de Células , Células Cultivadas , Colesterol/metabolismo , Enzima de Clivagem da Cadeia Lateral do Colesterol/metabolismo , Feminino , Mitocôndrias/metabolismo , Vacúolos/metabolismo
5.
Lab Chip ; 9(10): 1422-8, 2009 May 21.
Artigo em Inglês | MEDLINE | ID: mdl-19417909

RESUMO

Tauopathies such as Alzheimer's disease (AD) belong to the group of neurodegenerative diseases that are characterised by hyperphosphorylation of the protein tau. Hyperphosphorylation of tau is one of the salient events leading to neuronal cytotoxicity and cognitive impairments. In this context, inhibition of tau hyperphosphorylation by specific tau kinase inhibitors can provide an excellent drug target for the treatment of AD and other tau-related neurodegenerative diseases. To improve the identification, optimisation and validation during the high-cost hit-to-lead cycle of AD drugs, we established a fast and sensitive label-free technique for testing the efficacy of tau kinase inhibitors in vitro. Here, we report for the first time that microelectrode-based impedance spectroscopy can be used to detect the pathological risk potential of hyperphosphorylated tau in the human neuroblastoma cell line SH-SY5Y. Our findings provide a novel real-time recording technique for testing the efficiency of tau kinase inhibitors or other lead structures directed to tau hyperphosphorylation on differentiated SH-SY5Y cells.


Assuntos
Quinase 3 da Glicogênio Sintase , Procedimentos Analíticos em Microchip/métodos , Proteínas tau/metabolismo , Análise de Variância , Carbazóis , Linhagem Celular Tumoral , Impedância Elétrica , Inibidores Enzimáticos , Quinase 3 da Glicogênio Sintase/antagonistas & inibidores , Quinase 3 da Glicogênio Sintase/metabolismo , Humanos , Laminina , Microeletrodos , Neuroblastoma , Ácido Okadáico , Fosforilação , Estaurosporina
6.
Lab Chip ; 8(6): 879-84, 2008 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-18497906

RESUMO

Close to realistic responses to anti-cancer drugs are not adequately provided in monolayer or single cells assays. 3-dimensional multicellular cultures (spheroids) mimicking in vivo-like conditions are established as cell biological models for microtumors/metastases. For a non-invasive real-time monitoring of the electrical parameters of such spheroid cultures we designed, fabricated and tested a 3D multifunctional electrode-based microcavity array. In a non-adherent assay acute tests with tumor spheroids were done maintaining their spherical shape and cellular arrangement. The sensor chip with 15 individual square microcavities containing four gold electrodes each was used for impedance spectroscopy to analyze the tissue models in terms of morphological and structural changes. Cell type specific differences in the spectra and varying responses to several anti-tumor drugs were found. Further development of the prototype will provide a promising tool for the use in pharmacological high-throughput studies.


Assuntos
Técnicas Biossensoriais/métodos , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Imageamento Tridimensional/métodos , Procedimentos Analíticos em Microchip/métodos , Metástase Neoplásica , Esferoides Celulares/citologia , Animais , Automação , Impedância Elétrica , Eletrodos , Humanos , Modelos Biológicos , Metástase Neoplásica/patologia , Metástase Neoplásica/ultraestrutura , Análise Espectral/métodos , Esferoides Celulares/metabolismo , Células Tumorais Cultivadas
7.
J Neurochem ; 107(1): 96-104, 2008 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-18673446

RESUMO

The glial cell line-derived neurotrophic factor (GDNF) family consists of the four ligands GDNF, neurturin (NRTN), artemin and persephin, which bind to the four co-receptors GDNF family receptor alpha1-4 and control through the activation of the receptor tyrosin kinase Ret several developmental processes. The purpose of this study was to analyse the expression and the influence of NRTN in the developing retina. We used retinospheres, a three-dimensional model system of the developing chicken retina. The expression of NRTN and the GDNF family receptor alpha 2 increased during development. Furthermore, expression was comparable in retinae and retinospheres. Analysis of signalling pathways influenced by NRTN in retinospheres showed activation of phosphatidylinositol-3 kinase and mitogen-activated protein kinase (MAPK). Activation of MAPK could be localised in cells of the innermost rows of the inner nuclear layer which were predominantly acetylcholinesterase-positive cells. Exogenous application of NRTN increased the amount of acetylcholinesterase-positive cells within the retinospheres at late culture stages. Additionally, we could show that Müller glia cells did not express the GFRalpha2 receptor and were probably not involved in NRTN signalling. Therefore, we conclude that NRTN directly participates in regulatory processes concerning the differentiation of acetylcholinesterase-positive cells in the chicken retina.


Assuntos
Acetilcolinesterase/metabolismo , Neuroglia/metabolismo , Neurônios/metabolismo , Neurturina/metabolismo , Retina/embriologia , Retina/metabolismo , Acetilcolina/metabolismo , Animais , Diferenciação Celular/fisiologia , Embrião de Galinha , Receptores de Fator Neurotrófico Derivado de Linhagem de Célula Glial/metabolismo , Sistema de Sinalização das MAP Quinases/fisiologia , Modelos Biológicos , Neuroglia/citologia , Neurônios/citologia , Técnicas de Cultura de Órgãos , Organogênese/fisiologia , Fosfatidilinositol 3-Quinases/metabolismo , Retina/citologia , Esferoides Celulares/citologia , Esferoides Celulares/metabolismo
8.
Biosens Bioelectron ; 24(2): 253-9, 2008 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-18468883

RESUMO

Sensorchip based impedance spectroscopy can detect inhibitory effects of human neuropeptide Y (hNPY) on living cells in a non-invasive labelling free way in real time without the need of supporting reagents. Since the discovery that neoplasmatic transformations in breast cancer are correlated with a change of the receptor subtype expression of hNPY in the affected tissue, the hNPY receptor-ligand system has come to the fore of cancer research. Today there are different methods detecting hNPY receptor interactions like fluorescent and radioactive labelling or detecting hNPY-pathway activation like cyclic adenosine monophosphate (cAMP) and G protein-coupled receptor (GPCR)-assays. For all these assays it is necessary to either label related proteins with additional substances, which can affect the nature state of the cell, or the need of producing cell lysate which allows only a snapshot of the investigated cells. To overcome these problems we established a new method to detect hNPY-receptor interactions. Therefore, we monitor the complex electric resistance (impedance) of cells attached to a microelectrode over a wide frequency range. Cell alterations are detected as changes in the impedance spectra. After application of the adenylyl cyclase-stimulating reagent forskolin, impedance is decreased at 5 kHz frequency within minutes. This effect can be inhibited by preincubating the cells with hNPY for a time range of 20 min. The inhibitory effect of hNPY can be washed out and the same cells can be stimulated by forskolin again.


Assuntos
Biomarcadores Tumorais/análise , Neoplasias da Mama/metabolismo , Eletroquímica/instrumentação , Microeletrodos , Proteínas de Neoplasias/análise , Análise Serial de Proteínas/instrumentação , Receptores de Neuropeptídeo Y/análise , Linhagem Celular Tumoral , Sistemas Computacionais , Impedância Elétrica , Eletrônica/instrumentação , Desenho de Equipamento , Análise de Falha de Equipamento , Humanos , Reprodutibilidade dos Testes , Sensibilidade e Especificidade
9.
Biosens Bioelectron ; 23(10): 1473-80, 2008 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-18289841

RESUMO

Multicellular tumour spheroids that mimic a native cellular environment are widely used as model systems for drug testing. To study drug effects on three-dimensional cultures in real-time we designed and fabricated a novel type of sensor chip for fast, non-destructive impedance spectroscopy and extracellular recording. Precultured spheroids are trapped between four gold electrodes. Fifteen individual 100microm deep square microcavities with sizes from 200 to 400microm allow an optimised positioning during the measurement. Although apoptosis was induced in human melanoma spheroids by Camptothecin (CTT), treated cultures did not show disintegration but displayed increased impedance magnitudes compared to controls after 8h resulting from an altered morphology of the outer cells. Contractions in cardiomyocyte spheroids were monitored when the innovative chip was used for recording of extracellular potentials. The silicon-based electrode array is used as an acute test system for the monitoring of any kind of 3D cell cultures. Since no adherence of cells or labelling is necessary the multifunctional sensor chip provides a basis for improved drug development by high content screenings with reduced costs and assay times. Additional improvements for parallel testing of different substances on one chip are presented.


Assuntos
Bioensaio/instrumentação , Técnicas Biossensoriais/instrumentação , Técnicas de Cultura de Células/instrumentação , Avaliação Pré-Clínica de Medicamentos/instrumentação , Técnicas Analíticas Microfluídicas/instrumentação , Análise Espectral/métodos , Análise Serial de Tecidos/instrumentação , Bioensaio/métodos , Técnicas Biossensoriais/métodos , Técnicas de Cultura de Células/métodos , Avaliação Pré-Clínica de Medicamentos/métodos , Impedância Elétrica , Desenho de Equipamento , Análise de Falha de Equipamento , Técnicas Analíticas Microfluídicas/métodos , Neoplasias de Tecido Ósseo , Análise Serial de Tecidos/métodos
10.
Neurosci Lett ; 442(1): 10-4, 2008 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-18590797

RESUMO

Persephin (PSPN), a member of the glial cell line-derived neurotrophic factor family, and its implication in the retina is not well understood but might be an interesting therapeutic target for degenerative diseases. Although, PSPN is lost in the chicken during evolution, its target, the GDNF family receptor alpha 4 (GFRalpha4), is still expressed in a temporal and spatial pattern in the developing retina. We used this "knockout-precondition" to study the bioactivity and the effect of exogenous PSPN application and subsequent GFRalpha activation during retinal development in vitro without impairments of endogenous PSPN. Retinospheres, derived from dissociated chicken retina of embryonic day 6, were treated with PSPN and intracellular signalling was monitored. Additionally, PSPN was added during cultivation of the retinospheres and immunhistochemical stainings and Western blotting were performed to evaluate changes in proliferation, apoptosis and differentiation. Exogenous applied PSPN enhanced phosphatidylinositol-3-kinase (PI-3K) signalling and decreased signalling of mitogen-activated protein kinases (MAPK). Most importantly early retinal proliferation was enhanced and glutamine synthetase expression was decreased whereas differentiation of major retinal cell types was not changed. In contrast to GDNF, PSPN is exclusively influencing early progenitors whereas differentiation is not effected and seems to be regulated through PSPN-independent mechanisms. Since the binding site of PSPN and therefore the target of potential therapeuticals, is well conserved among species and is with high probability not able to bind other members of the GDNF-family, these results might be assigned to other species including mammals and humans.


Assuntos
Proliferação de Células/efeitos dos fármacos , Proteínas do Tecido Nervoso/farmacologia , Retina/efeitos dos fármacos , Retina/embriologia , Transdução de Sinais/efeitos dos fármacos , Animais , Western Blotting , Diferenciação Celular/efeitos dos fármacos , Embrião de Galinha , MAP Quinases Reguladas por Sinal Extracelular/efeitos dos fármacos , Receptores de Fator Neurotrófico Derivado de Linhagem de Célula Glial/metabolismo , Imuno-Histoquímica , Neurônios/citologia , Neurônios/efeitos dos fármacos , Fosfatidilinositol 3-Quinases/efeitos dos fármacos
11.
Invest Ophthalmol Vis Sci ; 48(11): 5306-14, 2007 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-17962487

RESUMO

PURPOSE: To investigate the role(s) of glial cell line-derived neurotrophic factor (GDNF) on expression of rod photoreceptor and dopaminergic amacrine cell-specific genes in an in vitro reaggregate model of the chick retina. METHODS: Retinal reaggregates derived from embryonic day (E)6 chicks (rosetted spheroids) were supplemented with 50 ng/mL GDNF, or, alternatively, endogenous GDNF expression was downregulated by transient transfection of spheroids with a pCMS-EGFP[GDNF] antisense vector. Using mainly semiquantitative RT-PCR analyses, expression of rhodopsin, four separate opsins, and tyrosine hydroxylase (THase) was analyzed after either treatment. RESULTS: Supplementation with GDNF accelerated rhodopsin mRNA expression and sustained it at an increased level, in contrast to untreated control subjects, where rhodopsin mRNA levels were lower and unmaintained. Expression of red, green, blue, and violet opsins were unaffected. Under these conditions, GDNF also massively increased the expression of tyrosine hydroxylase, the rate-limiting enzyme in the synthesis of dopamine. The expression of endogenous GDNF was blocked in spheroids by using antisense transfections, which resulted in both a significant decrease in rhodopsin mRNA expression and a complete suppression of THase expression, as determined by RT-PCR, Western blot analysis, and immunocytochemistry. CONCLUSIONS: GDNF supports expression of both rhodopsin and THase in vitro, two critical molecules involved in the production of rod photoreceptors and dopaminergic amacrine cells, respectively; however, the presence of GDNF does not affect cone production and survival.


Assuntos
Células Amácrinas/efeitos dos fármacos , Fator Neurotrófico Derivado de Linhagem de Célula Glial/farmacologia , Receptores Dopaminérgicos/metabolismo , Células Fotorreceptoras Retinianas Bastonetes/efeitos dos fármacos , Rodopsina/genética , Tirosina 3-Mono-Oxigenase/genética , Células Amácrinas/metabolismo , Animais , Elementos Antissenso (Genética) , Western Blotting , Contagem de Células , Embrião de Galinha , Regulação para Baixo , Técnica Indireta de Fluorescência para Anticorpo , Expressão Gênica , Fator Neurotrófico Derivado de Linhagem de Célula Glial/genética , Oligonucleotídeos Antissenso , RNA Mensageiro/metabolismo , Células Fotorreceptoras Retinianas Bastonetes/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Rodopsina/biossíntese , Esferoides Celulares/metabolismo , Transfecção , Tirosina 3-Mono-Oxigenase/biossíntese
12.
Trends Neurosci ; 25(3): 131-4, 2002 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-11852139

RESUMO

The reaggregate approach involves the regeneration of histotypical three-dimensional spheres from dispersed cells of a given tissue in suspension culture. Reaggregated spheres are used as tumour, genetic, toxicological, biohybrid and neurosphere models, and often replace animal experimentation. A particularly instructive example is the use of reaggregation to regenerate complete laminar tissue from avian embryonic retina. By revealing constraints of layered tissue formation, such retinal spheres could be instrumental for regenerative medicine, including stem cell-based tissue engineering.


Assuntos
Agregação Celular/fisiologia , Diferenciação Celular/fisiologia , Organoides/crescimento & desenvolvimento , Regeneração/fisiologia , Células-Tronco/citologia , Engenharia Tecidual/tendências , Animais , Humanos , Modelos Biológicos , Organoides/citologia , Organoides/metabolismo , Retina/citologia , Retina/embriologia , Retina/metabolismo , Células-Tronco/metabolismo , Engenharia Tecidual/métodos , Transplante de Tecidos/métodos , Transplante de Tecidos/tendências
13.
Biotechniques ; 41(4): 445-50, 2006 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-17068960

RESUMO

For a feasible and cost-effective impedance measurement of cellular alterations in real-time, we combined commercially available microelectrode arrays (MEAs), consisting of 60 microelectrodes, with a conventional impedance analyzer. For proof of principle, a breast carcinoma cell line (MCF-7) was cultured on MEAs, and cellular alterations were measured by impedance spectroscopy at a frequency ranging from 10 Hz to 1 MHz. Cells were stimulated with phorbol 12-myristate 13-acetate (PMA) at different concentrations to activate protein kinase C (PKC)-mediated extra- and intracellular changes. By addition of 0.03 microM PMA, an increase of the relative impedance (Z(rel)) was observed after 10 min with a maximum at 1 kHz. Moreover a gradual elevation of the impedance was measured 60 min after stimulation with PMA. If 0.3 microM PMA was applied, the maximal amplitude of the relative impedance after 60 min shifted from 1 kHz (0.03 microM PMA) to 150 Hz. Subsequently, the impedance was further increased up to 90 min after PMA application, after which the impedance reduced after 240 min. Since we could use MEAs for at least 10 times without affecting the sensitivity, our study revealed that commercially available MEAs comprising nanocolumnar titanium nitrite electrodes are suitable microstructures for a highly reproducible and cost-effective multisite measurement of intracellular processes by impedance spectroscopy.


Assuntos
Análise Espectral/métodos , Acetato de Tetradecanoilforbol/farmacologia , Ligas/química , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Impedância Elétrica , Epitélio/efeitos dos fármacos , Epitélio/metabolismo , Estudos de Viabilidade , Feminino , Fluoresceína-5-Isotiocianato , Corantes Fluorescentes , Humanos , Imuno-Histoquímica , Microeletrodos , Microscopia de Fluorescência , Nanotecnologia/métodos , Níquel/química , Análise de Sequência com Séries de Oligonucleotídeos , Titânio/química
14.
Invest Ophthalmol Vis Sci ; 47(6): 2716-25, 2006 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-16723491

RESUMO

PURPOSE: To determine the role of glial cell line-derived neurotropic factor family receptor alpha 4 (GFRalpha4) during retinogenesis in a three-dimensional histiotypic in vitro model of the embryonic chicken retina. METHODS: Retinal spheres were cultured from dissociated 6-day-old chicken retina under permanent rotation and transfected with GFRalpha4 siRNA at culture day 2. Alterations on proliferation, apoptosis, and differentiation were determined by semiquantitative RT-PCR, in situ hybridization, and immunohistochemistry after 24, 48, and 72 hours. RESULTS: In contrast to control cultures, retinal spheres transfected with GFRalpha4 siRNA showed reduced GFRalpha4 mRNA expression of only 38% after 24 hours, 3% after 48 hours, and 5% after 72 hours. Based on the suppression of GFRalpha4, a decline in proliferating cells from 10% to 4.8% even after 24 hours and a reduction of sphere size by up to 25% at later culture stages were observed. Moreover, the number of Pax 6-positive amacrine, ganglion, and horizontal cells was significantly decreased from 36% to 16% in GFRalpha4 siRNA-transfected retinal spheres 72 hours after transfection. Additionally, GFRalpha4 gene silencing affected the development of different types of photoreceptors, as revealed by a significant decrease of blue opsin mRNA expression from 29% to 2%, whereas green opsin mRNA and the number rho4D2-positive photoreceptors were significantly increased. CONCLUSIONS: These data showed for the first time that GFRalpha4 plays an essential role in regulating, at least in vitro, the development and differentiation of various cell types during retinogenesis.


Assuntos
Proteínas Aviárias/genética , Desenvolvimento Embrionário/fisiologia , Inativação Gênica/fisiologia , Receptores de Fator Neurotrófico Derivado de Linhagem de Célula Glial/genética , Glicoproteínas de Membrana/genética , RNA Interferente Pequeno/genética , Retina/embriologia , Células Amácrinas/citologia , Células Amácrinas/embriologia , Células Amácrinas/metabolismo , Animais , Apoptose , Contagem de Células , Diferenciação Celular , Proliferação de Células , Embrião de Galinha , Proteínas do Olho/metabolismo , Técnica Indireta de Fluorescência para Anticorpo , Proteínas de Homeodomínio/metabolismo , Hibridização In Situ , Fator de Transcrição PAX6 , Fatores de Transcrição Box Pareados/metabolismo , Sondas RNA , RNA Mensageiro/metabolismo , Proteínas Repressoras/metabolismo , Retina/citologia , Células Bipolares da Retina/citologia , Células Bipolares da Retina/metabolismo , Células Ganglionares da Retina/citologia , Células Ganglionares da Retina/metabolismo , Células Horizontais da Retina/citologia , Células Horizontais da Retina/embriologia , Células Horizontais da Retina/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Opsinas de Bastonetes/genética , Transfecção
15.
Tissue Eng ; 11(11-12): 1749-56, 2005.
Artigo em Inglês | MEDLINE | ID: mdl-16411820

RESUMO

The goal of this study was to establish a reliable three-dimensional culture system for the mammalian retina that allows the analysis of retinal function and dysfunction. To produce three-dimensional retinal tissues in vitro, dissociated retinal cells of neonatal rats were maintained in culture dishes on a self-made orbital shaker. On the basis of well-defined rotation conditions, dissociated free-floating cells reaggregate in the center of the culture dish to form a multicellular cluster. Subsequently, cells begin to proliferate, whereby they form spherelike retinal tissues that grow to a size of 180-210 microm. Immunohistochemical characterization of mature retinal spheres revealed the presence of ganglion cells, amacrine cells, Müller cells, and rod photoreceptors, which are arranged in different retina-like layers. Although a small number of cells undergo programmed cell death, retinal spheres remain viable for at least 35 days in culture as revealed by fluorescein diacetate and TUNEL staining. Because most biological processes involved in tissue organization such as proliferation, differentiation, apoptosis, and survival are also observable in retinal spheres, the presented novel mammalian three-dimensional culture system is not only an outstanding model for basic research but may also be of great benefit for stem cell tissue engineering and the pharmaceutical industry.


Assuntos
Apoptose/fisiologia , Diferenciação Celular/fisiologia , Proliferação de Células , Retina/fisiologia , Esferoides Celulares/fisiologia , Animais , Animais Recém-Nascidos , Técnicas de Cultura de Células/métodos , Ratos , Ratos Wistar , Retina/citologia , Esferoides Celulares/citologia , Técnicas de Cultura de Tecidos/métodos , Engenharia Tecidual/métodos
16.
Gene Expr Patterns ; 4(1): 59-63, 2004 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-14678829

RESUMO

GDNF family receptor alpha (GFRalpha) receptors are involved in the regulation of different aspects of embryonic development such as proliferation, migration, differentiation and survival. To determine the possible role of GFRalpha4 in retinal development, we analysed its expression in the developing chicken retina. We found that GFRalpha4 is temporally co-expressed with c-ret. Both, the temporal and spatial expression of GFRalpha4 is developmentally regulated during retinogenesis and is first detected in cells of the ganglion cell layer at E6. As development of the retina proceeds, the expression of GFRalpha4 extends to cells of the inner half of the inner nuclear layer and to cells of the outermost cell row of the inner nuclear layer. Later on, GFRalpha4 expression is also found in additional cells of the outer half of the inner nuclear layer and in a subpopulation of photoreceptors. A central-to-peripheral gradient of retinal differentiation is evident, as the onset of GFRalpha4 expression is first detectable in the central retina, while it is delayed by two days in its periphery.


Assuntos
Proteínas Aviárias , Perfilação da Expressão Gênica , Glicoproteínas de Membrana/genética , Receptores de Fator de Crescimento Neural/genética , Retina/metabolismo , Animais , Embrião de Galinha , Regulação da Expressão Gênica no Desenvolvimento , Receptores de Fator Neurotrófico Derivado de Linhagem de Célula Glial , Hibridização In Situ , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-ret , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores Proteína Tirosina Quinases/genética , Retina/embriologia
17.
Invest Ophthalmol Vis Sci ; 44(5): 2221-8, 2003 May.
Artigo em Inglês | MEDLINE | ID: mdl-12714664

RESUMO

PURPOSE: To investigate the role of glial-cell-line-derived neurotrophic factor (GDNF) on proliferation, differentiation, and apoptosis of different retinal cell types--in particular, photoreceptor cells. METHODS: Reaggregated histotypic spheres, derived from retinal cells of the E6 chicken embryo were used. Under rotation, so-called rosetted spheroids arose by aggregation of dissociated retinal cells, followed by the proliferation, migration, differentiation and programmed cell death of particular cell types. Rosetted spheroids were cultured under serum-reduced conditions, either in the absence or presence of 50 ng/mL GDNF. At appropriate stages, rosetted spheroids were analyzed by using conventional staining and immunolabeling with antibodies against different retinal cell types. RESULTS: At early stages of culture, the application of GDNF to rosetted spheroids significantly increased and sustained the rate of proliferation. In particular, a de novo production of rod photoreceptors was observed, whereas cone photoreceptors and amacrine, horizontal, ganglion, and Müller cells were not affected. In addition, in GDNF-treated cultures, rod photoreceptors differentiated earlier than in nontreated cultures. In older rosetted spheroids raised in absence of GDNF, rod but not cone photoreceptors underwent apoptosis. By supplementation with GDNF, the percentage of dying rod photoreceptors was dramatically reduced (31%-6% at 8 days in culture, 71%-3% at 10 days in culture). Both the mitogenic and survival promoting effect of GDNF were dose dependent. CONCLUSIONS: The results strongly suggest that GDNF, at least in vitro, affects rod photoreceptors. Depending on the developmental stage, GDNF regulates their proliferation, differentiation, and survival.


Assuntos
Fatores de Crescimento Neural/farmacologia , Fármacos Neuroprotetores/farmacologia , Células Fotorreceptoras Retinianas Bastonetes/embriologia , Animais , Apoptose/efeitos dos fármacos , Contagem de Células , Diferenciação Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Embrião de Galinha , Relação Dose-Resposta a Droga , Técnica Indireta de Fluorescência para Anticorpo , Fator Neurotrófico Derivado de Linhagem de Célula Glial , Células Fotorreceptoras Retinianas Bastonetes/efeitos dos fármacos , Esferoides Celulares
18.
Invest Ophthalmol Vis Sci ; 45(2): 655-61, 2004 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-14744911

RESUMO

PURPOSE: Application of ciliary neurotrophic factor (CNTF) can rescue mature photoreceptors from lesion-induced and hereditary degeneration. In the chick retina, expression of the CNTF receptor is present in a subpopulation of photoreceptor cells. The present study was undertaken to identify the CNTF receptor-expressing photoreceptors and to describe the subcellular localization of the receptor protein. METHODS: The localization of the CNTF receptor was analyzed by light and electron microscopic immunocytochemistry in chick retinal wholemount preparations, with an antibody for CNTF receptor alpha (CNTFRalpha). Immunoreactive cells were identified by double labeling with immunocytochemical markers for photoreceptor subpopulations. RESULTS: The CNTFRalpha antibody labeled evenly distributed outer segments (OS) of a photoreceptor subpopulation. CNTFRalpha-positive OS were associated with oil droplets of uniform size. Receptor immunoreactivity did not colocalize with markers for rods and red-green cones. Complete overlap was found after double labeling with the antibody CERN 933, which recognizes violet-sensitive cones in the chick retina. Ultrastructurally, the CNTFRalpha-immunoreactive OS showed rodlike properties: an elongated shape and stacks of membrane discs separated from the plasma membrane. Immunoreactivity was completely restricted to the plasma membrane of the OS and the inner membrane sheet of the photoreceptor calices present in avian retinas. CONCLUSIONS: CNTFRalpha expression identifies a unique type of photoreceptors in the avian retina which does not fit into the classic morphologic definition of rods and cones. The specific expression in violet-sensitive photoreceptors suggests that CNTF may have a neuroprotective role related to the specific function of these cells.


Assuntos
Galinhas/anatomia & histologia , Receptor do Fator Neutrófico Ciliar/metabolismo , Células Fotorreceptoras Retinianas Cones/citologia , Células Fotorreceptoras Retinianas Cones/metabolismo , Animais , Embrião de Galinha , Técnica Indireta de Fluorescência para Anticorpo , Hibridização In Situ , Luz , Microscopia Imunoeletrônica , Células Fotorreceptoras Retinianas Cones/embriologia
19.
Neurosci Lett ; 368(1): 68-72, 2004 Sep 16.
Artigo em Inglês | MEDLINE | ID: mdl-15342136

RESUMO

To investigate aspects of aging on rat oligodendrocytes, cells of an oligodendrocyte cell line, so-called OLN-93, were cultured either in the presence or absence of glucose. Our data demonstrated that glucose-induced aging in vitro caused an elongation and thickening of cell processes and significantly increased the expression of netrin reflecting a more mature state of oligodendrocyte development. A possible age-inducing effect of glucose is also supported by the decrease of ras protein expression and shortening of telomeres in glucose-treated oligodendrocytes. The present study clearly shows that OLN-93 cells are an exciting and suitable model system for the investigation of age-inducing molecules and the analysis of signaling pathways involved in cerebral aging and degenerations.


Assuntos
Envelhecimento/efeitos dos fármacos , Córtex Cerebral/citologia , Glucose/farmacologia , Oligodendroglia/efeitos dos fármacos , Telômero/ultraestrutura , Animais , Western Blotting , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/ultraestrutura , Meios de Cultura , DNA/genética , Ensaio de Imunoadsorção Enzimática , Imuno-Histoquímica , Fatores de Crescimento Neural/metabolismo , Netrina-1 , Oligodendroglia/metabolismo , Oligodendroglia/ultraestrutura , Ratos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Telômero/efeitos dos fármacos , Telômero/metabolismo , Proteínas Supressoras de Tumor , Proteínas ras/metabolismo
20.
Neurochem Res ; 33(2): 336-47, 2008 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-17940897

RESUMO

Glycogen is the major energy reserve in neural tissues including the retina. A key-enzyme in glycogen metabolism is glycogen phosphorylase (GP) which exists in three differentially regulated isoforms. By applying isozyme-specific antibodies it could be demonstrated that the GP BB (brain), but not the GP MM (muscle) isoform is expressed in the chicken retina in neuronal and glial (Müller) cells. In the embryonic chicken retina, GP showed a development-dependent expression pattern. Double-labeling experiments with cell type-specific antibodies revealed that GP is expressed in various layers of the retina some of which, e.g., the photoreceptor inner segments, are known to be sites of high energy consumption. This suggests important roles of GP BB, and therefore glycogen, in early differentiation, spontaneous wave generation and in formation and stabilization of synapses.


Assuntos
Glicogênio Fosforilase/metabolismo , Isoenzimas/metabolismo , Retina/enzimologia , Animais , Western Blotting , Galinhas , Imuno-Histoquímica , Microscopia de Fluorescência , Retina/crescimento & desenvolvimento
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