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PubMed2003

Induction of CD4+ T cell apoptosis as a consequence of impaired cytoskeletal rearrangement in UVB-irradiated dendritic cells.

Low dose UVB irradiation of dendritic cells (DC) dose-dependently decreases their allostimulatory capacity and inhibits alloreactive T cell proliferation. The reduction of the stimulatory capacity is not associated with a perturbation of CD28 costimulation. To examine the underlying mechanism, cell cycle analysis of T cells from cocultures with UVB-irradiated DC (UVB-DC) was performed, revealing no cell cycle arrest, but an increased number of apoptotic T cells in sub-G(0) phase. We confirmed T cells to undergo apoptosis after coincubation with UVB-DC by TUNEL staining and DNA laddering. To analyze whether T cell apoptosis requires the Fas/Fas ligand (FasL) pathway, MLRs were performed with Fas-, FasL-deficient, and wild-type DC and T cells. No differences were found on comparison of wild-type DC with Fas-/FasL-deficient DC or T cells. Likewise, addition of a neutralizing anti-TNF-alpha mAb to cocultures could not overcome inhibition of T cell proliferation by UVB-DC, excluding involvement of the TNF-alpha/TNF-alphaR pathway. FACS analysis of CD69 and CD25 revealed no up-regulation on T cells cocultured with UVB-DC, suggesting a perturbation of early T cell activation. Analysis of UVB-DC by confocal microscopy demonstrated impaired filamentous actin bundling, a process critical for T cell stimulation. To investigate the functional relevance of these observations, time lapse video microscopy was performed. Indeed, calcium signaling in CD4(+) T cells was significantly diminished after interaction with UVB-DC. In conclusion, UVBR of DC impairs their cytoskeletal rearrangement and induces apoptosis in CD4(+) T cells by disruption of early DC-T cell interaction, resulting in a reduced Ca(2+) influx in T cells.

Animals↗
PubMed1989

[Migration of monocytes and granulocytes in co-culture with endothelial cells].

The interaction of leucocytes and endothelial cells was analyzed in a co-culture system using time-lapse video microscopy. Isolated granulocytes or monocytes/macrophages were added to confluent endothelial cell cultures. The migratory behavior of the blood cells was subsequently analyzed using video direct visualization and morphometry. Using this technology the active migration of blood cells through the confluent endothelial layer in both directions could be shown. While granulocytes migrated several times through the endothelium in both directions, monocytes migrated from the top of the endothelial layer underneath the endothelial cells and remained there. The migratory speed of granulocytes was with 1166 microns/h about twice as fast as the speed of monocytes with 608 microns/h. The differentiation between the migratory speed of granulocytes on top or underneath the endothelial cells revealed a marked acceleration underneath the endothelial cells with a speed of 1519 microns/h compared to 671 microns/h on top of the endothelium. These results led to speculation that special extracellular matrix components produced by the endothelial cells activate the migratory behavior of blood cells.

Cell Movement↗
PubMed1990

Membrane electroporation--fast molecular exchange by electroosmosis.

Human and rabbit erythrocyte ghosts loaded with FITC-dextran (mol. mass = 10 kDa) and NBD-glucosamine (mol. mass = 342 Da) in buffers of different ionic strength and composition were subjected to electric pulses (intensity 0.7 kV/mm and decay half-time 1 ms) at 7-10 degrees C and 20-24 degrees C. The transfer of the fluorescent dyes from the interior of the ghosts through the electropores was observed by low light level video microscopy. The pulses caused the fluorescence to appear outside the membranes as a transient cylindrical cloud directed toward the negative electrode during the first video frame (17 ms). It was similar in both rabbit and human erythrocyte ghosts and at both temperatures but differs for the two dyes, the fluorescence cylinder is long and tall for the FITC-dextran and relatively short and thick for the NBD-glucosamine. The molecular exchange was 2-3 orders of magnitude faster within the first 17 ms after the pulse than the diffusional exchange. It decreased with increasing ionic strength. Formulae for the transfer of molecules by electroosmotic flow through the pores are in agreement with these observations. They allow estimation of the total area of pores with radii larger than that of the fluorescent dye during the pulse. The major conclusion is that electroosmosis is the dominating mechanism of molecular exchange in electroporation of erythrocyte ghosts.

4-Chloro-7-nitrobenzofurazan↗
PubMed1990

Inhibition of mural thrombus formation by novel nipecotoylpiperazine antiplatelet agents.

The effectiveness of two closely related nipecotoylpiperazine derivatives, BPAT-143 and BPAT-117, as antiplatelet agents was measured by their ability to inhibit the accumulation of human blood platelets on collagen-coated (type 1) glass in a parallel plate flow chamber. Whole human blood, with fluorescently labeled platelets, was perfused through the flow chamber, and epi-fluorescent video microscopy was used to visualize the dynamics of individual platelet adhesion and thrombus formation on the collagen-coated surface. Digital image processing was used to analyze the dynamics of thrombus growth on the surfaces. The collagen-coated surface serves as a model for the damaged blood vessel wall, as collagen is a primary component of the matrix beneath endothelial cells. At a concentration of 50 microM, BPAT-117 (the considerably more hydrophobic molecule) inhibited platelet accumulation by striking 90 +/- 2% (+/- S.E.), while it took 2- to 4-fold higher concentrations of BPAT-143 to register meaningful to comparable effects (52 +/- 6% and 80 +/- 4%, respectively). This further corroborates the substantial impact of hydrophobic features within the matrix of appropriately structured molecules on their ability to alter platelet function.

Blood Platelets↗
PubMed2004

Neurocan-GFP fusion protein: a new approach to detect hyaluronan on tissue sections and living cells.

Hyaluronan is an unsulfated glycosaminoglycan (GAG) that is ubiquitously expressed in the extracellular matrix (ECM) of all vertebrates, where hyaluronan rich matrices constitute a particular permissive environment for the development of complex biological structures and also for tumor progression. Because of its conserved structure and ubiquitous expression, antibodies for its histochemical detection cannot be produced. We have engineered a fusion protein, neurocan-GFP, and expressed it as a secreted molecule in mammalian cells. Neurocan-GFP fusion protein specifically binds to hyaluronan and directly visualizes hyaluronan on tissue sections, revealing a very detailed picture of hyaluronan distribution. The fluorescent fusion protein can be used in combination with antibodies and nuclear markers for double or triple staining. In addition, it is suitable to visualize hyaluronan on living cells by time-lapse video microscopy. The successful production and application of the neurocan-GFP fusion protein opens up new perspectives for using GFP fusion proteins as detection tools in histological and cytological studies complementing conventional antibody and biotin/avidin techniques.

Animals↗
PubMed1994

The function of c-fms in hairy-cell leukemia: macrophage colony-stimulating factor stimulates hairy-cell movement.

Hairy cells (HCs) and some activated B cells express high levels of macrophage colony-stimulating factor (M-CSF) (CSF-1) receptor, but the functional effects of the cytokine on B cells have not been previously identified. Using video microscopy, image analysis, and migration assays, M-CSF was shown to induce chemokinetic and chemotactic movement of HCs. This movement response involved transition to a highly mobile, rounded cell form and was accompanied by distinctive changes in F-actin polymerization and distribution. Furthermore, the M-CSF-induced motility was substantially modified by the adhesive protein used as a substratum and involved qualitative changes in the function of the alpha v beta 3 integrin of HCs. It is suggested that the findings are relevant to the pathophysiology of hairy-cell leukemia (HCL) in particular, and to the biology of B-cell migration in general.

Actins↗
PubMedDate not supplied

Neuron imaging with Neurolucida--a PC-based system for image combining microscopy.

Neurolucida is a new software package for performing 3-D neuron mapping and tracing to 0.5 micron precision through the oculars of a microscope. It incorporates computer controlled stage movement in three dimensions. Sections are limited in size only by the stage dimensions. An on-line, direct view image editor is provided as are dynamic image rotations in color and morphometric analyses. Neurolucida is written in 'C' and runs on PC ATs, XTs, and PS/2s using DOS and VGA, EGA, or NTSC graphics. Neurolucida has a video mode compatible with frame grabbers, thereby permitting its application to video microscopy.

Computer Graphics↗
PubMed1988

Video microscopic image processing facilitates the evaluation of light microscopic autoradiography at high magnification.

Light microscopic autoradiographs of 3H-thymidine labelled unstained semithin sections of Xenopus laevis embryonic nuclei were examined with conventional Nomarski differential interference contrast, phase-contrast and video microscopy. Whereas at low magnification it was possible to obtain a photograph of the nuclear structure and the silver grains in one focal plain, at high magnification, with small depths of focus, a satisfactory image was not attainable. Therefore, we stored the images of the two different focus levels with a digital image processing system and combined both images by an arithmetic operation. This video microscopic technique allows the use of high magnification light microscopy with oil immersion objectives and the application of additional electronic contrast enhancing methods for an adequate and rapid analysis of light microscopic autoradiographs.

Animals↗
PubMed1993

Growth cone dynamics during the migration of an identified commissural growth cone.

We have used time-lapse video microscopy to study the behavior of a neuron, Q1, that pioneers the posterior commissure of the embryonic grasshopper. Our goal is to use time-lapse video as a tool to acquire a precise picture of normal development over time, and thereby identify stereotypic activities that might indicate important interactions necessary for proper formation of the commissure. We have identified specific and reproducible behaviors that suggest the presence of underlying cellular interactions that may play a role in pathfinding. In particular, the Q1 growth cone undergoes several morphological changes as it contacts the midline. As a commissural neuron, the midline may be a target in its outgrowth; Q1's typical response upon contacting the midline with its filopodia, however, is a rapid retraction. This inhibitory reaction can be overridden by contact with filopodia of its contralateral homolog. Q1's growth cone can translocate across the midline at an accelerated rate by a process resembling "filopodial dilation" (O'Connor et al., 1990) once the two Q1 growth cones meet. Ablation of the contralateral Q1 blocks Q1's advance across the midline. We have also analyzed in detail the behavior of individual filopodia to identify behavioral differences that could indicate differences in substrate adhesivity. Except for instances of filopodial dilation seen only at the midline, we found no significant asymmetries in rates of filopodial extension and retraction, or in the survival times of individual filopodia. We suggest that either the adhesive signal used by Q1 is relatively weak, requiring the integration of many adhesive interactions by many filopodia to be resolved, or the guidance cues may not be adhesive in nature.

Animals↗
PubMed1989

Mathematical analysis of mural thrombogenesis. Concentration profiles of platelet-activating agents and effects of viscous shear flow.

The concentration profiles of adenosine diphosphate (ADP), thromboxane A2 (TxA2), thrombin, and von Willebrand factor (vWF) released extracellularly from the platelet granules or produced metabolically on the platelet membrane during thrombus growth, were estimated using finite element simulation of blood flow over model thrombi of various shapes and dimensions. The wall fluxes of these platelet-activating agents were estimated for each model thrombus at three different wall shear rates (100 s-1, 800 s-1, and 1,500 s-1), employing experimental data on thrombus growth rates and sizes. For that purpose, whole human blood was perfused in a parallel-plate flow chamber coated with type l fibrillar human collagen, and the kinetic data collected and analyzed by an EPl-fluorescence video microscopy system and a digital image processor. It was found that thrombin concentrations were large enough to cause irreversible platelet aggregation. Although heparin significantly accelerated thrombin inhibition by antithrombin lll, the remaining thrombin levels were still significantly above the minimum threshold required for irreversible platelet aggregation. While ADP concentrations were large enough to cause irreversible platelet aggregation at low shear rates and for small aggregate sizes, TxA2 concentrations were only sufficient to induce platelet shape change over the entire range of wall shear rates and thrombi dimensions studied. Our results also indicated that the local concentration of vWF multimers released from the platelet alpha-granules could be sufficient to modulate platelet aggregation at low and intermediate wall shear rates (less than 1,000 s-1). The sizes of standing vortices formed adjacent to a growing aggregate and the embolizing stresses and the torque, acting at the aggregate surface, were also estimated in this simulation. It was found that standing vortices developed on both sides of the thrombus even at low wall shear rates. Their sizes increased with thrombus size and wall shear rate, and were largely dependent upon thrombus geometry. The experimental observation that platelet aggregation occurred predominantly in the spaces between adjacent thrombi, confirmed the numerical prediction that those standing vortices are regions of reduced fluid velocities and high concentrations of platelet-activating substances, capable of trapping and stimulating platelets for aggregation. The average shear stress and normal stress, as well as the torque, acting to detach the thrombus, increased with increasing wall shear rate. Both stresses were found to be nearly independent of thrombus size and only weekly dependent upon thrombus geometry. Although both stresses had similar values at low wall shear rates, the average shear stress became the predominant embolizing stress at high wall shear rates.

Adenosine Diphosphate↗
PubMed1996

A role for phosphoinositide 3-kinase in the completion of macropinocytosis and phagocytosis by macrophages.

Phosphoinositide 3-kinase (PI 3-kinase) has been implicated in growth factor signal transduction and vesicular membrane traffic. It is thought to mediate the earliest steps leading from ligation of cell surface receptors to increased cell surface ruffling. We show here that inhibitors of PI 3-kinase inhibit endocytosis in macrophages, not by interfering with the initiation of the process but rather by preventing its completion. Consistent with earlier studies, the inhibitors wortmannin and LY294002 inhibited fluid-phase pinocytosis and Fc receptor-mediated phagocytosis, but they had little effect on the receptor-mediated endocytosis of diI-labeled, acetylated, low density lipoprotein. Large solute probes of endocytosis reported greater inhibition by wortmannin than smaller probes did, indicating that macropinocytosis was affected more than micropinocytosis. Since macropinocytosis and phagocytosis are actin-mediated processes, we expected that their inhibition by wortmannin resulted from deficient signaling from macrophage colony-stimulating factor (M-CSF) receptors or Fc receptors to the actin cytoskeleton. However, video microscopy showed cell surface ruffling in wortmannin-treated cells, and increased ruffling after addition of M-CSF or phorbol myristate acetate. Quantitative measurements of video data reported slightly diminished ruffling in wortmannin-treated cells. Remarkably, the ruffles that formed in wortmannin-treated macrophages all receded into the cytoplasm without closing into macropinosomes. Similarly, wortmannin and LY294002 did not inhibit the extension of actin-rich pseudopodia along IgG-opsonized sheep erythrocytes, but instead prevented them from closing into phagosomes. These findings indicate that PI 3-kinase is not necessary for receptor-mediated stimulation of pseudopod extension, but rather functions in the closure of macropinosomes and phagosomes into intracellular organelles.

Androstadienes↗
PubMed1994

Effect of controlled local acetylsalicylic acid release on in vitro platelet adhesion to vascular grafts.

Thrombosis is the most serious acute problem for small diameter arterial bypass grafts. In this research, small diameter expanded polytetrafluoroethylene (e-PTFE) vascular grafts were coated with acetylsalicylic acid (ASA) loaded poly (d,l-lactide) (PLA) by a solvent casting method. The feasibility and efficacy of this approach were evaluated by ASA release studies and platelet adhesion tests. First, the ASA release kinetics were evaluated from the ASA/PLA coated vascular grafts in an in vitro steady flow loop model. ASA release was measured by a spectrophotometric technique. Finally, the efficacy of local ASA release to reduce in vitro canine platelet adhesion to grafts was determined with epifluorescent video microscopy and quantitative image analysis. The steady state release rates from the 5%, 10%, and 15% ASA/PLA coated grafts were 13.2 x 10(-5), 32.0 x 10(-5), and 41.5 x 10(-5) micrograms/cm2.sec, respectively. Platelet adhesion to 10% and 15% ASA/PLA coated grafts was reduced with respect to the control and 5% grafts for 10 days. Platelet adhesion to 5% ASA/PLA coated grafts was reduced with respect to controls at 2 and 10 days, but not initially.

Animals↗
PubMed1998

Radiation kills human peripheral T cells by a Fas-independent mechanism.

The mechanism by which radiation induces human peripheral T cell apoptosis is not known. We examined sequential changes in post-irradiated peripheral blood mononuclear cells (PBMC(S)) taken from normal volunteers, by using flow-cytometer and an anti-CD3 monoclonal antibody, annexin V, propidium iodide, anti-Fas antibody, and anti-Fas ligand antibody. After 5 or 10 Gy of irradiation with a 60Co radiation therapy unit, most of the human peripheral T cells showed positivity against annexin V in 15 h, and positivity against propidium iodide in 23 h after irradiation. On a microscopy-video system, approximately 80% of mononuclear cells revealed apoptotic changes in 24 h after irradiation. Because of its proposed role in activation-induced cytotoxicity, we also examined the Fas (CD95/Apo-1) pathway in killing T cells by irradiation. Irradiated PBMC, displayed no increase in surface Fas expression and caspase-3 activity relative to non-irradiated cells. In addition, the anti-Fas ligand failed to eliminate the apoptotic death of PBMC, after irradiation. These results suggest that irradiation induces direct apoptosis of T cells by a Fas-independent mechanism.

Antibodies, Monoclonal↗
PubMed2002

VANQUIS, a system for the interactive semantic content analysis and spatio-temporal query by content of videos.

Using the video metadata descriptors and data model defined in the accompanying paper (Shotton, D. M. et al. (2002) A metadata classification schema for semantic content analysis of videos. J. Microsc. 205, 33-42), we discuss how analysis of the content of scientific videos, and subsequent query by content of the resulting semantic metadata, can be enhanced by the use of an object-relational database. We illustrate this by describing VANQUIS, a Web-based prototype video analysis and query interface system for the interactive spatio-temporal analysis and subsequent query by content of videos. Using VANQUIS to generate standard SQL (structured query language) statements that address complex data types stored in an object-relational database, relationships between characters and events contained within and between videos can be identified, and the appropriate video segments containing these characters and events can be retrieved for viewing. We give examples of analysis and query implementation by using VANQUIS to analyse a biological microscopy video, and discuss the wider potential of this methodology for the analysis and query by content of videos containing more general subject matter.

Comment↗
PubMed1997

Involvement of phosphorylation in doxorubicin-mediated myofibril degeneration. An immunofluorescence microscopy analysis.

Loss of myofilaments has been observed in both adaptive cardiac responses (i.e., hypertrophy) as well as in chemotheraputic use of antineoplastic drugs with cardiotoxic side effects (i.e., doxorubicin). An understanding of the degenerative process is a prerequisite for determining approaches to limit the cardiomyopathic changes associated with chronic heart disease or long-term chemotheraputic treatments. However, little is known about the specific events and molecular changes that initiate the degenerative process. To study this process, neonatal rat cardiomyocytes were treated with doxorubicin, which induced rapid and widespread thin-filament degeneration as observed by fluorescence confocal microscopy. Which demonstrated deterioration of sarcomeric thin-filament structure. Changes in the spontaneous beating of cardiomyocytes corresponding with myofibrillar degeneration were apparent using differential interference contrast video microscopy. After finding induction of kinase activity by doxorubicin in cultured cardiomyocytes, the protective effects of specific inhibitors of kinase activity were assessed for their ability to inhibit doxorubicin-induced myofibrillar break-down. Doxorubicin-induced changes appeared similar to the degeneration observed after treatment with a protein kinase activator (phorbol 12-myristate 13-acetate) or a serine-threonine protein phosphatase inhibitor (okadaic acid). Collectively, these results indicate that activation of protein kinase is an important event in the initiation of myofibrillar degeneration by doxorubicin. Further analyses of myofibrillar proteins with respect to biochemical modifications will be necessary to determine if phosphorylation events transmit signal(s) to initiate degeneration.

Animals↗
PubMed1991

Differential adhesion of Pseudomonas aeruginosa to human respiratory epithelial cells in primary culture.

Human nasal polyps in outgrowth culture were used to study the Pseudomonas aeruginosa adhesion to respiratory cells. By scanning electron microscopy, P. aeruginosa were seen associated with ciliated cells, but by transmission electron microscopy, bacteria were never seen at the interciliary spaces or attached along cilia, but were identified trapped at the extremities of cilia, usually as bacterial aggregates. A fibronectin-containing fibrillar material was seen associated with aggregated bacteria. By time-lapse video microscopy, bacteria were seen to aggregate in the culture medium following their addition to the culture wells. Progressively, these aggregates were trapped by cilia or attached to migrating cells of a lower cell layer that protruded beneath the upper layer cells, at the outgrowth periphery. P. aeruginosa adhesion to these lower cell layer migrating cells was significantly higher than to ciliated or nonciliated cells of the upper cell layer. Migrating cells were intensely labeled by the complexes Con A and arachis hypogea agglutinin (PNA)-FITC, in contrast to the other cells. The percentage of PNA-labeled cells with attached bacteria was significantly higher than that without bacteria. These results suggest that changes of cell surface glycoconjugates related with cell migration may favor P. aeruginosa adhesion to respiratory cells.

Bacterial Adhesion↗
PubMed1997

XMAP310: a Xenopus rescue-promoting factor localized to the mitotic spindle.

To understand the role of microtubule-associated proteins (MAPs) in the regulation of microtubule (MT) dynamics we have characterized MAPs prepared from Xenopus laevis eggs (Andersen, S.S.L., B. Buendia, J.E. Domínguez, A. Sawyer, and E. Karsenti. 1994. J. Cell Biol. 127:1289-1299). Here we report on the purification and characterization of a 310-kD MAP (XMAP310) that localizes to the nucleus in interphase and to mitotic spindle MTs in mitosis. XMAP310 is present in eggs, oocytes, a Xenopus tissue culture cell line, testis, and brain. We have purified XMAP310 to homogeneity from egg extracts. The purified protein cross-links pure MTs. Analysis of the effect of this protein on MT dynamics by time-lapse video microscopy has shown that it increases the rescue frequency 5-10-fold and decreases the shrinkage rate twofold. It has no effect on the growth rate or the catastrophe frequency. Microsequencing data suggest that XMAP230 and XMAP310 are novel MAPs. Although the three Xenopus MAPs characterized so far, XMAP215 (Vasquez, R.J., D.L. Gard, and L. Cassimeris. 1994. J. Cell Biol. 127:985-993), XMAP230, and XMAP310 are localized to the mitotic spindle, they have distinct effects on MT dynamics. While XMAP215 promotes rapid MT growth, XMAP230 decreases the catastrophe frequency and XMAP310 increases the rescue frequency. This may have important implications for the regulation of MT dynamics during spindle morphogenesis and chromosome segregation.

Animals↗
PubMed1994

Heterogeneity of red blood cell perfusion in capillary networks supplied by a single arteriole in resting skeletal muscle.

Flow heterogeneity within capillary beds may have two sources: (1) unequal distribution of red blood cell (RBC) supply among arterioles and (2) unique properties of RBC flow in branching networks of capillaries. Our aim was to investigate the capillary network as a source of both spatial and temporal heterogeneity of RBC flow. Five networks, each supplied by a single arteriole, were studied in frog sartorius muscle (one network per frog) by intravital video microscopy. Simultaneous data on RBC velocity (millimeters per second), lineal density (RBCs per millimeter), and supply rate (RBCs per second) were measured continuously (10 samples per second) from video recordings in 5 to 10 capillary segments per network for 10 minutes by use of automated computer analysis. To quantify heterogeneity, mean values from successive 10-second intervals were tabulated for each flow parameter in each capillary segment (ie, portion of capillary between successive bifurcations), and percent coefficient of variation (SD/mean.100%) was calculated for (1) spatial heterogeneity among vessels (CVs) every 10 seconds and for the entire 10-minute sample and (2) temporal heterogeneity within vessels for every capillary segment and for the mean flow parameter. Analysis of these data indicates that (1) capillary networks are a significant source of both spatial and temporal flow heterogeneity, and (2) continuous redistributions of flow occur within networks, resulting in substantial temporal changes in CVs, although a persistent spatial heterogeneity of perfusion still exists on a 10-minute basis. In most networks, CVs decreased as supply rate within the network increased, thus indicating that rheology plays a significant role in determining the perfusion heterogeneity.

Animals↗