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Results for “Cell Tracking”

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At least 235 records · Page 13Linked to original sources

PubMed2005

Activation of mitogen-activated protein kinase is required for migration and invasion of placental site trophoblastic tumor.

Placental site trophoblastic tumor (PSTT) is a gestational neoplasm derived from the extravillous (intermediate) trophoblast of the implantation site. PSTT is characterized by a highly invasive phenotype, but the molecular mechanisms are poorly understood. In this report, we demonstrate that PSTTs expressed the activated (phosphorylated) form of mitogen-activated protein kinase (MAPK) in 84% of cases, whereas the normal extravillous trophoblastic cells did not. To characterize the role of MAPK activation in PSTT, we established the first PSTT cell culture, IST-2, from a surgically resected PSTT. IST-2 cells expressed HLA-G and Mel-CAM but not E-cadherin, an immunophenotype characteristic of PSTT. IST-2 cells were highly motile and invasive in culture as compared to choriocarcinoma JEG-3 cells and normal extravillous trophoblastic cells. Based on wound assay, time-lapse videomicroscopy for cell tracking, and invasion chamber assays, we found that the motility and invasion of IST-2 cells were significantly reduced (P<0.01) after treatment with the MEK inhibitors CI-1040 and PD 59089, which prevent activation of MAPK. In contrast, neither compound had any effect on normal extravillous trophoblastic cells or JEG-3 cells. In conclusion, our findings demonstrate a functional role of MAPK activation in the motility and invasion of PSTT.

Benzamides↗

PubMed2003

Analysis of cell kinetics using a cell division marker: mathematical modeling of experimental data.

We consider an age-maturity structured model arising from a blood cell proliferation problem. This model is "hybrid", i.e., continuous in time and age but the maturity variable is discrete. This is due to the fact that we include the cell division marker carboxyfluorescein diacetate succinimidyl ester. We use our mathematical analysis in conjunction with experimental data taken from the division analysis of primitive murine bone marrow cells to characterize the maturation/proliferation process. Cell cycle parameters such as proliferative rate beta, cell cycle duration tau, apoptosis rate gamma, and loss rate micro can be evaluated from CarboxyFluorescein diacetate Succinimidyl Ester + cell tracking experiments. Our results indicate that after three days in vitro, primitive murine bone marrow cells have parameters beta = 2.2 day(-1), tau = 0.3 day, gamma = 0.3 day(-1), and micro = 0.05 day(-1).

Aging↗

PubMed1998

Cell division regulates the T cell cytokine repertoire, revealing a mechanism underlying immune class regulation.

Naive T lymphocytes have the potential to differentiate and produce a range of cytokines crucial for appropriate immune responses. How T lymphocytes vary their cytokine output during differentiation is unknown, although they are clearly influenced by the cytokines already present in the environment. Here we show that the number of divisions taken by the cells after activation is a critical element in T cell differentiation. Our experiments used the dye 5-(and 6-)carboxyfluorescein diacetate, succinimidyl ester to track cells in different divisions after activation by anti-CD3 in the presence of the differentiating cytokine interleukin (IL)-4. The patterns of acquisition or loss of secretion of IL-2, IL-3, IL-4, IL-5, IL-10, and interferon gamma all varied markedly with division number. These relationships were consistent regardless of the time-dependent variation in distribution of T cells among divisions. Thus, the observed combination of complex asynchronous T cell growth, overlaying a fixed probability of acquisition or loss of a cytokine at each division can explain why T cell differentiation displays the contradictory features of being both highly stochastic and highly controlled. Furthermore, these data reveal that T cells share a common regulatory strategy with B cells, whereby changes in the class of immune response are linked to the process of clonal expansion.

Animals↗

PubMed1994

Involvement of S100-related calcium-binding protein pEL98 (or mts1) in cell motility and tumor cell invasion.

We examined the relationship between cell motility and the expressions of pEL98 (mts1) mRNA and protein in various murine normal and transformed cells. The expression of pEL98 (mts1) in v-Ha-ras-transformed NIH3T3 cells and in normal rat kidney cells transformed by either v-Ha-ras or v-src was increased over that in the corresponding parental cells at both mRNA and protein levels. The expression in normal rat fibroblasts (3Y1) transformed by v-Ha-ras was also increased compared with that in 3Y1 cells. However, the expression of pEL98 (mts1) in 3Y1 cells transformed by v-src was increased in one clone (src 3Y1-K), but decreased in another clone (src 3Y1-H). The expression level of pEL98 (mts1) correlated well with cell motility, which was examined by measuring cell tracks by phagokinesis. In order to test direct involvement of the pEL98 (mts1) protein in cell motility, src 3Y1-H cells that showed low cell motility were transfected with pEL98 cDNA. The transfectants expressing large amounts of the pEL98 protein showed significantly higher cell motility than src 3Y1-H cells. The expression of pEL98 (mts1) was also found to be correlated with motile and invasive abilities in various clones derived from Lewis lung carcinoma. These results suggest that the pEL98 (mts1) protein plays a role in regulating cell motility and tumor cell invasiveness.

3T3 Cells↗

PubMed1998

The Rho GTPases in macrophage motility and chemotaxis.

The GTP-binding proteins, Rho, Rac and Cdc42 are known to regulate actin organisation. Rho induces the assembly of contractile actin-based microfilaments such as stress fibres, Rac regulates the formation of membrane ruffles and lamellipodia, and Cdc42 activation is necessary for the formation of filopodia. In addition, all three proteins can also regulate the assembly of integrin-containing focal adhesion complexes. The orchestration of these distinct cytoskeletal changes is thought to form the basis of the coordination of cell motility and we have investigated the roles of Rho family proteins in migration using a model system. We have found that in the macrophage cell line Bac1, the cytokine CSF-1 rapidly induces actin reorganisation: it stimulates the formation of filopodia, lamellipodia and membrane ruffles, as well as the appearance of fine actin cables within the cell. We have shown that Cdc42, Rac and Rho regulate the CSF-1 induced formation of these distinct actin filament-based structures. Using a cell tracking procedure we found that both Rho and Rac were required for CSF-1 stimulated cell translocation. In contrast, inhibition of Cdc42 does not prevent macrophages migrating in response to CSF-1, but does prevent recognition of a CSF-1 concentration gradient, so that cells now migrate randomly rather than up the gradient of this chemotactic cytokine. This implies that Cdc42, and thus probably filopodia, are required for gradient sensing and cell polarisation in macrophages.

Animals↗

PubMed1997

Chondrocyte transplantation to articular cartilage explants in vitro.

The transplantation of chondrocytes has shown promise for augmenting the repair of defects in articular cartilage. This in vitro study examined the efficiency of the transplantation of bovine chondrocytes onto articular cartilage disks and the ability of the transplanted chondrocytes to subsequently synthesize and deposit proteoglycan. The radiolabeling of chondrocyte cultures with [3H]thymidine, followed by 4 days of chase incubation, resulted in the incorporation of 98% of the radiolabel into DNA (as assessed by susceptibility to DNase). At the end of the culture period, the [3H]DNA was stable, with a half-life of radioactivity loss into the medium of 73 days. With use of radiolabeled chondrocytes for quantitation, the efficiency of transplantation onto a cartilage substrate was 93 +/- 4% for seeding densities of as much as 650,000 cells per cm2 and a seeding duration of 1 hour. These findings were confirmed both by tracking cells stained with 5-chlormethylfluorescein diacetate and by quantitating DNA. During the 16 hours after seeding onto a cartilage substrate (in which the endogenous cells had been lysed by lyophilization), the transplanted cells synthesized sulfated proteoglycan in direct proportion to the number of cells seeded. Most (83%) of the newly synthesized proteoglycan was released into the medium rather than retained within the layer of transplanted cells and the recipient cartilage substrate. Comparative studies with lyophilized-rehydrated or live cartilage as the recipient substrate indicated a similar efficiency of chondrocyte seeding and proteoglycan synthesis by the seeded chondrocytes. The transplanted cells retained the chondrocyte phenotype, as judged by a high proportion of the [35S]macromolecules being in the form of aggrecan that was capable of aggregating with hyaluronan and link protein, as well as by immunostaining within and around the transplanted cells for type-II, but not type-I, collagen. These results indicate that the number of chondrocytes transplanted onto a cut cartilage surface greatly affects the level of matrix synthesis; this in turn may affect repair.

Animals↗

PubMed2007

Cell labeling with the positive MR contrast agent Gadofluorine M.

The purpose of this study was to label human monocytes with Gadofluorine M by simple incubation for subsequent cell depiction at 1.5 and 3 T. Gadofluorine M displays a high r(1) relaxivity and is spontaneously phagocytosed by macrophages. Human monocytes were incubated with Gadofluorine M-Cy at varying concentrations and incubation times and underwent MR imaging at 1.5 and 3 T at increasing time intervals after the labeling procedure. R1-relaxation rates and r1 relaxivities of the labeled cells and non-labeled controls were determined. Cellular contrast agent uptake was examined by fluorescence microscopy and quantified by ICP-AES. Efficient cell labeling was achieved after incubation of the cells with 25 mM Gd Gadofluorine M for 12 h, resulting in a maximal uptake of 0.3 fmol Gd/cell without impairment of cell viability. Fluorescence microscopy confirmed internalization of the fluorescent contrast agent by monocytes. The r1 relaxivity of the labeled cells was 137 mM(-1)s(-1) at 1.5 T and 80.46 mM(-1)s(-1) at 3 T. Imaging studies showed stable labeling for at least 7 days. Human monocytes can be effectively labeled for MR imaging with Gadofluorine M. Potential in vivo cell-tracking applications include targeting of inflammatory processes with Gadofluorine-labeled leukocytes or monitoring of stem cell therapies for the treatment of arthritis.

Cells, Cultured↗

PubMed2006

Detection of cellular immunity to rabies antigens in human vaccinees.

A nonradioactive multi-parameter flow cytometry assay was developed to identify antigen-specific lymphocytes in human subjects previously vaccinated against rabies virus and was subsequently compared to the standard tritiated thymidine method. A cell tracking dye, carboxyfluorescein succinimidyl ester, was used in combination with surface label for CD4 and CD8 cells in order to determine the response of lymphocytes to killed rabies virus in an antigen recall assay. The rabies virus-specific lymphocyte response was compared to the humoral immune response in each of ten vaccinated and five non-vaccinated subjects. Lymphocyte responses to rabies virus were observed in all ten vaccinated subjects; some noted as early as 3 days after stimulation while others were not until 7 days after stimulation. There was good agreement between the proliferation index of the CFSE assay and the simulation index of the [3H]thymidine assay (kappa statistic=0.73). An inverse relationship was detected between the level of rabies virus neutralizing antibody (RVNA) and the lymphocyte response to inactivated rabies virus in the vaccinated subjects. The association between cytokines production and level of humoral and cellular response was investigated in four representative subjects. Two vaccinated subjects with high proliferation indices and low RVNA titers produced Th1 type cytokines to rabies virus stimulation, whereas two vaccinated individuals with low proliferation indices and high RVNA titers responses did not produce these cytokines.

Adult↗

PubMed1994

Ontogeny of serotonin-immunoreactive neurons in juvenile Aplysia california: implications for the development of learning.

Serotonin has been implicated in both nonassociative learning (sensitization and dishabituation) as well as associative learning (classical conditioning) in Aplysia californica. Dishabituation and sensitization, and their underlying physiological analogs, emerge according to different developmental timetables--sensitization develops 4 to 6 weeks after dishabituation (Rankin & Carew, 1988; Nolen & Carew, 1988; Wright, McCance, Lu, & Carew, 1991). Since the late emergence of sensitization could result from the delayed expression of facilitatory neurotransmitters, we have examined the ontogeny of serotonin immunoreactivity in juvenile A. californica by means of indirect immunohistofluorescence. The purpose of these experiments was to describe the developmental timetable for the expression of serotonin immunoreactivity and to correlate the emergence of immunoreactive neurons with the ontogenetic expression of different forms of learning. While the addition of serotonin-immunoreactive cells tracked the growth of the central nervous system, juveniles contained a relatively higher proportion of immunoreactive cells than adults. Immunoreactive cell bodies were present in the abdominal, cerebral, and pedal ganglia as early as juvenile Stage 9, prior to the emergence of dishabituation in Stage 10. The posterior cerebral cluster (PCC) contained four pairs of immunoreactive cells by Stage 9, including the facilitator CB1, which, as shown in adults, heterosynaptically facilitates siphon sensory neurons. The PCC reached the adult complement of five pairs of cells, by Stage 10, long before the development of sensitization, but at the time corresponding to the emergence of dishabituation. These results suggest that the late emergence of sensitization is not simply a consequence of the late expression of serotonergic facilitatory interneurons.

Animals↗

PubMed2006

Analyzing the cellular contribution of bone marrow to fracture healing using bone marrow transplantation in mice.

The bone marrow is believed to play important roles during fracture healing such as providing progenitor cells for inflammation, matrix remodeling, and cartilage and bone formation. Given the complex nature of bone repair, it remains difficult to distinguish the contributions of various cell types. Here we describe a mouse model based on bone marrow transplantation and genetic labeling to track cells originating from bone marrow during fracture healing. Following lethal irradiation and engraftment of bone marrow expressing the LacZ transgene constitutively, wild type mice underwent tibial fracture. Donor bone marrow-derived cells, which originated from the hematopoietic compartment, did not participate in the chondrogenic and osteogenic lineages during fracture healing. Instead, the donor bone marrow contributed to inflammatory and bone resorbing cells. This model can be exploited in the future to investigate the role of inflammation and matrix remodeling during bone repair, independent from osteogenesis and chondrogenesis.

Animals↗

PubMed2005

Bioluminescence imaging in living organisms.

Luciferase enzymes catalyze the emission of light from a substrate -- a phenomenon known as bioluminescence -- and have been employed as reporters of many biological functions. Luminescent reporters are much dimmer than fluorescent reporters, and therefore provide relatively modest spatial and temporal resolution. Yet, they are generally more sensitive and less toxic, making them particularly useful for long-term longitudinal studies of living cells, tissues and whole animals. Bioluminescence imaging has proven useful for detecting protein-protein interactions, for tracking cells in vivo, and for monitoring the transcriptional and post-transcriptional regulation of specific genes. Recent applications have included longitudinal monitoring of tumor progression in vivo, and monitoring circadian rhythms with single-cell resolution.

Animals↗

PubMed2004

Estimating human hematopoietic stem cell kinetics using granulocyte telomere lengths.

OBJECTIVE: To study in vivo behavior of hematopoietic stem cells (HSC). MATERIALS AND METHODS: Behavior of HSC is difficult to study because one cannot observe and track cells within the marrow microenvironment. Therefore, information must be obtained from indirect means, such as competitive repopulation assays or surrogate studies, such as observations of telomere shortening in granulocytes. In this article, we use granulocyte telomere length data and a novel approach, stochastic simulation, to derive replication rates of HSC. The approach is first applied to cats and then to humans. RESULTS: Human HSC replicate infrequently, on average once per 45 weeks (range: once per 23 to once per 67 weeks). CONCLUSIONS: This rate is substantially slower than the average replication rates estimated for murine (once per 2.5 weeks) and feline (once per 8.3-10 weeks) HSC in vivo.

Age Factors↗

PubMed1995

Heterogeneity in migration of smooth muscle cells from normal and injured rat thoracic aorta in primary culture.

OBJECTIVE: Aside from proliferation, migration of smooth muscle cells is an essential component of the arterial sclerotic reaction. The aim of this study was to define a model to study migration. METHODS: Primary cultures of smooth muscle cells were derived from normal or injured rat thoracic aorta. An image analysis system was used to track cells migrating out of the explants and measure the displacement of their centre of gravity. RESULTS: Migration speeds for smooth muscle cells randomly sampled from the normal whole media were very heterogeneous. The media were therefore separated into three vertical segments. Cells from the middle third migrated faster than those from the upper and lower thirds, regardless of whether they originated from the anterior and posterior parts of the segment (P = 0.001). Heparin (10 micrograms.ml-1) only inhibited smooth muscle cell migration from the middle segment (P < 0.001). Migration of smooth muscle cells from explants of aorta 3 and 14 d after injury was also studied using a balloon catheter. Three days after injury, cell velocity varied widely among the segments of the same media. In contrast, 14 d after injury cells from neointimal explants migrated homogeneously and at a slower rate than those obtained from normal media. CONCLUSIONS: These experiments show migratory variations among smooth muscle cells depending upon their position in the normal aorta and their state of activation after arterial injury. This variability must be taken into account when planning experiments to study smooth muscle cell migration.

Animals↗

PubMed1999

Flt3LP3nduces the ex-vivo amplification of umbilical cord blood committed progenitors and early stem cells in short-term cultures.

Umbilical cord blood (UCB) has been successfully used for haemopoietic stem cell transplantation, although its use has been cautiously limited to paediatric patients because of the reduced volume produced. The clinical results have confirmed that either engraftment or survival significantly correlate with cell dose infused. We have standardized a culture method providing in a short time a significant amplification of both committed progenitors and primitive stem cells for clinical use. Eight-day culture of UCB cells with flt3L/SCF/PIXY 321 induced a 10-fold amplification of CD34+ cells and the expansion of multipotent (CFU-GEMM) and committed (CFU-GM, BFU-E) progenitors respectively of 5-, 7- and 9-fold over input cells. As to the early stem cell pool, the primitive CD34+Thy-1+ cell fraction increased 6-fold and the LTC-IC were amplified 17-fold. Furthermore, the in vitro proliferation was detected by the gradual loss of fluorescence of the CD34+ cells tracked at day 0 with the dye PKH26. After 8 d of amplification >6% of the CD34+ cells remained intensely fluorescent. This subpopulation represents a deeply quiescent cell fraction unresponsive to cytokines and very enriched of primitive stem cells. These cells are most likely to be responsible for long-term reconstitution after transplant.

Antigens, CD34↗

PubMed1999

Functional hierarchy of simultaneously expressed adhesion receptors: integrin alpha2beta1 but not CD44 mediates MV3 melanoma cell migration and matrix reorganization within three-dimensional hyaluronan-containing collagen matrices.

Haptokinetic cell migration across surfaces is mediated by adhesion receptors including beta1 integrins and CD44 providing adhesion to extracellular matrix (ECM) ligands such as collagen and hyaluronan (HA), respectively. Little is known, however, about how such different receptor systems synergize for cell migration through three-dimensionally (3-D) interconnected ECM ligands. In highly motile human MV3 melanoma cells, both beta1 integrins and CD44 are abundantly expressed, support migration across collagen and HA, respectively, and are deposited upon migration, whereas only beta1 integrins but not CD44 redistribute to focal adhesions. In 3-D collagen lattices in the presence or absence of HA and cross-linking chondroitin sulfate, MV3 cell migration and associated functions such as polarization and matrix reorganization were blocked by anti-beta1 and anti-alpha2 integrin mAbs, whereas mAbs blocking CD44, alpha3, alpha5, alpha6, or alphav integrins showed no effect. With use of highly sensitive time-lapse videomicroscopy and computer-assisted cell tracking techniques, promigratory functions of CD44 were excluded. 1) Addition of HA did not increase the migratory cell population or its migration velocity, 2) blocking of the HA-binding Hermes-1 epitope did not affect migration, and 3) impaired migration after blocking or activation of beta1 integrins was not restored via CD44. Because alpha2beta1-mediated migration was neither synergized nor replaced by CD44-HA interactions, we conclude that the biophysical properties of 3-D multicomponent ECM impose more restricted molecular functions of adhesion receptors, thereby differing from haptokinetic migration across surfaces.

Antibodies, Monoclonal↗

PubMed2004

Transplantation of cultured salivary gland cells into an atrophic salivary gland.

Patients with dry mouth have been treated with salivary substitutes and/or medications such as pilocarpine or cevimeline hydrochloride. These treatments temporarily relieve their symptoms and induce salivation from residual tissue. However, no treatment is available for the purpose of regenerating an atrophic gland. In this study, the feasibility of a cell transplantation therapy for the atrophic submandibular glands was investigated in rats. Further, the potential of cell differentiation into a useful phenotype was assessed by immunohistochemistry together with cell tracking with the fluorescent dye PKH 26. Rat submandibular glands were excised, and the salivary gland epithelial cells were cultured for 3 weeks with 3T3 cells as a feeder layer. Ductal ligation of the submandibular gland was employed to generate an atrophic gland. One week after the operation, the ligation was removed, and the cultured cells labeled with PKH 26 were injected into the atrophic submandibular glands. As a control, the cultured cells were also injected into normal submandibular glands. Two weeks after cell transplantation, the transplanted cells were detectable in both the experimental and control groups. The cells were clustered in the connective tissue between the lobules. Four weeks after transplantation, the labeled cells were detectable in the experimental group but not in the control group. In the atrophic glands, the scattered transplanted cells were observed over a broad area of the gland but localized mainly around the acini and ductal region. Immunostaining results showed a possible involvement of the transplanted cells in ductal regeneration, while neither myoepithelial nor acinar differentiations were observed within the 4 weeks since transplantation. This study demonstrated that cell transplantation to the salivary gland is feasible, and that the transplanted cells were selectively attracted to and remained in the damaged area without affecting normal tissue.

3T3 Cells↗

PubMed1993

Needle biopsy of breast cancer. Appearance of tumour cells along the needle track.

Appearance of tumour cells along the needle track in patients with breast cancer diagnosed by SURECUT needle biopsy is found in two out of 47 consecutive cases. It is not known whether these cells are early implantation metastases or are harmless. To avoid any unnecessary risk to the patients, we recommend that needle biopsy is so performed, that it is possible to remove the track during the definite surgical procedure, and that penetration into the muscles of the thoracic wall during the biopsy procedure is avoided.

Biopsy, Needle↗

PubMed1999

Quantification of cellular properties from external fields and resulting induced velocity: cellular hydrodynamic diameter.

An experimental technique is discussed in which the size distribution of a population of cells is determined by calculating each cell's settling velocity. The settling velocity is determined from microscopically obtained images which were recorded on SVHS tape. These images are then computer imaged and processed, and the cell's location and velocity are determined using a computer algorithm referred to as cell tracking velocimetry (CTV). Experimental data is presented comparing the distribution of human lymphocytes and a human breast cancer cell line, MCF-7, determined using a Coulter counter and the CTV approach.

Algorithms↗