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At least 613 records · Page 34Linked to original sources
PubMed2000

Population code for tracking velocity based on cerebellar Purkinje cell simple spike firing in monkeys.

Velocity is an important determinant of the simple spike discharge of cerebellar Purkinje cells. In a previous study, Purkinje cells in the intermediate and lateral cerebellum recorded during manual tracking were found to be tuned to a combination of direction and speed, (i.e. preferred velocity). In this study a population analysis of this simple spike discharge was used to determine whether the velocity of tracking could be predicted. For the majority (30/32) of direction-speed combinations, the population response accurately specified the target velocity. A temporal analysis showed how the population response gradually converged to the required velocity 200 ms prior to the onset of tracking. Therefore, the simple spike discharge of a Purkinje cell ensemble contains sufficient information to reconstruct target velocity, providing support for the hypothesis that the cerebellum controls or signals movement velocity.

Animals↗
PubMed2006

Quantum dot labeling and tracking of human leukemic, bone marrow and cord blood cells.

Quantum dots (QDs) are nanometer scale fluorescent semiconductors that are increasingly used as labeling tools in biological research. These nanoparticles have physical properties, such as high quantum yield and resistance to photobleaching, that make them attractive molecular probes for tracking hematologic cells. Here, we show that QDs attached to a transporter protein effectively label all hematologic cells tested, including cell lines and malignant and non-malignant patient samples. We demonstrate that dividing cells can be tracked through at least four cell divisions. In leukemic cell lines, some cells remain labeled for 2 weeks. We show that QDs can be used to follow cells as they differentiate. QDs are seen in monocyte-like and neutrophil-like progeny of labeled HL-60 myeloblasts exposed to Vitamin D analogues and DMSO, respectively. QDs are also observed in monocytes generated from labeled CD34+ cells. In addition, QDs attached to streptavidin can target cells with differing cell surface markers, including CD33. In summary, QDs have the ability to bind to specific cells of interest, be taken up by a diverse range of hematologic cells, and followed through many divisions and through differentiation. These results establish QDs as extremely useful molecular imaging tools for the study of hematologic cells.

Antigens, CD↗
PubMed2003

CXCR4 chemokine receptors (CD184) and alpha4beta1 integrins mediate spontaneous migration of human CD34+ progenitors and acute myeloid leukaemia cells beneath marrow stromal cells (pseudoemperipolesis).

Marrow stromal cells play an important role in regulating the development and proliferation of haematopoietic stem cells (HSC) within the marrow microenvironment. However, the molecular mechanisms of stem cell-stromal cell interactions are not fully understood. We observed that mobilized peripheral blood and cord-blood-derived CD34+ progenitor cells, or CD34+ acute myeloid leukaemia (AML) cells spontaneously migrated beneath marrow stromal cells, an in vitro migration phenomenon termed pseudoemperipolesis. In contrast, the CD34+ myeloid leukaemia cell line, Kasumi-1, did not display pseudoemperipolesis. Cord blood CD34+ cells had a higher capacity than granulocyte-colony-stimulating-factor-mobilized CD34+ cells for pseudoemperipolesis (28.7 +/- 12%vs 18.1 +/- 6.1% of input cells within 24 h, mean +/- SD, n = 8), whereas 9.4 +/- 12.6% (mean +/- SD, n = 10) of input AML cells displayed this phenomenon. Pseudoemperipolesis of CD34+ progenitor and AML cells was significantly inhibited by pertussis toxin and antibodies to the CXCR4 chemokine receptor (CXCR4, CD184), but not control antibodies. Moreover, CD34+ and AML cell migration was significantly inhibited by a CS1 peptide that blocks alpha4beta1 integrin binding, but not by a control peptide, in which the fibronectin binding motif was scrambled. Pseudoemperipolesis was associated with an increased proliferation of migrated CD34+ progenitor cells but not AML cells within the stromal layer, demonstrated by cell cycle analysis and cell division tracking. We conclude that alpha4beta1 integrin binding and CXCR4 chemokine receptor activation are prerequisites for the migration of CD34+ haematopoietic progenitors and AML cells beneath marrow stromal cells. These observations suggest a central role of marrow stromal cells for HSC trafficking and homing within the marrow microenvironment.

Antigens, CD34↗
PubMed1974

Biochemical localization of alkaline phosphatase in the cell wall of a marine pseudomonad.

The various layers of the cell envelope of marine pseudomonad B-16 (ATCC 19855) have been separated from the cells and assayed directly for alkaline phosphatase activity under conditions established previously to be optimum for maintenance of the activity of the enzyme. Under conditions known to lead to the release of the contents of the periplasmic space from the cells, over 90% of the alkaline phosphatase was released into the medium. Neither the loosely bound outer layer nor the outer double-track layer (cell wall membrane) showed significant activity. A small amount of the alkaline phosphatase activity of the cells remained associated with the mureinoplasts when the outer layers of the cell wall were removed. Upon treatment of the mureinoplasts with lysozyme, some alkaline phosphatase was released into the medium and some remained with the protoplasts formed. Cells washed and suspended in 0.5 M NaCl were lysed by treatment with 2% toluene, and 95% of the alkaline phosphatase in the cells was released into the medium. Cells washed and suspended in complete salts solution (0.3 M NaCl, 0.05 M MgSO(4), and 0.01 M KCl) or 0.05 M MgSO(4) appeared intact after treatment with toluene but lost 50 and 10%, respectively, of their alkaline phosphatase. The results suggest that the presence of Mg(2+) in the cell wall is necessary to prevent disruption of the cells by toluene and may also be required to prevent the release of alkaline phosphatase by toluene when disruption of the cells by toluene does not take place.

Alkaline Phosphatase↗
PubMed2006

Nonperturbative chemical imaging of organelle transport in living cells with coherent anti-stokes Raman scattering microscopy.

Nonperturbative monitoring of intracellular organelle transport in unstained living cells was achieved with coherent anti-Stokes Raman scattering (CARS) microscopy. To avoid possible interference with the organelle transport introduced by laser radiation, we first examined different illumination conditions. Using a new photodamage criterion based on morphological changes of the cells, we determined the threshold values of both pulse energy and average power at relevant wavelengths. Under excitation conditions much milder than the threshold levels, we were able to monitor the motions of lipid droplet (LD) organelles in steroidogenic mouse adrenal cortical (Y-1) cells with CARS microscopy in real time without perturbations to the cells. Particle tracking analyses revealed subdiffusion as well as active transport of LDs along microtubules. Interestingly, LD active transport is only present in Y-1 cells that rounded up in culture, a morphological change associated with steroidogenesis, suggesting possible involvements of LD active transport in the latter. Simultaneous imaging of LDs and mitochondria with CARS and two-photon fluorescence microscopy clearly showed that interactions between the two organelles could be facilitated by high LD motility. These observations demonstrate CARS microscopy as a powerful noninvasive imaging tool for studying dynamic processes in living cells.

Animals↗
PubMed2006

Assessment of selective homing and contribution to vessel formation of cryopreserved peripherally injected bone marrow mononuclear cells following experimental myocardial damage.

In view of a potential clinical use we aimed this study to assess the selective homing to the injured myocardium and the definitive fate of peripherally injected labeled and previously cryopreserved Bone Marrow Mononuclear cells (BMMNCs). The myocardial damage (cryoinjury) was produced in 59 rats (45 treated, 14 controls). From 51 donor rats 4.4 x 10(9) BMMNCs were isolated and cryopreserved (slow-cooling protocols); the number of CD34+ and the viability of pooled cells was assessed by flow-cytometry analysis before and after cryopreservation and simulated delivery through a 23G needle. Seven days after injury, BMMNCs were thawed, labeled with PKH26 dye and peripherally injected (20 x 10(6) cells in 500 microl) in recipient rats. Two weeks after experimental injury, the heart, lungs, liver, kidneys, spleen and thymus were harvested to track transplanted cells. Except a small amount in the spleen, PKH26+ cells were found only in the infarcted myocardium of the treated animals. Typical vascular structures CD34+ were found in the infarcted areas of all animals; treated rats showed a significantly higher number of these structures if compared with untreated. Morphological ultra-structural examination of infarcted areas confirmed in treated rats the presence of early-stage PKH26+ vascular structures derived from injected BMMNCs. The estimated mean CD34+ cells loss due to the cryopreservation procedure and to the system of delivery was 0.24% and 0.1%, respectively, confirming the feasibility of the procedure. This study supports the possible therapeutic use of cryopreserved peripherally injecetd BMMNCs as a source of CD34+ independent vascular structures following myocardial damage.

Animals↗
PubMed1999

Adjuvant effect of IL-12: conversion of peptide antigen administration from tolerizing to immunizing for CD8+ T cells in vivo.

CD8+ T cells from TCR transgenic 2C mice, specific for SIYRYYGL peptide bound to H-2Kb, were adoptively transferred into C57BL/6 recipients to allow monitoring of their location, numbers, and phenotype upon peptide challenge. Recipients were primed by s.c. injection of SIYRYYGL alone or with CFA or IL-12, and the transferred cells then tracked by flow cytometry using the 1B2 mAb specific for the 2C TCR. Peptide alone induced a transient and weak expansion of 1B2+ cells in the draining lymph nodes (DLN) by day 3, but these cells were tolerant to secondary peptide challenge. In contrast, priming with CFA/peptide resulted in a large clonal expansion of 1B2+ cells in DLN by day 3, and the cells exhibited a CD25highCD44high phenotype, blast transformation, and lytic effector function. By day 5, 1B2+ cell numbers decreased in the DLN and increased in the spleen and blood. 1B2+ cells with a memory phenotype persisted through day 60 in the DLN, spleen, and blood and responded to secondary peptide challenge. Immunization with peptide, along with IL-12, mimicked the adjuvant effects of CFA with respect to phenotype, clonal expansion, effector function, and establishment of memory. IL-12 was not unique in providing this adjuvant effect however, since CFA/peptide immunization of IL-12-deficient recipient mice also resulted in 1B2+ T cell activation and clonal expansion. Thus, CFA or IL-12 can enhance Ag-specific CD8+ T cell responses to peptide, demonstrating that an inflammatory cytokine(s) can support activation and prevent tolerance induction.

Adjuvants, Immunologic↗
PubMed2005

alpha-Galactosylceramide therapy for autoimmune diseases: prospects and obstacles.

Autoimmune responses are normally kept in check by immune-tolerance mechanisms, which include regulatory T cells. In recent years, research has focused on the role of a subset of natural killer T (NKT) cells - invariant NKT (iNKT) cells, which are a population of glycolipid-reactive regulatory T cells - in controlling autoimmune responses. Because iNKT cells strongly react with a marine-sponge-derived glycolipid, alpha-galactosylceramide (alpha-GalCer), it has been possible to specifically target and track these cells. As I discuss here, although preclinical studies have shown considerable promise for the development of treatment with alpha-GalCer as a therapeutic modality for autoimmune diseases, several obstacles need to be overcome before moving alpha-GalCer therapy from the bench to the bedside.

Animals↗
PubMed1998

Insight into the origin and clonal history of B-cell tumors as revealed by analysis of immunoglobulin variable region genes.

Recombination of VH, DH and JH genes is a unique first step in normal B-cell development. Subsequent differentiation to a mature plasma cell is accompanied by further events in the Ig genes, including VL-JL joining, somatic hypermutation and isotype switching. Chromosomal changes leading to B-cell tumors can occur at many points in this sequence, and may be partly a consequence of the genetic mobility and mutability permitted in order to generate a diverse antibody repertoire. V genes of neoplastic B cells may reflect the point of maturation reached by the B cell of origin, prior to transformation. Analysis of tumors therefore provides useful information on V-gene patterns in normal B cells, and may add another dimension to classification of B-cell tumors. Transformation may also preserve cell populations normally destined to die by apoptosis. Tumor cells arrested in the site where somatic hypermutation and isotype switch are occurring can still be subject to these processes, and could be influenced by persisting antigen. However, mutation is silenced at the point of exit to the periphery, leading to fixed mutational patterns in tumors of mature B cells. V-gene analysis provides an invaluable tool for understanding the genesis of neoplastic change. It also has a clear clinical relevance in tracking tumor cells, measuring residual disease, and finally in offering the opportunity of developing vaccines for treatment.

Amino Acid Sequence↗
PubMed2003

Dynamic tracking of human hematopoietic stem cell engraftment using in vivo bioluminescence imaging.

The standard approach to assess hematopoietic stem cell (HSC) engraftment in experimental bone marrow transplantation models relies on detection of donor hematopoietic cells in host bone marrow following death; this approach provides data from only a single time point after transplantation for each animal. In vivo bioluminescence imaging was therefore explored as a method to gain a dynamic, longitudinal profile of human HSC engraftment in a living xenogeneic model. Luciferase expression using a lentiviral vector allowed detection of distinctly different patterns of engraftment kinetics from human CD34+ and CD34+CD38- populations in the marrow NOD/SCID/beta 2mnull mice. Imaging showed an early peak (day 13) of engraftment from CD34+ cells followed by a rapid decline in signal. Engraftment from the more primitive CD34+CD38- population was relatively delayed but by day 36 increased to significantly higher levels than those from CD34+ cells (P <.05). Signal intensity from CD34+CD38-engrafted mice continued to increase during more than 100 days of analysis. Flow cytometry analysis of bone marrow from mice after death demonstrated that levels of 1% donor cell engraftment could be readily detected by bioluminescence imaging; higher engraftment levels corresponded to higher image signal intensity. In vivo bioluminescence imaging provides a novel method to track the dynamics of engraftment of human HSC and progenitors in vivo.

Animals↗
PubMed1994

Lethality and mutagenesis of B lymphocyte progenitor cells following exposure to alpha-particles and X-rays.

B lymphocyte precursor cells are the target cells for the major subtype of paediatric cancer, acute lymphoblastic leukaemia. Using a murine IL-7-dependent clonogenic assay for normal B cell precursors as a model, we have investigated the sensitivity of these cells versus other normal and leukaemic haemopoietic cells to alpha-particle radiation. We find that B cell precursors are remarkably susceptible to the lethal effects of alpha-particles and have a very low probability of surviving a single alpha-track. B cell precursors are also very sensitive to the lethal effects of low LET X-rays. The mutation frequency in a marker gene (HPRT) does not, however, appear to be greater in B cell precursors that survive X-radiation than in other haemopoietic cells.

Alpha Particles↗
PubMed2002

Bone marrow as a source of endothelial cells and NeuN-expressing cells After stroke.

BACKGROUND AND PURPOSE: After an ischemic event, bone marrow-derived cells may be involved in reparative processes. There is increasing evidence that bone marrow-derived stem cells may be a source of endothelial cells and organ-specific cells. Our objectives were to determine whether bone marrow-derived cells were a source of endothelial cells and neurons after cerebral ischemia. METHODS: We transplanted bone marrow from male C57 BL/6-TgN (ACTbEGFP)1Osb mice, which express green fluorescent protein (GFP), into female C57 BL/6J mice. The recipient mice then underwent suture occlusion of the middle cerebral artery (MCA), and bone marrow- derived cells were tracked by GFP epifluorescence and Y chromosome probe. RESULTS: Within 3 days and at 7 and 14 days after MCA occlusion, bone marrow-derived cells incorporated into the vasculature in the ischemic zone and expressed an endothelial cell phenotype. Few bone marrow-derived cells incorporated into the vasculature 24 hours after MCA occlusion. Some bone marrow-derived cells also expressed the neuronal marker NeuN at 7 and 14 days after ischemia. CONCLUSIONS: Postnatal vasculogenesis occurs in the brain in the setting of a cerebral infarction. Bone marrow-derived cells are a source of endothelial cells and NeuN-expressing cells after cerebral infarction. This plasticity may be exploited in the future to enhance recovery after stroke.

Animals↗
PubMed1999

A null-mutated lysyl hydroxylase gene in a compound heterozygote British patient with Ehlers-Danlos syndrome type VI.

A British patient with EDS VI had two novel null mutations in the lysyl hydroxylase gene, one nucleotide deletion in the acceptor splice site of intron 4 in one allele, and an insertion of a C nucleotide in exon 2 of the other allele. The abnormal alleles lead to a markedly decreased lysyl hydroxylase mRNA levels, the finding making the affected cells different from the normal cells. In addition to the mutation analysis, we have revealed many exon-deleted splicing variants for lysyl hydroxylase mRNA which were first discovered in the affected cells, but tracks of similarly spliced mRNA species are also found in the cytoplasm of normal human skin fibroblasts. The data suggest that the splicing machinery of the cell is leaky generating differently spliced transcripts in the cell but only in a small amounts.

Alternative Splicing↗
PubMed1998

Radiation-induced genomic instability in haemopoietic cells.

PURPOSE: To review studies of radiation-induced genomic instability in haemopoietic cells. MAJOR FINDINGS: Studies have demonstrated a high frequency of non-clonal, cytogenetic abnormalities in the clonal descendants of alpha-particle-irradiated (approximately one track per cell) primary murine and human haemopoietic stem cells in vitro. The induction of this phenomenon has a strong dependence on the genetic characteristics of the cells and is transmissible in vivo following transplantation of alpha-irradiated mouse bone marrow. In clonogenic cell cultures of alpha-irradiated haemopoietic cells, there is also an increased incidence of hprt mutations and an increased incidence of apoptosis. These effects may be regarded as the consequences of a destabilization of the genome collectively termed radiation-induced genomic instability. Instability is induced at very high frequencies suggesting that epigenetic changes may be a common underlying mechanism. Consistent with this suggestion is the finding of an enhanced and persisting oxy-radical activity in the descendants of irradiated stem cells, which is consistent with oxidative stress being an important feature of the mechanism(s) underlying the persistence of instability in haemopoietic cells. Recent studies have revealed that more clonogenic cells than are actually traversed by an alpha-particle are able to express the instability phenotype. These data are consistent with unexpected interactions between irradiated and non-irradiated cells but the mechanism of initiation of instability is not understood.

Alpha Particles↗
PubMed2006

X inactivation patterns of closely, but not distantly, related cells are highly correlated: little evidence for stem cell plasticity in normal females.

The early, random nature of X inactivation should cause related cells to have similar, but distinctive, active X chromosomes. We assessed the frequency of stem cell plasticity using X inactivation proportions (XIPs), determined at the human androgen receptor locus, in paired tissue samples from healthy individuals. Tissues sampled were stomach (n = 18 informative females), duodenum (n = 18), colon (n = 10) with corresponding peripheral blood samples (n = 33), and varicose veins (n = 28) with corresponding T cells (n = 26) and peripheral blood granulocytes (n = 25). XIPs from samples thought to have common stem cell origins were highly correlated: multiple samples from single vein, r = .80 (n = 24); T cells versus granulocytes, r = .67 (n = 23); duodenum versus stomach, r = .63 (n = 12). Blood cells and vessels are derived from a common hemangioblast, but XIP correlations were moderate or poor: vein versus T cells, r = .42 (n = 26); vein versus granulocytes, r = .11 (n = 25). X inactivation is believed to be a late process in gut, especially hind-gut, with corresponding independence from blood precursors. Correlations with blood cells were low: stomach, r = .23 (18); duodenum, r = .21 (18); colon, r = .034 (10). Any crossover of stem cells between different organs during adult life should increase correlations with age; no such increase was seen. This study confirms that XIPs can be used to track stem cell populations, provides a theoretical basis for the power of such studies, and indicates that hemopoietic stem cell plasticity is, at most, uncommon in normal humans.

Adult↗
PubMed2003

Homing of peripherally injected bone marrow cells in rat after experimental myocardial injury.

BACKGROUND AND OBJECTIVES: Significant progress has been achieved during the past 10 years in cell transplantation and recent research has focused on the possibility of improving ventricular function after myocardial infarction. Most studies in the field of cardiac tissue repair are performed by direct intramyocardial injection of cells of different origin. Since this approach requires a surgical intervention, in this study we investigated the feasibility of non-invasive administration of bone marrow mononuclear cells (BMMNCs) by assessing the fate of peripherally injected, purified, labeled cells in cryodamaged hearts. DESIGN AND METHODS: Ten donor and ten recipient inbred isogenic adult (4 weeks old) Fisher rats were used as models to mimic autologous transplantation. Myocardial damage was obtained in recipient rats by placing a frozen metal probe on the anterior left ventricular wall for 15 seconds (freeze-thaw injury technique). BMMNCs were purified and labeled with a red fluorescent cell dye. Seven days after the injury about 15-25x10(6) cells were infused through the femoral vein of recipient rats. Seven days after the infusion, the heart, lungs, liver, kidneys, spleen and thymus were harvested to track transplanted cells. RESULTS: Labeled cells were found only in the injured area of the heart and not in the normal tissue, and a limited number of cells were identified in the spleen of all the animals. Most of the labeled cells in the infarcted area were Thy-1(+) and some were CD34(+). INTERPRETATION AND CONCLUSIONS: Our data suggest that peripherally injected BMMNCs can traffic through the circulation to the site of damage; we hypothesize that tissue injury leads to the priming of a cytokine cascade acting as chemoattractant for the infused cells.

Animals↗
PubMed2004

Radionuclide imaging of mesenchymal stem cells transplanted into spinal cord.

In vivo tracking of stem cells implanted to spinal cord by radionuclide imaging was investigated. The high expression of transferrin receptor on human mesenchymal stem cells (hMSCs) was verified by flow cytometry, radioligand binding and immunofluorescence. Radiolabelled transferrin was chosen as a tracer for scintigraphic imaging of the hMSCs transplanted into spinal cord of rabbits. Comparative experiments with radiolabelled human serum albumin as tracer and PBS as graft as well as ex vivo autoradiography demonstrated the specific uptake of radiolabelled transferrin of hMSCs. hMSCs could be detected in vivo with radiolabelled transferrin targeting at cellular transferrin receptors at an early stage after transplantation into spinal cord.

Animals↗