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Results for “Cell Tracking”

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At least 631 records · Page 35Linked to original sources

PubMed1989

Phase tracking: an improved phase detection technique for cell membrane capacitance measurements.

We describe here a technique called phase tracking that greatly improves the accuracy of measurements of the membrane capacitance of single cells. We have modified the original phase detection technique to include a method for creating calibrated changes in the resistance in series with the cell. This provides a method to automate the adjustment of the phase detector to the appropriate phase angle for measuring membrane capacitance. The phase determination depends only on the cell's electrical parameters and does not require matching of the cell impedance with that of the slow capacitance cancellation circuitry of the patch-clamp amplifier. We show here that phase tracking can accurately locate the phase of the capacitance signal and can keep the detector aligned with this signal during measurements of exocytosis in mast cells, irrespective of the large drifts which occur in cell membrane resistance, membrane capacitance, or series resistance. The phase tracking technique is a valuable tool for quantifying exocytosis and endocytosis in single cells.

Animals↗

PubMed2000

Cocultivation of umbilical cord blood cells with endothelial cells leads to extensive amplification of competent CD34+CD38- cells.

OBJECTIVE: In this report, methods to expand the number of human cord blood hematopoietic stem cells were explored. MATERIALS AND METHODS: CD34+ cord blood cells were expanded in the presence of various cytokine combinations in either a stroma-free cell culture system or a preformed porcine microvascular endothelial cell layer. After 7 to 21 days, stem cell number and function were monitored. In addition, the replicative history of stem cells was tracked using the fluorescent dye, PKH26. RESULTS: With the addition of various cytokine combinations, total cellular expansion was equivalent for both culture systems, although the endothelial cell-based system contained statistically greater numbers of CD34+ cells. By day 21, the endothelial-based system receiving the FLT3L, SCF, IL-6, and GM-CSF cytokine combination contained five-fold greater numbers of CD34+ than the stroma cell-free culture cell system. Endothelial-based cultures receiving these four cytokines plus megakaryocyte growth and development factor produced a 640-fold expansion of CD34+CD38- cells as compared to a four-fold expansion in the stroma-free system. The number of progenitor cells generated was similar with both systems, yet the greatest degree of expansion of cobblestone area-forming cells was observed in the endothelial based cultures (11-fold vs four-fold). Virtually all CD34+ and CD34+CD38+ cells expanded in the presence of endothelial cells had undergone self replication by day 10, yet stromal cell-free cultures contained a significant number (4.8%) of quiescent cells. Identical numbers of re-isolated cord blood CD34+ cells expanded in both systems exhibited a similar ability to engraft and generate cells belonging to multiple hematopoietic lineages in human fetal bones implanted in immunodeficient mice. CONCLUSIONS: These results suggest that the use of preformed endothelial cell monolayers might permit the ex vivo generation of sufficient numbers of cord stem cells to serve as successful grafts for adult transplant recipients.

ADP-ribosyl Cyclase↗

PubMed2003

Visualizing T cell migration in vivo.

Ever since the realization that T lymphocytes are key players in the defense against pathogens and tumors, a major aim of immunologists has been to understand the relationship between the functional and migratory properties of antigen-specific T cells. The current paradigm proposes that T cells follow organ-specific trafficking pathways to exit from blood into the extravascular compartment. T cell homing is regulated at the level of adhesion molecules and chemokine receptors, whose expression is linked tightly to the differentiation state of the cell. Naïve T lymphocytes follow relatively uniform recirculation routes through secondary lymphoid organs, the molecular cues of which are fairly well understood. As effector and memory T cells must be capable of reaching virtually any site in the body, their migratory behavior is considerably more heterogeneous. During the past few years, innovative approaches for tracking T cells in vivo have emerged. Here, we review recent technical developments in experimental methods for the visualization of T cells both at the population and single cell level in vivo, and discuss what these methods have taught us about T cell trafficking.

Animals↗

PubMed2006

The extrapulmonary origin of fibroblasts: stem/progenitor cells and beyond.

Although intrapulmonary progenitor cells are traditionally believed to be the source for regenerating cells in response to lung injury, recent mounting evidence indicates that a significant proportion of the mesenchymal cells involved in this repair/remodeling process may be derived from extrapulmonary sources, such as the recently described circulating fibrocyte as well as other bone marrow-derived progenitor cells. Studies tracking CD34 and/or CD45 markers of fibrocytes show their presence in injured murine lung tissue. Moreover, bone marrow chimeric mice with green fluorescence protein (GFP)-expressing marrow cells show abundant GFP-expressing fibroblasts in their lungs in response to lung injury. However, although fibrocytes express CD34 and CD45, and appear to have the capacity to differentiate to myofibroblasts, these properties are not evident in the bone marrow-derived fibroblasts. Induction of CCL21 (SLC) and CXCR12 (SDF1alpha) in injured lung tissue, and their respective cognate receptors, CCR7 and CXCR4, in fibroblasts from injured lungs, suggests recruitment of these extrapulmonary progenitor cells via these chemokines. This is supported by evidence that antibody neutralization of CXCR12 reduces recruitment of fibrocytes and pulmonary fibrosis. In contrast, other studies suggest a protective effect for bone marrow-derived cells. Thus, although suggesting that influx of extrapulmonary fibroblast progenitor cells occurs in response to lung injury, these recent studies do not yet provide clear insight as to the actual phenotype and fate of the recruited cells, the identity of the progenitor cell population in bone marrow, and most important, the function or role of these cells in pathogenesis of the idiopathic interstitial pneumonias.

Animals↗

PubMed1994

Selection of retrovirally transduced hematopoietic cells using CD24 as a marker of gene transfer.

We have investigated the use of a cell surface antigen as a dominant selectable marker to facilitate the detection and selection of retrovirally infected target cells. The small coding region of the human cell surface antigen CD24 (approximately 240 bp) was introduced into a myeloproliferative sarcoma virus (MPSV)-based retroviral vector, which was then used to infect day 4 5-fluorouracil (5-FU)-treated murine bone marrow cells. Within 48 hours of termination of the infection procedure CD24-expressing cells were selected by fluorescent-activated cell sorting (FACS) with an antibody directed against the CD24 antigen. Functional analysis of these cells showed that they included not only in vitro clonogenic progenitors and day 12 colony-forming unit-spleen but also cells capable of competitive long-term hematopoietic repopulation. Double-antibody labeling studies performed on recipients of retrovirally transduced marrow cells showed that some granulocytes, macrophages, erythrocytes, and, to a lesser extent, B and T lymphocytes still expressed the transduced CD24 gene at high levels 4 months later. No gross abnormalities in hematopoiesis were detected in mice repopulated with CD24-expressing cells. Our results show that the use of the CD24 cell surface antigen as a retrovirally encoded marker enables the rapid, efficient, and nontoxic selection in vitro of infected primary cells, facilitates tracking and phenotyping of their progeny, and should provide a unique tool to identify elements that regulate the expression of transduced genes in the most primitive hematopoietic cells.

Animals↗

PubMedDate not supplied

[Evaluation of the survival of bone marrow mononucleate cells transplanted in a rat model of striatal lesion with quinolinic acid].

INTRODUCTION: Transplant is one of the alternatives available for the treatment of neurodegenerative diseases aimed at replacing the cells lost during the course of the disease. One promising source of cells for the development of transplants could be the mononucleate cells from bone marrow. AIMS. The purpose of this study was to study the capacity of bone marrow mononucleate cells to survive the transplant process, and to search for a method that enables tracking of these cells in vivo once they have been implanted. MATERIALS AND METHODS: Bone marrow mononucleate cells were extracted from the femur of rats by means of a Ficoll-Hypaque gradient. The cells under study were modified genetically with an adenovirus that expresses the PFV or which are marked with Hoechst dye. The marked cells were implanted in the striatum of rats with lesions caused by quinolinic acid. RESULTS: The viability of the genetically modified cells was low, whereas that of the cells marked with Hoechst dye was above 90%. The implanted cells survived the transplant at least a month and dispersed away from the site of entry towards the corpus callosum and cortex. CONCLUSIONS: We consider that the use of Hoechst dye offers more advantages for tracking these cells in vivo. Mononucleate cells have a number of characteristics that allow them to be included as candidate sources of cells for the treatment of neurodegenerative diseases.

Animals↗

PubMed2003

T cell receptor gene rearrangement lineage analysis reveals clues for the origin of highly restricted antigen-specific repertoires.

Due to ordered, stage-specific T cell receptor (TCR)-beta and -alpha locus gene rearrangements and cell division during T cell development, a given, ancestral TCR-beta locus VDJ rearrangement might be selected into the mature T cell repertoire as a small cohort of "half-sibling" progeny expressing identical TCR-beta chains paired with different TCR-alpha chains. The low frequency of such a cohort relative to the total alphabeta TCR repertoire precludes their direct identification and characterization in normal mice. We considered it possible that positive selection constraints might limit the diversity of TCR-alpha chains selected to pair with beta chains encoded by an ancestral VDJ-beta rearrangement. If so, half-sibling T cells expressing structurally similar, but different TCR-alpha chains might recognize the same foreign antigen. By single cell polymerase chain reaction analysis of antigen-specific TCRs selected during a model anti-tumor response, we were able to identify clusters of T cells sharing identical VDJ-beta rearrangements but expressing different TCR-alpha chains. The amplification of residual DJ-beta rearrangements as clonal markers allowed us to track T cells expressing different TCR-alpha chains back to a common ancestral VDJ-beta rearrangement. Thus, the diversity of TCR-alpha's selected as partners for a given VDJ-beta rearrangement into the mature TCR repertoire may indeed be very limited.

Amino Acid Sequence↗

PubMedDate not supplied

Isolation and characterization of residual undifferentiated mouse embryonic stem cells from embryoid body cultures by fluorescence tracking.

The differentiation of mouse embryonic stem (ES) cells can be induced in vitro after leukemia inhibitory factor (LIF) withdrawal and further enhanced by the formation of "embryoid body" (EB) aggregates. This strategy is being used in order to optimize differentiation protocols that would result in functional cells for experimental cell replacement therapies. However, this study presents the possibility for residual undifferentiated cells to survive after standard in vitro procedures. Mouse ES cells were stably transfected with the enhanced green fluorescent protein (EGFP), under the control of the Oct4 promoter, a transcription factor that is expressed in undifferentiated ES cells but down-regulated on differentiation. Residual fluorescent cells were isolated from EBs that were cultured in standard conditions in absence of LIF. These residual cells displayed recurrent gain of chromosomes 8 and 9. Residual fluorescent cells, further expanded in absence of LIF and cultured as EBs, still displayed a significant Oct4 expression in comparison with parental transfected ES cells. Consequently, these residual cells have an intrinsic resistance to differentiate. The behavior of these cells, observed in vitro, can be overcome in vivo, as they were able to induce teratomas in subcutaneously injected nude mice. Residual undifferentiated cells displayed slight levels of VASA and DAZL expression. These results demonstrate that mouse ES cells cultured in vitro, in standard conditions, can spontaneously acquire recurrent karyotypical changes that may promote an undifferentiated stage, being selected in standard culture conditions in vitro.

Animals↗

PubMed2004

Blastomere transplantation in human embryos may be a treatment for single gene diseases.

OBJECTIVE: To determine whether human embryos accept blastomere transplants and integrate them normally into the architecture of the developing embryo. DESIGN: A human blastomere transplantation model, involving 44 cryopreserved embryos that were specifically donated to research. SETTING: Academically affiliated private infertility center. PATIENT(S): Forty-four human embryos. INTERVENTION(S): In 21 experiments, one, two, or three blastomeres were transplanted, using standard microsurgical techniques that are widely used in preimplantation genetic diagnosis (PGD). Embryos were thawed and gender was determined, using established PGD techniques. Male (xy) blastomeres were then transplanted into female (xx) day 3 embryos, and the xy cells were tracked through blastocyst stage (days 5-6) and into the hatching period (day 6), using fluorescent in situ hybridization (FISH). MAIN OUTCOME MEASURE(S): Degree and location of xy cell integration into xx embryos. RESULT(S): High-quality recipient embryos (with 4 to 10 cells) developed uniformly into normal blastocyst stage embryos in 12 of 12 experiments (100%) and integrated donor blastomeres into their architecture, with apparently even distribution of daughter cells; this integration was documented in inner cell mass as well as in trophoectoderm. The intensity of this distribution appeared to correlate with the number of blastomeres transferred. Among nine abnormally developing embryos, only three (33%) demonstrated a normal distribution of offspring donor cells. CONCLUSION(S): High-quality embryos appear to have the ability to integrate donor blastomeres. Because the treatment of single gene diseases does not require successful treatment of all cells, blastomere transplantation could be explored as a treatment option, which also would greatly enhance efficiency and utilization of preimplantation genetic diagnosis.

Blastocyst↗

PubMed2001

Effects of doxorubicinol on excitation--contraction coupling in guinea pig ventricular myocytes.

The cardiotoxicity of the anticancer drug doxorubicin may be related to its main metabolite doxorubicinol. In this study, the acute effects of doxorubicinol on excitation-contraction coupling in isolated guinea pig ventricular myocytes were investigated and compared with doxorubicin using the whole-cell patch-clamp-, fura-2 fluorescence- and cell-edge tracking techniques. Both drugs were applied intracellularly by diffusion from the patch electrode for 15--20 min. Doxorubicin (100 microM) prolonged the action potential duration (APD) by 31% and enhanced cell shortening by 26%. Contrary to doxorubicin, doxorubicinol (10 microM) shortened APD by 25% and decreased cell shortening by 31%. APD shortening by doxorubicinol was due to an increase of the delayed rectifier K(+) current. Neither the inward rectifier K(+) current nor the L-type Ca(2+) current was influenced by doxorubicinol. The decline in cell shortening induced by doxorubicinol was not exclusively due to APD shortening because doxorubicinol reduced the peak Ca(2+) transient by 23% in cells clamped with an action potential of constant duration. Despite opposite effects on APD and contractility, both doxorubicin and doxorubicinol produced a considerable delay in the activation and inactivation of contraction and Ca(2+) transient, compatible with an impaired function of the sarcoplasmic reticulum. It is suggested that doxorubicinol-induced APD shortening may amplify the detrimental effects of both doxorubicin and doxorubicinol on sarcoplasmic reticulum Ca(2+) load and hence on contractile function. The accumulation of doxorubicinol in the cardiac myocytes may play an important role in the time-dependent development of doxorubicin-induced ventricular dysfunction.

Action Potentials↗

PubMed2002

Cell adhesion-related proteins as specific markers of sponge cell types involved in allogeneic recognition.

Sponge immunocyte identification is of interest to comparative immunologists since characterizing these cells will allow investigations into the mechanisms of non-self recognition in the oldest animal phylum. Here, we report that polyclonal antibodies raised against the core protein of a proteoglycan involved in cell adhesion in the marine sponge Microciona prolifera are specific markers for archaeocytes, the totipotent sponge cells. Archaeocytes are mobilized upon allogeneic contact and they accumulate in the contact zone. A second type of cell, the gray cells, are specifically recognized by monoclonal antibodies raised against CD44, a hyaluronan receptor. Gray cells do also accumulate in the contact area. Specific staining of a third sponge cell type, the rhabdiferous cells, shows that these do not accumulate upon allografting. These specific cell markers allow tracking of archaeocytes and gray cells, and show that they play an active role in sponge allogeneic reactions.

Animals↗

PubMed2003

Intracytoplasmic tagging of cells with ferumoxides and transfection agent for cellular magnetic resonance imaging after cell transplantation: methods and techniques.

BACKGROUND: Superparamagnetic iron oxides (SPIO) are being used to label cells for in vivo monitoring by magnetic resonance imaging (MRI). The purpose of this study is to present protocols using SPIO and a polycationic transfection agent for magnetic labeling of cells as a basis for cellular MRI. METHODS: Various concentrations of ferumoxides (FE)-poly-l-lysine (PLL) complexes were used to magnetically label cells. Iron incorporation into cells along with cell viability and short- and long-term toxicity were evaluated. RESULTS: Rapidly growing cell suspension and adherent cells were effectively labeled by means of endocytosis into endosomes at low concentrations of FE (25 microg/mL media) and PLL (0.75 microg/mL media). Hematopoietic stem cells and lymphocytes required higher concentrations of PLL (1.5 microg/mL) in serum-free media during initial FE-PLL complex formation before labeling the cells in culture. Total iron concentration in cells depended on the cell type, concentration of FE-PLL complexes in media, cellular density, and incubation time. Iron concentrations after overnight incubation with given FE at 25 microg/mL media resulted in, for example, T cells being labeled with 1 to 3 pg/cell of intracytoplasmic endosomal iron and 15 to 20 pg/cell of intracytoplasmic iron in mesenchymal stem cells compared with 0.01 to 0.1 pg/cell for unlabeled cells. Protocols developed for this study demonstrated no adverse effect on the cell viability, functional capacity, or toxicity. CONCLUSION: This technique can be used to label cells for in vivo MRI tracking of stem cells and lymphocytes. FE at a concentration of 25 to 50 microg/mL with a ratio of SPIO to PLL of 1:0.03 to 1:0.06 would be sufficient to label cells for cellular MRI.

Animals↗

PubMed1983

Perineural spread by squamous carcinomas of the head and neck: a morphological study using antiaxonal and antimyelin monoclonal antibodies.

Perineural spread has been demonstrated histologically in 65/180 (36%) major surgical resections for squamous carcinomas of the head and neck; the incidence in a smaller necropsy series was 18/20 (90%). Perineural infiltration was observed most commonly in the vicinity of carcinomas arising in the buccal cavity (31/63, 50%) and, at all sites, it was most commonly encountered near tumours less than or equal to 2.5 cm in diameter. Perineural spread near cervical node metastases was, by contrast, uncommon in the surgical series. Tumour within perineural spaces tends to be concentrated at the margin of the nerve and shows only limited extension inwards, but cells may track upwards and downwards within the spaces. Distant spread for greater than 2 cm is unusual, and interval sampling of involved nerves in necropsy material indicates that most perineural tumour cells are confined to the distal 1 cm of the affected nerve. Infiltrated nerves regularly show varying degrees of myelin and axonal degeneration, probably anoxic in origin, and segmental infarction of nerve trunks was observed in three patients. Fine changes in axons and myelin have been regularly demonstrated with two monoclonal antibodies, and the use of these new reagents is described.

Aged↗

PubMed2004

Human memory B cells transferred by allogenic bone marrow transplantation contribute significantly to the antibody repertoire of the recipient.

The bone marrow is an important source of Abs involved in long-term protection from recurrence of infections. Allogenic bone marrow transplantation (BMT) fails to restore this working memory. Attempts to overcome this immunodeficiency by immunization of the donor have not been very successful. More needs to be known about transfer of B cell memory by BMT. We tracked memory B cells from the donor to the recipient during BMT of a girl with leukocyte adhesion deficiency. Vaccination of her HLA-identical sibling donor 7 days before harvest induced Haemophilus influenzae type b (Hib) capsular polysaccharide (HibCP)-specific B cells readily detectable in marrow and blood. BMT did not lead to spontaneous production of HibCP Abs, but the recipient responded well to booster immunizations 9 and 11 mo after BMT. HibCP-specific B cells were obtained 7 days after the vaccinations, and their V(H) genes were sequenced and analyzed for rearrangements and unique patterns of somatic hypermutations identifying clonally related cells. Ninety (74%) of 121 sequences were derived from only 16 precursors. Twelve clones were identified in the donor, and representatives from all of them were detected in the recipient where they constituted 61 and 68% of the responding B cells after the first and second vaccinations, respectively. No evidence for re-entry of memory clones into the process of somatic hypermutation was seen in the recipient. Thus, memory B cells were transferred from the donor, persisted for at least 9 mo in the recipient, and constituted the major part of the HibCP-specific repertoire.

Amino Acid Sequence↗

PubMed2002

Induction of glioblastoma apoptosis using neural stem cell-mediated delivery of tumor necrosis factor-related apoptosis-inducing ligand.

Current therapies for gliomas fail to address their highly infiltrative nature. Standard treatments often leave behind microscopic neoplastic reservoirs, resulting in eventual tumor recurrence. Neural stem cells (NSCs) are capable of tracking disseminating glioma cells. To exploit this tropism to develop a therapeutic strategy that targeted tumor satellites, we inoculated human glioblastoma xenografts with tumor necrosis factor-related apoptosis-inducing ligand-secreting NSCs. This resulted in the dramatic induction of apoptosis in treated tumors and tumor satellites and was associated with significant inhibition of tumor growth. These results add credence to the potential of NSCs as therapeutically effective delivery vehicles for the treatment of intracranial glioma.

Animals↗

PubMed1980

Cellular and molecular mechanisms in angiogenesis.

Low concentrations of copper sulphate, Dispirin, or Walker carcinoma extract elicit intraocular vascularization when tested by anterior chamber implants in rats. The response is markedly depressed by pre-treatment of animals with methylprednisolone acetate, suggesting that such induced vascularization is mediated by leucocytes. Since many agents inducing vascularization also induce migration of cultured endothelial cells, it raises the problem of how to isolate and study the action of possible leucocyte-derived angiogenic factors. Regardless of the identity of the natural angiogenic factor or factors, it is proposed that during blood vessel formation specialized endothelial cells migrate in response to an angiogenic signal and deposit fibronectin on which other cells can track and subsequently adhere, forming an endothelium.

Animals↗

PubMedDate not supplied

Millennium fat-cell lipolysis reveals unsuspected novel tracks.

Adipose tissue lipolysis, i.e., the catabolic process leading to the breakdown of triglycerides into fatty acids and glycerol, is often considered as a simple and well-understood metabolic pathway. However, progress on the hormonal regulation and molecular mechanism of fat-cell lipolysis is opening new avenues and points to a number of unanswered questions. Recent studies on the lipolytic beta- and antilipolytic alpha2-adrenergic control of lipolysis has allowed a better understanding of the relative contribution of the two types of receptors and provide strong evidence for the in vivo implication of alpha2-adrenoceptors in the physiological control of subcutaneous adipose-tissue lipolysis. A novel lipolytic system has been characterized in human fat cells. Natriuretic peptides stimulate lipolysis through a cGMP-dependent pathway. The molecular details of the lipolytic reaction are not fully understood. Translocation of hormone-sensitive lipase, the rate-limiting enzyme of lipolysis, to the lipid droplet seems to be an important step during lipolytic activation. Reorganization of the lipid droplet coating by perilipins may also facilitate the access of the enzyme. Unexpectedly, hormone-sensitive lipase-deficient mice are not obese and show residual adipose-tissue lipolysis, which suggests the existence of another triglyceride lipase. Whether the expression of this uncharacterized neutral lipase is compensatory for the lack of hormone-sensitive lipase is an important question yet to be resolved. In humans, alterations of hormone-sensitive lipase expression are associated with changes in lipolysis in various physiological and pathological states. Genetic studies show that beta2-adrenoceptor and hormone-sensitive lipase genes may participate in the polygenic background of obesity.

Adipose Tissue↗

PubMed1999

Pertussis toxin treatment differentially affects cholinergic and dopaminergic receptor stimulation of midbrain dopaminergic neurons.

Extracellular single-unit recordings and iontophoresis were used to compare the effect of a single administration of pertussis toxin (PTX, 1 microg), into midbrain dopamine (DA) nuclei (A9 and A10 regions), on the muscarinic, NMDA and DA receptor responses of midbrain DA cells in the anesthetized rat. Iontophoretic applications of DA, or apomorphine (50 microg/kg, i.v.), markedly reduced the firing of DA cells in control rats. In PTX-treated animals, these inhibitory responses were totally abolished, indicating that, in both DA nuclei, the inhibitory DA receptors are coupled to Gi/o proteins. In parallel, there was a significant decrease in the number of active DA cells per track which returned to baseline 5 weeks after the treatment. Applications of the muscarinic agonist oxotremorine M (OXO M) or of NMDA produced a potent increase in the firing of DA cells in control rats. DA neurons treated with PTX were still responsive to OXO M, although their sensitivity to the agonist was significantly reduced by 40%. In contrast, NMDA-induced activation remained unchanged, indicating that PTX did not non-selectively dampen all excitatory responses. Applications of cell-permeable cAMP derivatives did not change the basal firing of DA neurons. On the other hand, the phospholipase C inhibitors neomycin and ET-18-OCH3 (200 microg, i.c.v.), reduced significantly the activation of DA cells induced by OXO M. These data suggest that muscarinic activation of DA cells involves an M1-like receptor, possibly coupled to Gq/11 proteins, but also the participation of a PTX substrate.

Action Potentials↗