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1.
Am J Physiol Renal Physiol ; 302(10): F1305-12, 2012 May 15.
Article in English | MEDLINE | ID: mdl-22357919

ABSTRACT

Endothelial progenitor cells (EPCs) protect the kidney from acute ischemic injury. The aim of this study was to analyze whether pretreatment of murine "early outgrowth" EPCs (eEPCs) with the hormone melatonin increases the cells' renoprotective effects in the setting of murine acute ischemic renal failure. Male (8-12 wk old) C57Bl/6N mice were subjected to unilateral ischemia-reperfusion injury postuninephrectomy (40 min). Postischemic animals were injected with either 0.5×10(6) untreated syngeneic murine eEPCs or with cells, pretreated with melatonin for 1 h. Injections were performed shortly after reperfusion of the kidney. While animals injected with untreated cells developed acute renal failure, eEPC pretreatment with melatonin dramatically improved renoprotective actions of the cells. These effects were completely reversed after cell pretreatment with melatonin and the MT-1/-2 antagonist luzindole. In vitro analysis revealed that melatonin reduced the amount of tumor growth factor-ß-induced eEPC apoptosis/necrosis. Secretion of vascular endothelial growth factor by the cells was markedly stimulated by the hormone. In addition, migratory activity of eEPCs was enhanced by melatonin and supernatant from melatonin-treated eEPCs stimulated migration of cultured mature endothelial cells. In summary, melatonin was identified as a new agonist of eEPCs in acute ischemic kidney injury.


Subject(s)
Acute Kidney Injury/drug therapy , Endothelial Cells/cytology , Melatonin/pharmacology , Reperfusion Injury/drug therapy , Stem Cells/drug effects , Acute Kidney Injury/pathology , Acute Kidney Injury/physiopathology , Animals , Antioxidants/pharmacology , Apoptosis/drug effects , Cell Movement/drug effects , Cells, Cultured , Male , Mice , Mice, Inbred C57BL , Necrosis , Neovascularization, Physiologic/drug effects , Recovery of Function/drug effects , Reperfusion Injury/pathology , Reperfusion Injury/physiopathology , Stem Cells/cytology , Stem Cells/physiology , Transforming Growth Factor beta/metabolism
2.
Cytometry A ; 77(5): 420-8, 2010 May.
Article in English | MEDLINE | ID: mdl-20151452

ABSTRACT

In recent years, slide-based cytometry has become a key technology for polychromatic cytometric investigations, and many efforts have been made to increase the number of measurable fluorochromes for multiparametric analysis. Sequential photobleaching of fluorochromes next to very photostable dyes is one approach for this technology. As the ALEXA dyes are known to be photostable as compared to the conventional fluorochromes FITC, PE (Riggs et al., Am J Pathol 1958;34:1081-1097), and APC, a differentiation within a fluorochrome pair is possible. Here, we have analyzed the newly available NorthernLights secondary antibodies for use in slide-based cytometry and microscopy. Currently, these fluorochrome-conjugates are now available with three distinct excitation- and emission maxima (NL493, NL557, NL637). Their spectral properties are similar to the frequently used fluorochromes FITC, PE, and APC and can, therefore, be used with most common excitation sources of cytometers or microscopes. As the NorthernLights are bright, resistant to photobleaching, stable in alcohols and xylene and of affordable price, these dyes are promising candidates for use with most laser- and HBO/XBA-based fluorescence microscopy-like techniques.


Subject(s)
Antibodies/analysis , Flow Cytometry/methods , Fluorescent Dyes/analysis , Microscopy, Fluorescence/methods , Antibodies/chemistry , Cell Line, Tumor , Fluorescein-5-isothiocyanate/metabolism , Humans , Leukocytes/cytology , Leukocytes/radiation effects , Photobleaching/radiation effects , Phycoerythrin/metabolism , Staining and Labeling , Ultraviolet Rays
3.
Am J Physiol Renal Physiol ; 298(1): F78-85, 2010 Jan.
Article in English | MEDLINE | ID: mdl-19906949

ABSTRACT

Endothelial progenitor cells (EPCs) protect kidneys from acute ischemic damage. The aim of this study was to identify "treatment parameters" that optimize an EPC-based therapy of acute ischemic renal failure. Male C57BL/6N mice underwent unilateral nephrectomy with simultaneous contralateral renal artery clamping for 30, 35, and 40 min. Tagged murine EPCs were systemically injected at the time of reperfusion. In some experiments, EPCs were pretreated with the Epac (exchange protein directly activated by cAMP-1) activator 8-pCPT-2'-O-Me-cAMP (Epac-1 Ac) and the integrin binding antagonist cyclic Arg-Gly-Asp peptide (cRGD). Injections of 10(6) EPCs after 30 and 35 min of renal ischemia protected animals from acute renal failure. The same effect occurred with 0.5 x 10(6) EPCs after a 35-min period of ischemia. If ischemia lasted for 40 min, 0.5 x 10(6) cells mice did not prevent acute renal failure. To analyze whether EPC integrin receptor activation would modify the cells' renoprotective activity, EPCs were pretreated with Epac-1 Ac. Such animals did not develop acute renal failure, even if ischemia lasted for 40 min. This effect was negated if the cells were pretreated with both Epac-1 Ac and cRGD. In kidneys from those animals medullopapillary EPCs were significantly accumulated. In vitro Epac-1 Ac preactivation of EPCs did not increase the overall expression intensity but induced a redistribution of beta(1)-integrins toward the cell membranes. We conclude that EPC pretreatment with the integrin receptor activator 8-pCPT-2'-O-Me-cAMP augments the anti-ischemic potential of the cells.


Subject(s)
Acute Kidney Injury/prevention & control , Cyclic AMP/analogs & derivatives , Endothelial Cells/metabolism , Endothelial Cells/transplantation , Guanine Nucleotide Exchange Factors/metabolism , Reperfusion Injury/prevention & control , Stem Cells/metabolism , Acute Kidney Injury/metabolism , Acute Kidney Injury/pathology , Animals , Cyclic AMP/therapeutic use , Disease Models, Animal , Endothelial Cells/pathology , Integrin beta Chains/metabolism , Kidney/blood supply , Kidney/metabolism , Kidney/pathology , Male , Mice , Mice, Inbred C57BL , Nephrectomy , Peptides, Cyclic/therapeutic use , Reperfusion Injury/metabolism , Reperfusion Injury/pathology , Stem Cell Transplantation/methods , Stem Cells/pathology , Transplantation, Homologous
4.
Cell Prolif ; 41(2): 365-74, 2008 Apr.
Article in English | MEDLINE | ID: mdl-18336479

ABSTRACT

OBJECTIVE: The aim of this study was to establish a preclinical mouse model to study metastases of paediatric rhabdomyosarcoma at the macroscopic and cellular levels, with different imaging methods. EXPERIMENTAL DESIGN: The alveolar rhabdomyosarcoma cell line Rh30 was stably transfected with the red fluorescent protein (DsRed2) then was xenotransplanted (intravenous injection [n = 8], and footpad injection [n = 8]) into nude mice (NMRI nu/nu). Macroscopic imaging of metastases was performed using DsRed2-fluorescence and flat-panel volumetric computed tomography scan. In a further series of animals (n = 8), in vivo cell trafficking of rhabdomyosarcoma cells using cellular imaging with an Olympus OV100 variable-magnification small-animal imaging system was used. RESULTS: Metastases in the pelvis, thoracic wall and skin were visualized by fluorescence imaging. Pelvic metastases were found after tail vein injection and at other metastatic sites after footpad injection. Flat-panel volumetric computed tomography scan data allowed highly specific analysis of contrast between tumour and surrounding tissue. Correlation between fluorescence and flat-panel volumetric computed tomography scan imaging data was observed. Single-cell imaging visualized tumour cells in the vessels and demonstrated the arrest of tumour cells at vessel junctions followed by extravasation of the tumour cells. CONCLUSION: We established a model for visualization of experimental metastatic invasion and describe relevant tools for imaging childhood rhabdomyosarcoma metastases at the macroscopic and cellular levels. Imaging of cell trafficking visualized the behaviour of tumour cells and development of metastases by accumulation and extravasation of rhabdomyosarcoma cells.


Subject(s)
Image Interpretation, Computer-Assisted/methods , Imaging, Three-Dimensional/methods , Rhabdomyosarcoma/pathology , Rhabdomyosarcoma/secondary , X-Ray Intensifying Screens , Animals , Cell Movement , Cell Proliferation , Disease Models, Animal , Humans , Image Interpretation, Computer-Assisted/instrumentation , Imaging, Three-Dimensional/instrumentation , Luminescent Proteins/chemistry , Mice , Mice, Nude , Microscopy, Fluorescence , Neoplasm Invasiveness , Neoplasm Metastasis , Neovascularization, Pathologic/pathology , Sensitivity and Specificity , Tumor Cells, Cultured , Red Fluorescent Protein
5.
Cytometry A ; 71(8): 542-9, 2007 Aug.
Article in English | MEDLINE | ID: mdl-17598185

ABSTRACT

The multiparametric molecular cell and tissue analysis in vitro and in vivo is characterized by rapid progress in the field of image generation technologies, sensor biotechnology, and computational modeling. Fascinating new potentials in unraveling the detailed functions of single cells, organs, and whole organisms are presently emerging and permit the close monitoring i.e. tumor development or basic cell development processes with an unprecedented multiplicity of promising investigative possibilities. To answer basic questions of in vivo tumor development and progression fluorescence based imaging techniques provide new insights into molecular pathways and targets. Genetic reporter systems (eGFP, DsRED) are available and high sensitive detection systems are on hand. These techniques could be used for in vitro assays and quantified e.g. by microscopy and CCD based readouts. The introduction of novel fluorescent dyes emitting in the near infrared range (NIR) combined with the development of sensitive detector systems and monochromatic powerful NIR-lasers for the first time permits the quantification and imaging of fluorescence and/or bioluminescence in deeper tissues. Laser based techniques particularly in the NIR-range (like two-photon microscopy) offer superb signal to noise ratios, and thus the potential to detect molecular targets in vivo. In combination with flat panel volumetric computed tomography (fpVCT), questions dealing e.g. with tumor size, tumor growth, and angiogenesis/vascularization could be answered noninvasively using the same animal. The resolution of down to 150 microm/each direction can be achieved using fpVCT. It is demonstrated by many groups that submillimeter resolutions can be achieved in small animal imaging at high sensitivity and molecular specificity. Since the resolution in preclinical small animal imaging is down to approximately 10 microm by the use of microCT and to subcellular resolutions using ( approximately 1 microm) microscope based systems, the advances of different techniques can now be combined to "multimodal" preclinical imaging and the possibilities for in vivo intravital cytometry now become within one's reach.


Subject(s)
Diagnostic Imaging/methods , Neoplasms, Experimental/pathology , Animals , Imaging, Three-Dimensional , Magnetic Resonance Imaging , Neoplasms, Experimental/diagnosis , Neoplasms, Experimental/diagnostic imaging , Tomography, X-Ray Computed , Whole Body Imaging
6.
J Immunother ; 23(5): 536-48, 2000.
Article in English | MEDLINE | ID: mdl-11001547

ABSTRACT

Human gammadelta T lymphocytes play an important role in nonadaptive reactions to infection and early tumor defense. This is the first report that freshly isolated, native gammadelta T cells of some healthy donors can kill human neuroblastoma cells to varying degrees. Their killing ability was increased and maintained during expansion and cultivation with interleukin-2 (IL-2; 400 IU/mL) for as long as 30 days (100% specific lysis at an effector-to-target cell (E:T) ratio of 20:1). gammadelta T lymphocytes without this spontaneous killing ability gained a specific cytolytic activity of 81% +/- 10.4% SD after stimulation with IL-2 for 24 hours. gammadelta cells were isolated from peripheral blood by positive enrichment (using a magnetic cell sorting system; purity, 95.2% +/- 3.2% SD, n = 21). High natural cytotoxic activity against human neuroblastoma cell lines (>50% specific lysis at an E:T ratio of 20:1) was exhibited by one of 11 donors, whereas two of 11 showed medium cytotoxicity (30% to 50% specific lysis). Eight of 11 donors showed very slight or no lytic activity against human neuroblastoma cells (<30% specific lysis). gammadelta T cells were also cytotoxic against Daudi (32.7% specific lysis at an E:T ratio of 20:1), Raji (10.3%), Colo 205 (23.1%), A 204 (54%), K 562 (100%), and SK-N-MC (100%) cells. Isolated gammadelta T cells were grown in Iscove modified Dulbecco medium with IL-2 (400 IU/mL). Increased cell proliferation (38.5% to 182%) was induced with phytohemagglutinin, IL-15, Clodronat, OKT3, or various combinations of these. Results of cold target inhibition assays suggest a natural killer-like activity of the gammadelta T-cell killing mechanism. Peptidase or papain render neuroblastoma cells unsusceptible to gammadelta T-cell killing, suggesting the involvement of antigen peptide(s) in the process of neuroblastoma cell killing. Treatment with acid phosphatase reduced specific lysis by 66.5% +/- 34.1% SD, which suggests a binding to phosphorylated neuroblastoma cell-surface structures in the killing mechanism of gammadelta T cells. Heat shock did not affect the extent of neuroblastoma killing by gammadelta cells. Recognition of neuroblastoma cells by gammadelta cytotoxic T lymphocytes is negatively regulated by major histocompatibility complex I receptors. Evidence for natural and inducible cell cytotoxicity of gammadelta T cells against human neuroblastoma cells, easy propagation, purification, and missing alloreactivity in mixed lymphocytes cultures indicates a role for this subpopulation of T lymphocytes in adoptive immunotherapy.


Subject(s)
Antibodies, Monoclonal/immunology , Antibodies, Neoplasm/immunology , Interleukin-2/immunology , Neuroblastoma/immunology , T-Lymphocytes/immunology , Adult , Apoptosis , Cell Division , Clodronic Acid/pharmacology , Clone Cells , Flow Cytometry , Humans , Immunotherapy , Interleukin-2/pharmacology , Neuroblastoma/pathology , Neuroblastoma/therapy , Sensitivity and Specificity , T-Lymphocytes/classification , T-Lymphocytes/drug effects , Tumor Cells, Cultured
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