PubMed Health⌕ Search

SEARCH · PubMed Health

Results for “Cell Tracking”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 649 records · Page 36Linked to original sources

Solving the binding problem of the brain with bi-directional functional connectivity.

We propose a neural network model which gives one solution to the binding problem on the basis of 'functional connectivity' and bidirectional connections. Here, 'functional connectivity' is dynamic neuronal connectivity peculiar to temporal spike coding neural networks with coincidence detector neurons. The model consists of a single primary map and two higher modules which extract two different features shown on the primary map. There exist three layers in each higher module and the layers are connected bi-directionally. An object in the outer world is represented by a 'global dynamical cell assembly' which is organized across the primary map and the two higher modules. Detailed, but spatially localized, information is coded in the primary map, whereas coarse, but spatially extracted information or globally integrated information is coded in the higher modules. Computer simulations of the proposed model show that multiple cell assemblies sharing the same neurons partially can co-exist. Furthermore, we introduce a three-dimensional J-PSTH (Joint-Peri Stimulus Time Histogram) which is capable of tracking such cell assemblies, altering its constituent neurons as in our proposed model.

Action Potentials↗

Dendritic cells endocytose Bacillus anthracis spores: implications for anthrax pathogenesis.

Phagocytosis of inhaled Bacillus anthracis spores and subsequent trafficking to lymph nodes are decisive events in the progression of inhalational anthrax because they initiate germination and dissemination of spores. Found in high frequency throughout the respiratory track, dendritic cells (DCs) routinely take up foreign particles and migrate to lymph nodes. However, the participation of DCs in phagocytosis and dissemination of spores has not been investigated previously. We found that human DCs readily engulfed fully pathogenic Ames and attenuated B. anthracis spores predominately by coiling phagocytosis. Spores provoked a loss of tissue-retaining chemokine receptors (CCR2, CCR5) with a concurrent increase in lymph node homing receptors (CCR7, CD11c) on the membrane of DCs. After spore infection, immature DCs displayed a mature phenotype (CD83(bright), HLA-DR(bright), CD80(bright), CD86(bright), CD40(bright)) and enhanced costimulatory activity. Surprisingly, spores activated the MAPK cascade (ERK, p38) within 30 min and stimulated expression of several inflammatory response genes by 2 h. MAPK signaling was extinguished by 6 h infection, and there was a dramatic reduction of secreted TNF-alpha, IL-6, and IL-8 in the absence of DC death. This corresponded temporally with enzymatic cleavage of proximal MAPK signaling proteins (MEK-1, MEK-3, and MAP kinase kinase-4) and may indicate activity of anthrax lethal toxin. Taken together, these results suggest that B. anthracis may exploit DCs to facilitate infection.

Anthrax↗

In vivo tracking of bone marrow stromal cells transplanted into mice cerebral infarct by fluorescence optical imaging.

Recent experimental studies have indicated that bone marrow stromal cells (BMSC) improve neurological deficits when transplanted into the animal models of various neurological disorders, although precise mechanism still remains unclear. In this study, we developed a new in vivo fluorescence optical imaging protocol to sequentially track the transplanted into the brain of the living animals subjected to cerebral infarct. Mice BMSC were harvested from transgenic mice expressing green fluorescent protein (BMSC-GFP). They were stereotactically transplanted into the ipsilateral striatum of mice subjected to permanent middle cerebral artery occlusion after 7 days of ischemia (n=12). During 12 weeks after transplantation, the skull was exposed and the green fluorescence emitted from the brain surface was sequentially observed, using in vivo fluorescence optical microscopy. As the results, regional green fluorescence was detected in the ipsilateral parietal region 4-12 weeks after transplantation in all animals and became more apparent over the time. The images obtained through the skull were very similar to those acquired by thinning or removing the skull. Immunohistochemistry evaluation revealed that the transplanted cells migrated towards the ischemic boundary zone and expressed the neuronal or astrocytic marker, supporting the findings on fluorescence optical images. Sequential visualization of the BMSC transplanted into the brain of living animals would be valuable for monitoring the migration, growth and differentiation of the transplanted cells to explore the fate and safety of stem cell transplantation for various neurological disorders.

Animals↗

'Fast-tracking' cancer referrals: application for periocular basal cell carcinoma.

AIM: To study the feasibility of a modified fast-track protocol for periocular basal cell carcinoma (BCC). METHODS: A modified protocol was set up with an aim to examine all periocular BCCs within 6 weeks of referral to our oculoplastic clinic. An audit of this protocol was performed over a 2-year period. RESULTS: A total of 65 patients were referred as a 'possible BCC' over the 2-year period. In all, 32 of these patients were referred by dermatologists (49%), followed by fellow consultant ophthalmologists (20 patients, 31%) and general practitioners (11 patients, 17%). The clinical suspicion in the oculoplastic clinic agreed with the referral diagnosis in 71% (46 of the 65) of patients. This figure was particularly high for referrals from dermatologists (84%). In all, 44 out of the 46 'clinically suspected BCC' underwent surgical excision. Histopathology confirmed BCC in 39 of these 44 patients, a diagnostic accuracy of 89%. Among the subgroup of patients referred by the dermatologists, the largest source of referrals, 24 out of 30 patients that underwent surgical excision had histologically proven BCC; a diagnostic accuracy of 80%. CONCLUSION: Our study shows that the modified fast-track protocol for periocular BCCs is practical and feasible. Such a practice is highly desirable since it prevents a long wait for patients who are aware of a possible malignant periocular lesion.

Basal Cell Carcinoma↗

Efficient non-viral transfection of adult neural stem/progenitor cells, without affecting viability, proliferation or differentiation.

BACKGROUND: Neurogenesis occurs in defined areas of the adult mammalian brain, including the dentate gyrus of the hippocampus. Rat neural stem/progenitor cells isolated from this region retain their multipotency in vitro and in vivo after grafting into the adult brain. Molecular signalling and lineage selection in these cells may be examined using genetic manipulation. However, valid analysis requires that this manipulation should not affect cellular viability, proliferation or differentiation. METHODS: We screened several transfection protocols to develop a method which met these criteria. We then tested the effects of transfection on viability, proliferation and differentiation into the three neural lineages: neurons, astrocytes and oligodendrocytes. RESULTS: In initial testing, ExGen500 and FuGene6 efficiently transfected adult neural stem/progenitor cells, in vitro. After optimisation, these agents transfected 16% and 11% of cells, respectively. FuGene6-treated cells did not differ from untransfected cells in their viability or rate of proliferation, whereas these characteristics were significantly reduced following ExGen500 transfection. Importantly, neither agent affected the pattern of differentiation following transfection. Both agents could be used to genetically label cells, and track their differentiation into the three neural lineages, after grafting onto ex vivo organotypic hippocampal slice cultures. CONCLUSIONS: These data demonstrate that non-viral transfection may be used to genetically manipulate neural stem/progenitor cells, without adversely affecting their growth or perturbing lineage selection. Such a method is valuable for examining the molecular mechanisms of cell fate determination in vitro. Furthermore, this protocol may be exploited in the development of cell-based gene therapy strategies.

Animals↗

A fast online hit verification method for the single ion hit system at GSI.

For a single ion hit facility built to irradiate specific targets inside biological cells, it is necessary to prove that the ions hit the selected targets reliably because the ion hits usually cannot be seen. That ability is traditionally tested either indirectly by aiming at pre-etched tracks in a nuclear track detector or directly by making the ion tracks inside cells visible using a stain coupled to special proteins produced in response to ion hits. However, both methods are time consuming and hits can be verified only after the experiment. This means that targeting errors in the experiment cannot be corrected during the experiment. Therefore, we have developed a fast online hit verification method that measures the targeting accuracy electronically with a spatial resolution of +/-1 microm before cell irradiation takes place.

Cell Culture Techniques↗

In situ characterization of CD4+ T cell behavior in mucosal and systemic lymphoid tissues during the induction of oral priming and tolerance.

The behavior of antigen-specific CD4+ T lymphocytes during initial exposure to antigen probably influences their decision to become primed or tolerized, but this has not been examined directly in vivo. We have therefore tracked such cells in real time, in situ during the induction of oral priming versus oral tolerance. There were marked contrasts with respect to rate and type of movement and clustering between naive T cells and those exposed to antigen in immunogenic or tolerogenic forms. However, the major difference when comparing tolerized and primed T cells was that the latter formed larger and longer-lived clusters within mucosal and peripheral lymph nodes. This is the first comparison of the behavior of antigen-specific CD4+ T cells in situ in mucosal and systemic lymphoid tissues during the induction of priming versus tolerance in a physiologically relevant model in vivo.

Administration, Oral↗

Binding and endocytosis of thrombospondin and thrombospondin fragments in endothelial cell cultures analyzed by cuprolinic blue staining, colloidal gold labeling, and silver enhancement techniques.

We investigated the distribution of thrombospondin-specific binding sites and the uptake of thrombospondin-gold conjugates in cultured porcine endothelial cells by light and electron microscopy. Colloidal gold marker and silver enhancement techniques were applied for cytochemical detection of monomeric thrombospondin and fragments of thrombospondin. Thrombospondin binds to granular and fibrillar structures and to sites of cell-cell contact on the cell surface, as indicated by many proteoglycan-cuprolinic blue precipitates. Cell migration tracks on the culture dish bottom are most heavily stained. Labeling of intact thrombospondin and of proteolytic fragments of thrombospondin with colloidal gold followed by silver intensification enables one to detect its binding and uptake in endothelial cells. Binding to the cell surface and uptake of thrombospondin-gold particles was inhibited by heparin but not by hyaluronic acid or chondroitin sulfate. The heparin binding region at the N-terminal end of the thrombospondin molecule proved to be essential for cell surface binding. Gold-conjugated thrombospondin fragments devoid of the heparin binding region were not internalized. After 60 min incubation at 37 degrees C, thrombospondin-gold particles accumulated in the lysosomal compartment close to the nucleus. In the presence of monensin and ammonium chloride, vesicles in this area are swollen and the concentration of particulate marker is reduced. Binding and uptake of thrombospondin by vascular endothelial cells appears to require linkage of the heparin binding region of the thrombospondin molecule to coated pits and heparan sulfate-rich molecules as receptors. Colloidal gold conjugation of thrombospondin fragments proved to be useful for cytochemical characterization of molecular domains.

Ammonium Chloride↗

Microdosimetry of rat alveolar type II cells irradiated with alpha particles from 239PuO2.

The alveolar type II cell is one of the critical cells for radiation damage in the lungs after inhalation of radioactive aerosols. With the aid of a Quantimet-970 image analyzer and a VAX-11/780 computer, we calculated the radiation dose to rat alveolar type II cells from alpha particles emitted by 239PuO2. A series of dosimetric parameters for type II cells, including track length distribution, linear energy transfer (LET), values of the specific energy for a single hit of a spherical target (z1), cellular dose, hit number, and their spatial distributions were calculated. By comparing the volume density of type II cells and lung tissue with energy deposited in alveolar type II cells, we found that the energy deposited per unit volume of type II cells was larger than that of lung tissue excluding type II cells. The z1 for spherical targets and the LET across type II cells were less than those in lung tissue excluding type II cells. The age of the rat and damage to lung by inhalation may significantly influence some of the parameters. The neoplastic transformation probability for type II cells is also discussed. The results suggest that the type II cell is an important target cell in the rat lung for exposure to inhaled 239PuO2.

Aerosols↗

Flow cytometric analysis of fluorescence in situ hybridization with dye dilution and DNA staining (flow-FISH-DDD) to determine telomere length dynamics in proliferating cells.

BACKGROUND: Telomeres shorten during DNA replication; extensive erosion of telomeres likely promotes replicative senescence and chromosomal instability. Telomere length in individual cells has been quantified by flow cytometric analysis of fluorescence in situ hybridization (flow-FISH). To determine the rate of telomere attrition (telomere erosion per cell division), we combined flow-FISH with dye dilution and DNA staining (flow-FISH-DDD) and measured telomere-specific fluorescence in proliferating cells identified by cell generation and cell cycle phase. METHODS: Peripheral blood mononuclear cells (PBMC) were stained with the cell division tracking dye carboxyfluorescein diacetate succinimidyl ester (CFSE), stimulated with phytohemagglutinin (PHA), grown for 5-6 days, hybridized with a telomere sequence-specific peptide nucleic acid fluorescent probe (PNA-Cy5), counterstained with DAPI, and analyzed by flow cytometry. The cell cycle distribution and cell division generations were respectively identified by analysis of DAPI emission and deconvolution of CFSE emission, and Cy5 emission was used to determine telomere-specific fluorescence, an indicator of telomere length, in each cell. RESULTS: In stimulated PBMC, in each cell cycle phase, the telomere-specific fluorescence diminished with increasing cell generation. The rate of decline of the telomere-specific fluorescence per cell generation did not significantly differ between cell cycle phases. CONCLUSIONS: Application of flow-FISH-DDD to measure mean telomere length and the rate of telomere attrition in proliferating cells may find use in studies of ageing and disease, the effects of telomere-modifying agents, and variability between individuals.

Carbocyanines↗

Primitive, quiescent, Philadelphia-positive stem cells from patients with chronic myeloid leukemia are insensitive to STI571 in vitro.

In clinical trials, the tyrosine kinase inhibitor STI571 has proven highly effective in reducing leukemic cell burden in chronic myeloid leukemia (CML). The overall sensitivity of CML CD34(+) progenitor cells to STI571 and the degree to which cell death was dependent on cell cycle status were determined. Stem cells (Lin(-)CD34(+)) from the peripheral blood of patients with CML in chronic phase and from granulocyte-colony-stimulating factor-mobilized healthy donors were labeled with carboxy-fluorescein diacetate succinimidyl diester dye to enable high-resolution tracking of cell division. Then they were cultured for 3 days with and without growth factors +/- STI571. After culture, the cells were separated by fluorescence-activated cell sorting into populations of viable quiescent versus cycling cells for genotyping. For healthy controls, in the presence of growth factors, STI571 affected neither cell cycle kinetics nor recovery of viable cells. In the absence of growth factors, normal cells were unable to divide. For CML samples, in the presence or absence of growth factors, the response to STI571 was variable. In the most sensitive cases, STI571 killed almost all dividing cells; however, a significant population of viable CD34(+) cells was recovered in the undivided peak and confirmed to be part of the leukemic clone. STI571 also appeared to exhibit antiproliferative activity on the quiescent population. These studies confirm that CML stem cells remain viable in a quiescent state even in the presence of growth factors and STI571. Despite dramatic short-term responses in vivo, such in vitro insensitivity to STI571, in combination with its demonstrated antiproliferative activity, could translate into disease relapse after prolonged therapy.

Antigens, CD34↗

Simultaneous quantification of cell motility and protein-membrane-association using active contours.

We present a new method for the quantification of dynamic changes in fluorescence intensities at the cell membrane of moving cells. It is based on an active contour method for cell-edge detection, which allows tracking of changes in cell shape and position. Fluorescence intensities at specific cortical subregions can be followed in space and time and correlated with cell motility. The translocation of two GFP tagged proteins (CRAC and GRP1) from the cytosol to the membrane in response to stimulation with the chemoattractant cAMP during chemotaxis of Dictyostelium cells and studies of the spatio-temporal dynamics of this process exemplify the method: We show that the translocation can be correlated with motility parameters and that quantitative differences in the rate of association and dissociation from the membrane can be observed for the two PH domain containing proteins. The analysis of periodic CRAC translocation to the leading edge of a cell responding to natural cAMP waves in a mound demonstrates the power of this approach. It is not only capable of tracking the outline of cells within aggregates in front of a noisy background, but furthermore allows the construction of spatio-temporal polar plots, capturing the dynamics of the protein distribution at the cell membrane within the cells' moving co-ordinate system. Compilation of data by means of normalised polar plots is suggested as a future tool, which promises the so-far impossible practicability of extensive statistical studies and automated comparison of complex spatio-temporal protein distribution patterns.

Algorithms↗

Real time computer tracking of free-swimming and tethered rotating cells.

A computerized image processing system has been developed that tracks individual free-swimming cells and rotating bacterial cell bodies tethered by their flagella in real time. Free-swimming bacteria of Rhodobacter sphaeroides, Rhodospirullum rubrum, and Salmonella typhimurium have been tracked swimming at speeds from 0 to over 120 microns s-1. A high level of discrimination is exerted against noncellular objects, allowing analysis of stopped as well as moving cells. This enabled detection of both speed and qualitative change in the swimming patterns of R. sphaeroides WS8 upon tactic stimulation. Comparison with darkfield microscopy indicated that the two techniques were in substantial agreement. The unidirectional rotation of cells of R. sphaeroides WS8 could be detected when the cells were either parallel to the microscope slide or end on. Frequencies of rotation of up to 10 Hz were monitored before image blurring became a problem. True rods would be easier to analyze at higher speeds of rotation. Although developed for photosynthetic bacteria, a wide range of bacteria, eucaryotic organisms, and subcellular organelles could be tracked with this system. Minor modifications to the software allow customization to different types of motility analysis.

Bacterial Physiological Phenomena↗

Expansion of hematopoietic stem cells in vitro as a model system for human tissue engineering.

The authors have taken a new approach to finding optimal conditions for stimulating conservative division of single isolated CD34(+)lin(-) hematopoietic stem cell candidates from human umbilical cord blood. The approach required the design and development of a novel multi-well single cell combinatorial culture system. This system incorporates the use of a multi-well tissue culture plate in which each well receives a single hematopoietic stem cell candidate. During an experiment lasting several days to weeks, each cell-containing well is moved sequentially and serially to a microscopic imaging system. This movement is facilitated by computer control of a motorized stage and stabilization of the experiment in an environmentally controlled Biobox built on the microscopic stage. New image analysis software facilitates tracking of cell movement, recording the time of cell division, and immunophenotyping of multiple, individual, or recently doubled cells in real time by a robotically controlled pipetting station. The principles of single cell culture should help solve many problems in human hematopoietic stem cell expansion and may be applicable to a wide range of other systems of interest in tissue engineering.

Animals↗

Tracking of green fluorescent protein (GFP)-labeled LAK cells in mice carrying B16 melanoma metastases.

In adoptive immunotherapy, in vivo trafficking of adoptively transferred cells, including their accumulation at tumor sites, remains to be further investigated. Tracking of these cells by visualization is useful to clarify antitumor mechanisms and develop new modalities to enhance antitumor capacities. In the present study, an in vivo tracking study was performed using an adoptive transfer model of lymphokine-activated killer (LAK) cells induced from green mice into C57/BL6 mice with B16 melanoma metastases. Green mice are green fluorescent protein (GFP) transgenic mice originating from C57/BL6 mice. All of the tissues, except for erythrocytes and hair, express green fluorescence under excitation light. Although LAK cells in combination with IL-2 potently suppressed pulmonary metastases with survival prolongation, very few LAK cells accumulated in tumor tissues compared to those localized in the spleen, as visualized by fluorescent microscopy and quantitated by flow cytometry. The present method using transfer of green mice-derived cells into parental tumor-bearing mice is simple because there is no need for in vitro labeling and is feasible for the in vivo tracking of effector cells in an adoptive immunotherapy model.

Animals↗

A dynamic fate map of the forebrain shows how vertebrate eyes form and explains two causes of cyclopia.

Mechanisms for shaping and folding sheets of cells during development are poorly understood. An example is the complex reorganisation of the forebrain neural plate during neurulation, which must fold a sheet into a tube while evaginating two eyes from a single contiguous domain within the neural plate. We, for the first time, track these cell rearrangements to show that forebrain morphogenesis differs significantly from prior hypotheses. We postulate a new model for forebrain neurulation and demonstrate how mutations affecting two signalling pathways can generate cyclopic phenotypes by disrupting normal cell movements or introducing new erroneous behaviours.

Animals↗

Inhibitors of histone deacetylases promote hematopoietic stem cell self-renewal.

BACKGROUND: Histone deacetylases (HDAC) are associated with a variety of transcriptional repressors that control cellular differentiation and proliferation. HDAC inhibitors such as trichostatin A, trapoxin and chlamydocin could be useful tools to modulate these cellular processes. We investigated their effect on the self-renewal of hematopoietic stem cells (HSC) during ex vivo culture. METHODS: Purified murine HSC with the phenotype c-Kit+,Thy-1.1(lo), Lin(-/lo), Sca-1+ were cultured for 4 days with IL-3, IL-6 and c-Kit ligand without or with HDAC inhibitors, after which their degree of phenotypic differentiation in culture was assessed by flow cytometric analysis. To explore whether HDAC inhibitors could have a beneficial role in human HSC transplantation, mobilized peripheral blood CD34+ cells were cultured with thrombopoietin mimetic peptide, flt3 ligand, and c-Kit ligand, without or with various HDAC inhibitors. The fluorescent dye, carboxyfluorescein-diacetate succinimidylester (CFSE), was used to track division of cell subsets, and engrafting ability was evaluated in a non-obese diabetic (NOD) -SCID xenotransplantation model. RESULTS: Murine HSC cultured with HDAC inhibitors maintained a more primitive phenotype than control cultures. The number of human HSC expressing Thy-1 increased up to seven-fold during a 5-day culture with HDAC inhibitors compared with control cultures. Chlamydocin was the most effective of the HDAC inhibitors tested at promoting Thy-1 expression on human cells. CFSE tracking showed that the increase in Thy-1+ cells resulted from cell division. In a NOD-SCID repopulation assay, cells exposed to chlamydocin for 24 h displayed an average four-fold higher engrafting ability over control cells. DISCUSSION: Our studies suggest that HDAC inhibitors can induce ex vivo expansion of human HSC, and may improve engraftment in hematopoietic transplant patients when cell dose is limiting.

Animals↗

Non-cultured cell transplantation in an ovine model of non-ischemic heart failure.

OBJECTIVE: Cell therapy may be a promising alternative or adjunct to current treatment modalities for ischemic heart failure. But little is known on the impact of myogenic cell transplantation in large animal models of non-ischemic cardiomyopathy. The aim of the present study was to explore whether an ovine model of toxin-induced heart disease could benefit from non-cultured skeletal muscle cell transplantation. METHODS: Sequential intracoronary injections of doxorubicin (0.75 mg/kg) were carried out every 2 weeks until echocardiographic detection of myocardial dysfunction. Sheep were then randomly assigned to either non-cultured cell transplantation (n=8) or placebo injection (n=5). For the cell therapy group, a skeletal muscle biopsy (about 10 g) was explanted from each animal approximately 3h before grafting. After thoracotomy, 20 epicardial injections were carried out. The animals were assessed one last time before sacrifice, 2 months after the thoracotomy. Cells were tracked with cmDiI (red fluorescence) and characterized with immunohistochemistry with monoclonal antibodies to a fast skeletal isoform of myosin heavy chain. RESULTS: Two months after intramyocardial grafting, tissue Doppler imaging and conventional echocardiographic assessment of the groups showed a marked improvement in the non-cultured cell therapy group. Ejection fraction (EF) (p<0.05) as well as systolic endocardial velocities (p<0.01) improved versus the placebo group. CmDiI and skeletal myosin heavy chain expression was detected in all animals at 2 months after implantation confirming engraftment of skeletal muscle cells. CONCLUSIONS: In conclusion, our data indicate that non-cultured muscle cell transplantation is feasible and may translate into a functional benefit in an ovine model of dilated heart failure.

Animals↗