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1.
Cell Prolif ; 44(3): 244-53, 2011 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-21535265

RESUMO

OBJECTIVES: Multipotential human hair follicle stem cells can differentiate into various cell lineages and thus are investigated here as potential autologous sources for regenerative medicine. Towards this end, we have attempted to expand these cells, directly isolated from minimal amounts of hair follicle explants, to numbers more suitable for stem-cell therapy. MATERIALS AND METHODS: Two types of human follicle stem cells, commercially available and directly isolated, were cultured using an in-house developed medium. The latter was obtained from bulge areas of hair follicles by mechanical and enzymatic dissociation, and was magnetically enriched for its CD200(+) fraction. Isolated cells were cultured for up to 4 weeks, on different supports: blank polystyrene, laminin- and Matrigel(TM) -coated surfaces. RESULTS: Two-fold expansion was found, highlighting the slow-cycling nature of these cells. Flow cytometry characterization revealed: magnetic enrichment increased the proportion of CD200(+) cells from initially 43.3% (CD200+, CD34: 25.8%; CD200+, CD34+: 17.5%) to 78.2% (CD200+, CD34: 41.5%; CD200+, CD34+: 36.7%). Enriched cells seemed to have retained and passed on their morphological and molecular phenotypes to their progeny, as isolated CD200(+) presenting cells expanded in our medium to a population with 80% of cells being CD200(+): 51.5% (CD200(+), CD34(-)) and 29.6% (CD200(+), CD34(+)). CONCLUSIONS: This study demonstrates the possibility of culturing human hair follicle stem cells without causing any significant changes to phenotypes of the cells.


Assuntos
Técnicas de Cultura de Células/métodos , Folículo Piloso/citologia , Células-Tronco/citologia , Antígenos CD/biossíntese , Linhagem da Célula , Terapia Baseada em Transplante de Células e Tecidos/métodos , Colágeno/química , Meios de Cultura/metabolismo , Combinação de Medicamentos , Humanos , Imunofenotipagem , Laminina/química , Fenótipo , Poliestirenos/química , Proteoglicanas/química , Medicina Regenerativa , Fatores de Tempo
2.
Br J Cancer ; 101(2): 303-11, 2009 Jul 21.
Artigo em Inglês | MEDLINE | ID: mdl-19568241

RESUMO

BACKGROUND: Cancerous stem-like cells (CSCs) have been implicated as cancer-initiating cells in a range of malignant tumours. Diverse genetic programs regulate CSC behaviours, and CSCs from glioblastoma patients are qualitatively distinct from each other. The intrinsic connection between the presence of CSCs and malignancy is unclear. We set out to test whether tumour stem-like cells can be identified from benign tumours. METHODS: Tumour sphere cultures were derived from hormone-positive and -negative pituitary adenomas. Characterisation of tumour stem-like cells in vitro was performed using self-renewal assays, stem cell-associated marker expression analysis, differentiation, and stimulated hormone production assays. The tumour-initiating capability of these tumour stem-like cells was tested in serial brain tumour transplantation experiments using SCID mice. RESULTS: In this study, we isolated sphere-forming, self-renewable, and multipotent stem-like cells from pituitary adenomas, which are benign tumours. We found that pituitary adenoma stem-like cells (PASCs), compared with their differentiated daughter cells, expressed increased levels of stem cell-associated gene products, antiapoptotic proteins, and pituitary progenitor cell markers. Similar to CSCs isolated from glioblastomas, PASCs are more resistant to chemotherapeutics than their differentiated daughter cells. Furthermore, differentiated PASCs responded to stimulation with hypothalamic hormones and produced corresponding pituitary hormones that are reflective of the phenotypes of the primary pituitary tumours. Finally, we demonstrated that PASCs are pituitary tumour-initiating cells in serial transplantation animal experiments. CONCLUSION: This study for the first time indicates that stem-like cells are present in benign tumours. The conclusions from this study may have applications to understanding pituitary tumour biology and therapies, as well as implications for the notion of tumour-initiating cells in general.


Assuntos
Adenoma/patologia , Células-Tronco Neoplásicas/patologia , Neoplasias Hipofisárias/patologia , Adenoma/genética , Adenoma/metabolismo , Animais , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Expressão Gênica , Glioblastoma/genética , Glioblastoma/metabolismo , Glioblastoma/patologia , Humanos , Hormônios Hipotalâmicos/biossíntese , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Células-Tronco Multipotentes/metabolismo , Células-Tronco Multipotentes/patologia , Transplante de Neoplasias , Células-Tronco Neoplásicas/metabolismo , Hormônios Hipofisários/biossíntese , Neoplasias Hipofisárias/genética , Neoplasias Hipofisárias/metabolismo , Células Tumorais Cultivadas
3.
Oncogene ; 20(34): 4650-64, 2001 Aug 02.
Artigo em Inglês | MEDLINE | ID: mdl-11498788

RESUMO

Members of the Myc oncoprotein network (c-Myc, Max, and Mad) play important roles in proliferation, differentiation, and apoptosis. We expressed chimeric green fluorescent protein (GFP) fusions of c-Myc, Max, and three Mad proteins in fibroblasts. Individually, c-Myc and Mad proteins localized in subnuclear speckles, whereas Max assumed a homogeneous nuclear pattern. These distributions were co-dominant and dynamic, however, as each protein assumed the pattern of its heterodimeric partner when the latter was co-expressed at a higher level. Deletion mapping of two Mad members, Mad1 and Mxi1, demonstrated that the domains responsible for nuclear localization and speckling are separable. A non-speckling Mxi1 mutant was also less effective as a transcriptional repressor than wild-type Mxi1. c-Myc nuclear speckles were distinct from SC-35 domains involved in mRNA processing. However, in the presence of co-expressed Max, c-Myc, but not Mad, co-localized to a subset of SC-35 loci. These results show that Myc network proteins comprise dynamic subnuclear structures and behave co-dominantly when co-expressed with their normal heterodimerization partners. In addition, c-Myc-Max heterodimers, but not Max-Mad heterodimers, localize to foci actively engaged in pre-mRNA transcription/processing. These findings suggest novel means by which Myc network members promote transcriptional activation or repression.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas I-kappa B , Fosfoproteínas/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Proteínas Repressoras/metabolismo , Fatores de Transcrição/metabolismo , Células 3T3 , Animais , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos , Fatores de Transcrição Hélice-Alça-Hélice Básicos , Fatores de Transcrição de Zíper de Leucina Básica , Western Blotting , Células COS , Compartimento Celular , Proteínas de Ciclo Celular , Linhagem Celular , Núcleo Celular/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Proteínas de Fluorescência Verde , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Camundongos , Inibidor de NF-kappaB alfa , Proteínas Nucleares , Fosfoproteínas/química , Fosfoproteínas/genética , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas c-myc/genética , Processamento Pós-Transcricional do RNA , Ratos , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Repressoras/química , Proteínas Repressoras/genética , Fatores de Transcrição/química , Fatores de Transcrição/genética , Transcrição Gênica , Proteínas Supressoras de Tumor
4.
J Mol Cell Cardiol ; 33(8): 1421-33, 2001 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-11448131

RESUMO

This study aims to characterize the pattern of membrane disintegration during myocardial ischemia and reperfusion. Intracellular volumes were measured by 1H and 59Co NMR in isolated rat hearts during 10, 30 and 60 min of total ischemia and 30 min of reperfusion at normothermia. Perfusion with hypo-osmotic medium (210 mosm/l) increased intracellular water from 2.50+/-0.06 to 3.07+/-0.07 ml/g dry weight (P<0.001) during pre-ischemia. Hypo-osmotic swelling decreased by 16+/-3, 32+/-6 and 44+/-11% of the pre-ischemic value after 10, 30 and 60 min of ischemia (n.s., P<0.005, P<0.001) respectively, indicating that membrane permeabilization facilitated efflux of osmolytes and counterbalanced the osmotic driving force for water influx. Hypo-osmotic swelling decreased during 30 min of reperfusion by 18+/-5% in all groups (P<0.0.005 v post-ischemia). The volume of distribution of the extracellular marker cobalticyanide increased by more than 3.2+/-0.4 and 5.8+/-0.5% of the intracellular space after 30 and 60 min of ischemia respectively (P<0.001), and by an additional 2% after reperfusion. During 30 min of reperfusion, hearts released 1.6+/-0.2 and 3.2+/-0.4% of the intracellular creatine kinase contents after 30 and 60 min of ischemia, respectively (P<0.001). In addition to the correlation between ischemia duration and membrane permeability, evident from the analysis of each probe, the data showed a progressive increase in severity of membrane injury over time and permeabilization to larger molecules. 23Na NMR spectroscopy in conjunction with an extracellular shift reagent (SR) showed formation of a resonance at an intermediate chemical shift in between the intra and extracellular Na+ peaks, suggesting penetration of SR into cells with disrupted membranes. The constant chemical shift and narrow line shape of this resonance, characteristic of a homogeneous chemical environment, suggested that the distribution of SR was contained within the cytosol of cardiomyocytes. We propose that sarcolemmal membranes are gradually permeabilized to larger molecules by ischemia, and the evolving chemical instability is spatially contained within the myocyte.


Assuntos
Permeabilidade da Membrana Celular , Líquido Intracelular/fisiologia , Espectroscopia de Ressonância Magnética , Isquemia Miocárdica/metabolismo , Reperfusão Miocárdica , Miocárdio/metabolismo , Sarcolema/metabolismo , Animais , Transporte Biológico/fisiologia , Tamanho Celular/fisiologia , Creatina Quinase/metabolismo , Espaço Extracelular/metabolismo , Espaço Extracelular/fisiologia , Técnicas In Vitro , Masculino , Miocárdio/citologia , Concentração Osmolar , Perfusão , Ratos , Ratos Sprague-Dawley , Sódio/metabolismo , Fatores de Tempo
5.
Pigment Cell Res ; 14(1): 2-8, 2001 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-11277490

RESUMO

Light-based imaging has extensive applications for medicine and biology, and recent advances in optical imaging modalities, such as confocal and multi-photon scanning fluorescence microscopy, bioluminescence, optical coherence tomography, and spectral imaging, have opened new avenues for visualizing and recording over time dynamic changes in genetic, developmental, and disease mechanisms that cannot be captured by conventional light microscopy. In the present article, we focus on spectral imaging, and using human melanoma and its precursor lesions as an example, we describe the ability of spectral imaging to detect early-stage disease, capture gene expression profiles in tissue specimens, and visualize gene functions in tumors growing in living animals.


Assuntos
Diagnóstico por Imagem/métodos , Melanoma/diagnóstico , Melanoma/patologia , Microscopia/instrumentação , Microscopia/métodos , Neoplasias Cutâneas/diagnóstico , Neoplasias Cutâneas/patologia , Animais , Humanos , Camundongos , Lesões Pré-Cancerosas/diagnóstico , Lesões Pré-Cancerosas/patologia
6.
Cell Calcium ; 29(4): 217-27, 2001 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-11243930

RESUMO

Both theoretical and experimental results are presented for in vivo calibration of the dissociation constant K(Ca)(d)of the calcium-sensitive fluorescent dye Rhod(2)in the perfused mouse heart, using manganese quenching of fluorescence transients. An analytical model is derived, based on the biochemical equilibrium of manganese competition with calcium for Rhod(2)binding. Expressing the differential of the changes between systole and diastole in fluorescence transient (delta Delta F(sys-dia)). delta DeltaF(sys-dia)in a beating heart as a function of the perfusate manganese concentration [Mn(2+)](p)allows correlation of the measured differential transient changes delta Delta F(sys-dia)with the calcium dissociation constant K(Ca)(d)of Rhod(2)and the calcium concentration in the heart. Numerical modeling indicates that the K(Ca)(d)predominantly affects the asymptotic slope of the delta Delta F(sys-dia)versus [Mn(2+)](p)curve at certain manganese concentrations, which suggests that the K(Ca)(d)can be inversely calculated by partially fitting the delta Delta F(sys-dia)distribution as a function of the perfusate manganese concentration. The feasibility of this approach is confirmed by quenching of calcium transients by manganese infusion into isolated perfused beating mouse hearts. The resulting calculated dissociation constant K(Ca)(d)of Rhod(2)is 720nM. Using the same approach, we are able to also estimate intracellular calcium concentrations of 700nM at peak systole and 300nM in diastole. This is in good agreement with values obtained by calibration of fluorescence values with a calcium saturation tetanization procedure in the same perfused mouse heart model.


Assuntos
Cálcio/análise , Cálcio/metabolismo , Corantes Fluorescentes/análise , Modelos Químicos , Miocárdio/metabolismo , Animais , Ligação Competitiva , Cálcio/química , Calibragem , Corantes Fluorescentes/química , Fluorometria , Compostos Heterocíclicos com 3 Anéis , Técnicas In Vitro , Cinética , Manganês/análise , Manganês/química , Manganês/metabolismo , Camundongos , Miocárdio/química , Perfusão
7.
J Biomed Opt ; 6(1): 23-30, 2001 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-11178577

RESUMO

We have demonstrated a method of measuring intracellular calcium in the perfused mouse heart with the red fluorescent dye rhod-2. In Langendorff perfused isolated mouse hearts, rhod-2 is bolused through the perfusate, resulting in a 6.2+/-1.9-fold increase in fluorescence over background, and calcium transients with a transient amplitude to diastolic fluorescence ratio of 33+/-9%. Quantification of the relative amount of rhod-2 in the heart was done by taking the ratio of absorbance at 524 nm (rhod-2 sensitive) to 589 nm (rhod-2 insensitive). Maximal calcium saturated fluorescence was measured during tetanization of the heart with calcium chloride (20 mM) and cyclopiazonic acid (10 microM). Electron microscopy was used to determine the subcellular localization of rhod-2, by fixing rhod-2 in the heart with a carbodiimide compound, and then using a double antibody technique to stain rhod-2. These images demonstrated prominent cytosolic rhod-2 localization. Fluorescence and confocal fluorescence microscopy were consistent with the electron microscopy data. Endothelial cell uptake of rhod-2 was shown with fluorescence microscopy, though functional studies with bradykinin infusion (3 microM), which increases endothelial cell calcium, had no effects on mean fluorescence (N=4, p=NS), suggesting that endothelial uptake was small relative to total fluorescence. Calculated values of intracellular calcium were 686+/-237 nM at peak systole, and 360+/-101 nM in diastole, and with high perfusate calcium (3.5 mM) were 1199+/-215 and 544+/-53 nM, respectively. Thus, this appears a valid method of measuring cytosolic calcium in the perfused mouse heart, which will help determine the mechanisms of altered contractility in genetically engineered mice.


Assuntos
Cálcio/metabolismo , Corantes Fluorescentes , Membranas Intracelulares/metabolismo , Miocárdio/metabolismo , Animais , Cálcio/farmacologia , Endotélio Vascular/citologia , Endotélio Vascular/metabolismo , Compostos Heterocíclicos com 3 Anéis , Técnicas In Vitro , Masculino , Camundongos , Microscopia Confocal , Microscopia Eletrônica , Microscopia de Fluorescência , Contração Miocárdica/fisiologia , Perfusão , Frações Subcelulares/metabolismo , Distribuição Tecidual
8.
Biophys J ; 80(1): 549-61, 2001 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-11159425

RESUMO

Both theoretical and experimental results are presented for the quantitative detection of calcium transients in the perfused mouse heart loaded with the calcium-sensitive fluorescent dye Rhod-2. Analytical models are proposed to calculate both the reflected absorbance and fluorescence spectra detected from the mouse heart. These models allow correlation of the measured spectral intensities with the relative quantity of Rhod-2 in the heart and measurement of the changes in quantum yield of Rhod-2 upon binding calcium in the heart in which multiple scattering effects are predominant. Theoretical modeling and experimental results demonstrate that both reflected absorbance and fluorescence emission are attenuated linearly with Rhod-2 washout. According to this relation, a ratiometric method using fluorescence and absorbance is validated as a measure of the quantum yield of calcium-dependent fluorescence, enabling determination of the dynamics of cytosolic calcium in the perfused mouse heart. The feasibility of this approach is confirmed by experiments quantifying calcium transients in the perfused mouse heart stimulated at 8 Hz. The calculated cytosolic calcium concentrations are 368 +/- 68 nM and 654 +/- 164 nM in diastole and systole, respectively. Spectral distortions induced by tissue scattering and absorption and errors induced by the geometry of the detection optics in the calcium quantification are shown to be eliminated by using the ratio method. Methods to effectively minimize motion-induced artifacts and to monitor the oxygenation status of the whole perfused heart are also discussed.


Assuntos
Cálcio/metabolismo , Miocárdio/metabolismo , Animais , Fenômenos Biofísicos , Biofísica , Citosol/metabolismo , Estimulação Elétrica , Corantes Fluorescentes , Compostos Heterocíclicos com 3 Anéis , Técnicas In Vitro , Manganês/farmacologia , Camundongos , Modelos Cardiovasculares , Oxigênio/metabolismo , Perfusão , Fótons , Espalhamento de Radiação , Espectrometria de Fluorescência , Espectrofotometria
9.
Curr Protoc Cytom ; Chapter 2: Unit 2.4, 2001 May.
Artigo em Inglês | MEDLINE | ID: mdl-18770698

RESUMO

Because of its sensitivity and specificity, fluorescence-based detection is one of the foremost methods for microscopic imaging of biological tissues and cells. Dramatic improvements in filter system design and implementation coupled with development of an ever-widening range of sensitive fluorescent dyes have made multicolor imaging a powerful tool for structural and functional analysis. This revised and expanded unit reviews some of the main principles and developments of optical filtering.


Assuntos
Microscopia/instrumentação , Microscopia/métodos , Óptica e Fotônica , Animais , Corantes Fluorescentes/farmacologia , Humanos , Lasers , Luz , Reprodutibilidade dos Testes , Sensibilidade e Especificidade
10.
Med Phys ; 28(12): 2432-40, 2001 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-11797946

RESUMO

Optical coherence tomography (OCT) is a novel technique that enables noninvasive cross-sectional imaging of biological tissues. Because of its high resolution (approximately 10 microm), superior dynamic range (140 dB in our case) and up to 2-3 mm penetration depth, OCT is potentially useful for noninvasive screening of superficial lesions. Bladder cancer arises within the transitional epithelium. Despite the ability to visualize the epithelium via cystoscopy, it is often difficult to detect early epithelial cancers and to determine their penetration to the underlying layers. To investigate the potential of OCT to enhance imaging of bladder cancers and other epithelial lesions, we applied OCT to normal and diseased bladder epithelium, and correlated the results with histological findings. OCT images of porcine bladder (a close homolog of human bladder) confirm the ability of this method to image human tissues. To determine whether OCT can track the course of bladder cancer, a standard rat model of bladder cancer in which Fisher rats are exposed to methyl-nitroso-urea (MNU), was followed both with OCT and histological studies. Our results show that the micro morphology of porcine bladder such as the urothelium, submucosa and muscles is identified by OCT and well correlated with the histological evaluations. OCT detected edema, inflammatory infiltrates, and submucosal blood congestion as well as the abnormal growth of urothelium (e.g., papillary hyperplasia and carcinomas). By contrast, surface imaging, which resembles cystoscopy, provided far less sensitivity and resolution than OCT. This is the first OCT study of any tumor documented in a systematic fashion, and the results suggest the potential of OCT for the noninvasive diagnosis of both bladder inflammatory lesions and early urothelial abnormalities, which conventional cystoscopy often misses, by imaging characterization of the increases in urothelial thickening and backscattering. However, because of the depth limitation, OCT may have limited applications in staging the invasion of higher-state urothelial cancers, especially for papillary carcinomas.


Assuntos
Óptica e Fotônica , Tomografia/métodos , Neoplasias da Bexiga Urinária/diagnóstico , Neoplasias da Bexiga Urinária/patologia , Animais , Ratos , Ratos Endogâmicos F344 , Suínos , Fatores de Tempo
11.
Cytometry ; 42(6): 347-56, 2000 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-11135288

RESUMO

Laser scanning cytometry (LSC) is a relatively new slide-based technology developed for commercial use by CompuCyte (Cambridge, MA) for performing multiple fluorescence measurements on individual cells. Because techniques developed for performing four or more measurements on individual lymphoid cells based on light scatter as a triggering parameter for cell identification are not suitable for the identification of fixed epithelial tumor cells, an alternative approach is required for the analysis of such cells by LSC. Methods for sample preparation, event triggering, and the performance of multiple LSC measurements on disaggregated fixed human cells were developed using normal lymphocytes and two human breast cancer cell lines, JC-1939 and MCF-7, as test populations. Optimal conditions for individual cell identification by LSC were found to depend on several factors, including deposited cell density (cells per unit area), the dynamic range of probe fluorescence intensities, and intracellular distribution of the fluorescent probe. Sparsely deposited cells exhibited the least cell overlap and the brightest immunofluorescent staining. Major advantages of using DNA probes over a cytoplasmic immunofluorescent protein marker such as tubulin for event triggering are that the former exhibit greater fluorescence intensity within a relatively sharply demarcated nuclear region. The DNA-binding dye LDS-751 was found to be suboptimal for quantitative DNA measurements but useful as a triggering measurement that permits the performance of simultaneous fluorescein isothiocyanate-, phycoerythrin-, and indodicarbocyanine-based measurements on each cell. A major potential advantage of LSC over flow cytometry is the high yields of analyzable cells by LSC, permitting the performance of multiple panels of multicolor measurements on each tumor. In conclusion, we have developed and optimized a technique for performing multiple fluorescence measurements on fixed epithelial cells by LSC based on event triggering using the DNA-binding dye LDS 751. Although not ideal for quantitative measurements of cell DNA content, the large Stokes shift of this dye permits the performance of three or more additional fluorescence measurements on each cell.


Assuntos
Carcinoma/química , Citometria de Fluxo/métodos , Corantes Fluorescentes/química , Microscopia Confocal/métodos , Neoplasias da Mama/química , Carbocianinas/química , Núcleo Celular/química , DNA de Neoplasias/análise , Feminino , Fluoresceína-5-Isotiocianato/química , Fluorimunoensaio , Humanos , Compostos Orgânicos , Ficoeritrina/química , Sensibilidade e Especificidade , Fixação de Tecidos , Células Tumorais Cultivadas
12.
Clin Plast Surg ; 26(4): 529-35, vii, 1999 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-10553210

RESUMO

The Pittsburgh Tissue Engineering Initiative (PTEI) is the prime catalyst for tissue-engineering research and the development of a viable for-profit tissue-engineering industry in southwestern Pennsylvania. PTEI operates targeted programs that support commercializable research; provide professional and community education; and facilitate interactions between academic medical centers, regional government, and industry to support the development of a vibrant tissue-engineering industry. This article discusses the types of research and education programs provided by PTEI and provides a brief summary of some of the tissue-engineering-related research in the region.


Assuntos
Biotecnologia , Técnicas de Cultura de Células , Transplante de Células , Humanos , Pennsylvania , Pesquisa
13.
Mol Med ; 5(1): 11-20, 1999 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-10072444

RESUMO

BACKGROUND: In the setting of familial melanoma, the presence of atypical nevi, which are the precursors of melanoma, is associated with a nearly 100% risk of developing primary melanoma by age 70. In patients with sporadic melanoma, it is estimated that 40-60% of melanomas develop in contiguous association with atypical nevi. Currently, the only way to prevent atypical nevi from progressing to melanoma is to monitor and excise them as soon as they exhibit changes in their clinical features. Activation of the transcription factor, Stat3, has been linked to abnormal cell growth and transformation as well as to interferon alpha (IFN-alpha)-mediated growth suppression in vitro. MATERIALS AND METHODS: To determine whether IFN-alpha, used for adjuvant therapy of high-risk, resected melanoma, induces changes in Stat3 in atypical nevi, patients with a clinical history of melanoma who have multiple atypical nevi were treated for 3 months with low-dose IFN-alpha. Thereupon, the new technology of microscopic spectral imaging and biochemical assays such as electrophoretic mobility shift assays (EMSAs) and immunoblot analysis were used for the study of atypical nevi, obtained before and after IFN-alpha treatment. RESULTS: The results of the investigations provided evidence that, as a result of systemic IFN-alpha treatment, Stat1 and Stat3, which are constitutively activated in melanoma precursor lesions, lose their ability to bind DNA, and as shown in the case of Stat3, become dephosphorylated. CONCLUSIONS: Unlike primary and metastatic melanomas, melanoma precursor lesions cannot be established as cell cultures. Thus, the only way to explore pathways and treatment regimens that might help prevent progression to melanoma is within the context of a melanoma precursor lesion study conducted prospectively. The findings presented here suggest that down-regulation of the transcription factors Stat1 and Stat3 by systemic IFN-alpha treatment may represent a potential pathway to prevent the activation of gene(s) whose expression may be required for atypical nevus cells to progress to melanoma.


Assuntos
Proteínas de Ligação a DNA/antagonistas & inibidores , Interferon-alfa/uso terapêutico , Melanoma/metabolismo , Melanoma/terapia , Lesões Pré-Cancerosas/metabolismo , Lesões Pré-Cancerosas/terapia , Neoplasias Cutâneas/metabolismo , Neoplasias Cutâneas/terapia , Transativadores/antagonistas & inibidores , Idoso , DNA de Neoplasias/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Regulação para Baixo , Humanos , Imuno-Histoquímica , Técnicas In Vitro , Interferon alfa-2 , Melanoma/genética , Fosforilação , Lesões Pré-Cancerosas/genética , Proteínas Recombinantes , Fator de Transcrição STAT1 , Fator de Transcrição STAT3 , Neoplasias Cutâneas/genética , Transativadores/genética , Transativadores/metabolismo
14.
Mol Med ; 5(12): 785-94, 1999 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-10666478

RESUMO

BACKGROUND: The stages of melanocytic progression are defined as atypical (dysplastic) nevus, melanoma in situ, melanoma in the radial growth phase (RGP), melanoma in the vertical growth phase (VGP), and melanoma in the metastatic growth phase (MGP). Melanoma in situ and RGP melanoma often develop in contiguous association with atypical nevi. This frequently poses a problem with respect to their early detection. Furthermore, unlike cells obtained from VGP and MGP melanomas, cells derived from melanoma in situ and RGP melanoma do not proliferate in vitro. Thus, compared to the late stages of the disease, less information is available regarding genes expressed in the early stages. MATERIALS AND METHODS: To determine whether spectral imaging, a recently developed optical imaging technique, can detect melanoma in situ and RGP melanoma arising in melanoma precursor lesions, atypical nevi in patients with a clinical history of melanoma were subjected to noninvasive macroscopic spectral imaging. To determine at what stage in the progression pathway of melanoma genes having important biological functions in VGP and MGP melanomas are activated and expressed, lesions of melanoma in situ were analyzed by immunohistochemistry and in situ hybridization for expression of some of these known molecular and immunologic markers. RESULTS: The present study demonstrates the capability of noninvasive spectral imaging to detect melanoma in situ and RGP melanoma that arise in contiguous association with atypical nevi. Furthermore, the study provides evidence that genes and antigens expressed in VGP and MGP melanoma are also expressed in melanoma in situ. CONCLUSIONS: Because of the dark and variegated pigmentation of atypical nevi, melanoma in situ and RGP melanoma that arise in these melanoma precursor lesions are often difficult to recognize and thus frequently go unnoticed. The application of new optical screening techniques for early detection of melanoma and the identification of genes expressed in the early stages of melanoma development are two important avenues in the pursuit of melanoma prevention. The investigations presented here document that macroscopic spectral imaging has the potential to detect melanoma in its early stage of development and that genes essential for the proliferation and cell adhesion of VGP and MGP melanoma are already expressed in melanoma in situ.


Assuntos
Regulação Neoplásica da Expressão Gênica/genética , Processamento de Imagem Assistida por Computador/métodos , Melanoma/genética , Lesões Pré-Cancerosas/genética , Biomarcadores Tumorais/biossíntese , Carcinoma in Situ/genética , Carcinoma in Situ/patologia , Humanos , Imuno-Histoquímica , Melanoma/química , Melanoma/patologia , Nevo/química , Nevo/genética , Nevo/patologia , Lesões Pré-Cancerosas/patologia , Neoplasias Cutâneas/química , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/patologia
15.
Comput Med Imaging Graph ; 22(2): 89-102, 1998.
Artigo em Inglês | MEDLINE | ID: mdl-9719850

RESUMO

Light is a most versatile tool for investigating biological systems and phenomena; the range, non-destructiveness, spatial discrimination and speed of optical imaging are all important for investigating structure and function at the cellular, tissue or even whole organism level. In live biological imaging, where the technological requirements are heightened, other features of light, such as coherence and wavelength, are used to generate the additional contrast and resolution needed. We report here recent improvements in our ability to image biological specimens optically, focusing on (a) spectral resolution and the related image processing issues, and (b) tomographic three-dimensional fluorescence imaging in vivo.


Assuntos
Processamento de Imagem Assistida por Computador , Microscopia , Óptica e Fotônica , Fluorescência , Análise Espectral , Tomografia
16.
Cancer Detect Prev ; 22(3): 251-7, 1998.
Artigo em Inglês | MEDLINE | ID: mdl-9618048

RESUMO

Monoclonal antibodies to two different targetable antigens were conjugated to each of four commercially available cyanine fluorochromes. Equal amounts of all four antibodies were coinjected into tumor-bearing animals and imaged. Small, superficial tumors were adequately labeled using all four fluorochromes. Large tumors were labeled well only by Cy7, probably due to self-masking and dilution effects. Cy7 was superior to other cyanine fluorochromes for visualizing structures located deep within the animal.


Assuntos
Anticorpos Monoclonais , Carbocianinas , Corantes Fluorescentes , Animais , Antígenos de Neoplasias/metabolismo , Benzotiazóis , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Fosfoproteínas/metabolismo , Proteínas de Ligação a RNA/metabolismo , Teratocarcinoma/química , Teratocarcinoma/patologia , Células Tumorais Cultivadas , Nucleolina
17.
Nature ; 391(6667): 540-1, 1998 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-9468132
19.
J Biomed Opt ; 3(4): 446-55, 1998 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-23015145

RESUMO

We demonstrate the potential of optical coherence-domain tomography (OCT) for noninvasive imaging of living skin simultaneously at two wavelengths in the near infrared range (830 and 1285 nm). The technical details of a prototype monomode fiber-optic coherence tomographic scanner providing rapid two-dimensional (2D) and three-dimensional (3D) imaging of biological tissues are described. The effects of both instrumentation parameters and the dynamic characteristics of living tissue on image contrast and resolution and on speckle reduction are discussed. The impact of imaging speed on OCT image quality is studied by a comparison between a single scan and the corresponding frame-averaged OCT images, with the latter resulting in decreased speckle noise as well as loss of some subtle structures. Both theoretical predictions and experimental results in human skin imaging show that longer wavelength can minimize the influence of multiple scattering on image contrast and resolution and thus increase the effective penetration depth of OCT imaging to about 2 mm. Some high-resolution 2D and 3D images of microscopic anatomic structures of living human skin are presented and analyzed, illustrating the unique capability of OCT for in depth, noninvasive visualization of living skin microscopic morphology in vivo. © 1998 Society of Photo-Optical Instrumentation Engineers.

20.
Biotechnol Prog ; 13(5): 649-58, 1997.
Artigo em Inglês | MEDLINE | ID: mdl-9336985

RESUMO

Tumor localization using fluorescence has been made practical by current improvements in tumor targeting molecules, especially monoclonal antibodies and their derivatives, by the development of convenient near-infrared emitting fluorochromes and by the availability of digital cameras having high sensitivity in this spectral region. Recent studies in animals have demonstrated that fluorochrome labeling of monoclonal antibodies confers adequate sensitivity and improved resolution. Distribution and catabolism of fluorochrome-labeled and radiolabeled antibodies are similar. Simultaneous localization of multiple reagents is made possible by labeling with several different near-infrared emitting fluorochromes; thus background subtraction and differential labeling of multiple tumor-associated components can be performed. Difficulties in using the fluorochrome labels are mainly related to light scattering and absorption in tissues, but detection of small tumors at depths of several millimeters is feasible. The major medical use of this new technology is likely to be endoscopic location of tumors. Scientific uses include studies of tumor metastasis, uptake and distribution of drugs and tumor-targeting molecules by tumors, and migration patterns of near-infrared labeled cells in vivo.


Assuntos
Anticorpos Monoclonais , Carbocianinas , Corantes Fluorescentes , Neoplasias/diagnóstico , Animais , Humanos , Sensibilidade e Especificidade
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