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Neutrophil degranulation detected by right angle light scattering: spectroscopic methods suitable for simultaneous analyses of degranulation or shape change, elastase release, and cell aggregation.

We have used simultaneous spectrometric analysis of right angle scattering and elastase release from human neutrophils to demonstrate the similarity of these two measures of degranulation. Both responses depend on the presence of cytochalasin B, and are similar in kinetics, dose-response, and dependence on receptor occupancy at the formyl peptide receptor. This scattering response is shown to be largely independent of cell aggregation. In the absence of cytochalasin B, a rapid and transient right angle scatter response of a different character, probably associated with cell ruffling, is detected. Either right angle response can be detected by flow cytometry.

Cell Aggregation↗

Correlation between relaxation and automaticity in embryonic heart cell aggregates.

Diastolic depolarization in cardiac muscle is due to a decline in potassium permeability that has been ascribed to removal of intracellular free calcium. A continued decline in tension during the pacemaker potential might therefore occur. In this study, contractile responses of chicken embryonic heart cell aggregates are recorded with a photodiode. Photodiode output is well correlated with the position of the aggregate's edge. Movements of different edges are synchronous, and their amplitude and duration vary appropriately during experimental maneuvers that alter the magnitude and duration of contractile force. Edge movement during relaxation has two phases, a rapid phase lasting about 100 msec and a slow phase that may last over 10 sec. The slow phase is not due to viscoelasticity because its time course does not depend on the magnitude or duration of the initial deformation. The rate of relaxation is correlated with the rate of depolarization during the pacemaker potential. Reduction in automaticity during cooling, spontaneous variation, and overdrive pacing are associated with impairment of the slow component of relaxation. Electrophysiological evidence suggests that the diastolic potassium permeability of the aggregates is controlled by intracellular calcium. A possible explanation for the correlation between the slope of the pacemaker potential and the slow component of relaxation is that both phenomena reflect a common physiological process-i.e., the removal of free calcium from the cytoplasm.

Action Potentials↗

Human CD100, a novel leukocyte semaphorin that promotes B-cell aggregation and differentiation.

Herein we describe the molecular characterization of the human leukocyte activation antigen CD100 and identify it as the first semaphorin, to our knowledge, in the immune system. Semaphorins have recently been described as neuronal chemorepellants that direct pioneering neurons during nervous system development. In this study we demonstrate that CD100 induces B cells to aggregate and improves their viability in vitro. We show that CD100 modifies CD40-CD40L B-cell signaling by augmenting B-cell aggregation and survival and down-regulating CD23 expression. Thus, these results suggest that semaphorins as exemplified by CD100 also play a functional role in the immune system.

Amino Acid Sequence↗

Effects of 2-methoxyestradiol on proliferation, apoptosis and PET-tracer uptake in human prostate cancer cell aggregates.

The purpose of this study was to investigate the potential use of PET in vivo to record cytotoxic effects of 2-methoxyestradiol (2-ME), an endogenous metabolite of 17beta-estradiol. The anti-proliferative and pro-apoptotic effects of 2-ME on human prostate cancer cell (PC3) aggregates in vitro, were correlated with the uptake of fluoro-deoxy-D-glucose, FMAU and choline labelled with 18F, 11C, or 3H. 2-ME clearly reduced growth of PC3 aggregates and induced apoptosis in a dose-dependent manner. However, the uptake of the putative proliferation markers 11C-FMAU or 3H-choline failed to record the growth inhibitory effects of 2-ME on PC3 cell aggregates. The uptake of 18F-FDG was used as a marker for effects on cellular metabolism and also failed to show any dose-dependent effects in PC3 aggregates. The use of these PET-tracers in vivo is therefore not recommended in order to evaluate the cytotoxic effects of 2-ME on human prostate cancer cells.

2-Methoxyestradiol↗

Studies of baby hamster kidney natural cell aggregation in suspended batch cultures.

Microcarrier cultures of animal cells of industrial relevance are known to shed aggregates into the suspension phase. For a BHK cell line, which is known to be prone to aggregate naturally, microcarrier and aggregate forms of culture are compared in spinner culture. In microcarrier cultures, it is shown that increasing initial microcarrier concentration yields decreasing concentration of smaller aggregates in suspension; roughly equivalent concentrations of total cells and single cells in suspension are obtained. In the absence of Cytodex 3, aggregate final size is hydrodynamically controlled in batch and semicontinuous suspension culture. Rate of agitation is the main variable controlling aggregate size in batch cultures. The range of agitation rates studied (20 to 70 rpm in 250 mL spinner flasks) produced aggregates with maximum sizes of 200 microns. Necrotic centers were not observed; this was confirmed by Trypan blue viability measurements after mechanical dissociation of aggregates and also by the constant productivity obtained from different aggregate sizes. Comparing aggregate and microcarrier culture conditions, it is shown that at 100 rpm maximum total cell concentration is larger in the absence of microcarriers; dead cell concentrations, most of which exist in suspension, are slightly larger in microcarrier culture. Total viable cell concentrations in aggregate, hydrodynamically controlled culture, are almost one order of magnitude higher than in microcarrier cultures. These results suggest that there might be advantages in using aggregate cultures under hydrodynamic control of aggregate size in lieu of microcarrier cultures for naturally aggregating cell lines.

Animals↗

Depletion-mediated red blood cell aggregation in polymer solutions.

Polymer-induced red blood cell (RBC) aggregation is of current basic science and clinical interest, and a depletion-mediated model for this phenomenon has been suggested; to date, however, analytical approaches to this model are lacking. An approach is thus described for calculating the interaction energy between RBC in polymer solutions. The model combines electrostatic repulsion due to RBC surface charge with osmotic attractive forces due to polymer depletion near the RBC surface. The effects of polymer concentration and polymer physicochemical properties on depletion layer thickness and on polymer penetration into the RBC glycocalyx are considered for 40 to 500 kDa dextran and for 18 to 35 kDa poly (ethylene glycol). The calculated results are in excellent agreement with literature data for cell-cell affinities and with RBC aggregation-polymer concentration relations. These findings thus lend strong support to depletion interactions as the basis for polymer-induced RBC aggregation and suggest the usefulness of this approach for exploring interactions between macromolecules and the RBC glycocalyx.

Animals↗

Interactions of B16F10 melanoma cells aggregated on a cellulose substrate.

There is evidence that the shape of cells and their contact with a matrix direct the growth and the differentiation of both normal and cancer cells. Cells in 3D culture resemble the in vivo situation more closely than do those in conventional 2D cultures. We have studied the interactions and functions of B16F10 mouse melanoma cells, which spread and grow well on tissue culture polystyrene (tPS), when they were made to aggregate on cellulose-coated Petri dishes (CEL). This aggregation of melanoma cells on CEL was Ca2+ dependent and mediated by N-cadherins. The levels of N-cadherin and beta-catenin transcripts in cells cultured on CEL and tPS were similar, but those on CEL contained less beta-catenin protein. Immunoprecipitation and immunostaining showed that both N-cadherins and beta-catenins were present at the membranes of cells on CEL. Cells proliferated significantly more slowly after 48 h on CEL and the cellulose coating caused most of them to arrest in G1. We also compared the melanin contents and tyrosinase activity of cells on CEL and controls grown on tPS. Melanogenesis was induced in cells aggregated on CEL. A cellulose substrate thus appears to be an outstanding tool for studying cell-cell interactions and cell functions in 3D cultures.

Animals↗

Influence of cell-specific factors on red blood cell aggregation.

The reversible aggregation of red blood cells (RBC) into linear and three-dimensional structures continues to be of basic science and clinical interest: RBC aggregation affects low shear blood viscosity and microvascular flow dynamics, and can be markedly enhanced in several clinical states. Until fairly recently, most research efforts were focused on relations between suspending medium composition (i.e., protein levels, polymer type and concentration) and aggregate formation. However, there is now an increasing amount of experimental evidence indicating that RBC cellular properties can markedly affect aggregation, with the term "RBC aggregability" coined to describe the cell's intrinsic tendency to aggregate. Variations of aggregability can be large, with some changes of aggregation substantially greater than those resulting from pathologic states. The present review provides a brief overview of this topic, and includes such areas as donor-to-donor variations, polymer-plasma correlations, effects of RBC age, effects of enzymatic treatment, and current developments related to the mechanisms involved in RBC aggregation.

Adult↗

Alteration of red cell aggregability and shape during blood storage.

BACKGROUND: Storage of blood units (for 35-42 days, depending on the preservative solution) has been reported to induce changes (e.g., reduction of sialic acid level) in red cells that are expected to alter their aggregability. STUDY DESIGN AND METHODS: The aggregability of stored red cells was monitored in their autologous plasma and compared to that obtained with washed cells in dextran-containing buffer throughout the storage period. Red cell aggregability was determined by using a computerized image analyzer of cell flow properties. RESULTS: Blood storage induced changes in red cells that are associated with continuous increase of their aggregability. At the same time, blood storage was associated with a reduction in the level of plasma fibrinogen, the major aggregating agent in plasma. Accordingly, the increased red cell aggregability was observed in red cells stored in dextran-containing buffer, but not in red cells stored in autologous plasma. CONCLUSION: Because blood transfusion is routinely given to patients with normal or high fibrinogen level, the transfusion of stored red cells has the potential to induce increased aggregation in vivo, depending on the storage period. This should be taken into account when blood transfusion is considered, particularly for patients with microcirculatory disorders.

Blood Preservation↗

The mechanical activity of chick embryonic myocardial cell aggregates.

1. Simultaneous recordings of membrane potential and edge movement were obtained in spontaneously beating chick embryonic myocardial cell aggregates, which are known to behave as an isopotential syncytium.2. The time course of edge movement was similar in different aggregates, and in different regions of the same aggregate.3. Peak amplitude was increased by 10(-6)m-ouabain, and by rapid reduction of the external sodium concentration.4. Peak amplitude was decreased during single premature action potentials, but sustained rapid pacing produced an ascending staircase.5. Depolarizing current pulses increased both the amplitude and duration of the contraction, and caused potentiation of the next spontaneous beat. Edge displacement during a series of pulses was a monotonic function of membrane potential.6. Edge movement between action potentials (diastolic movement) was well fitted by an exponential with a mean time constant of 69 msec. Diastolic edge movement was due to a weak, slowly decaying contractile force, which was demonstrated in cells grown on a linear-elastic nylon bristle.7. The time course of diastolic edge movement remained constant, or nearly constant, during variations in peak amplitude that resulted from prematurity of the action potential, exposure to 10(-6)m-ouabain, spontaneous mechanical alternans, or prolongation of the action potential by current pulses.8. In contrast, reduction of the external sodium concentration produced marked, selective slowing of the diastolic edge movement. Similar slowing occurred during cooling and during staircase. Diastolic edge movement was selectively accelerated when the preceding interbeat interval was prolonged by a hyperpolarizing current pulse.9. The above observations are consistent with the hypothesis that edge displacement is a monotonic function of contractile force.10. The slow relaxation between action potentials probably reflects removal of intracellular calcium across the surface membrane in exchange for sodium. Changes in the rate of calcium removal may play a role in the regulation of contractility in this tissue.

Action Potentials↗

Some effects of positively charged surface groups on cell aggregation.

A study was made of the effects of 2,3-dimethylmaleic anhydride (DMA), a reagent removing positive charges, on the aggregation and surface charge of embryonic chick neural retina cells. Neural retina cells, recovered from the dissociation procedure, were cultured on a gyratory shaker and the aggregate dimaeters formed in the presence of DMA or DMA-serum dialysate, following DMA-pretreatment, or in appropriate control cultures measured. The electrophoretic mobilities of similarly treated cells were also determined. In addition, cellulose acetate electrophoresis was carried out on samples of serum containing DMA, and the incorporation of 14C-amino acids into DMA-treated cells studied. Aggregates formed in the presence of DMA, or following DMA-pretreatment, were significantly smaller than aggregates from control cultures. The electrophoretic mobility of DMA-treated cells was significantly increased in serum-containing medium, but not serum-free Hanks' solution. At 24 h after removal of DMA-containing medium, the mobilities of pretreated cells were similar to those of controls. The electrophoretic pattern of DMA-treated serum was changed only with concentrations of DMA many times that affecting cell aggregation or mobility. DMA-serum dialysate did not significanlty reduce aggregate size. The incorporation of 14C-amino acids in DMA-treated cells and the structure of aggregates were unchanged from controls. It is concluded that positively charged consituents of the cell periphery play a demonstrable, but not limiting, role in cell aggregation, while a minor role for positive charges on serum protein cannot be totally excluded.

Amino Acids↗

Rheological effects of red blood cell aggregation in the venous network: a review of recent studies.

It has long been recognized that understanding the rheological properties of blood is essential to a full understanding of the function of the circulatory system. Given the difficulty of obtaining carefully controlled measurements in vivo, most of our current concepts of the flow behavior of blood in vivo are based on its properties in vitro. Studies of blood rheology in rotational and tube viscometers have defined the basic properties of blood and pointed to certain features that may be especially significant for understanding in vivo function. At the same time, differences between in vivo and in vitro systems combined with the complex rheological properties of blood make it difficult to predict in vivo blood rheology from in vitro studies. We have investigated certain flow properties of blood in vivo, using the venular network of skeletal muscle as our model system. In the presence of red blood cell aggregation, venous velocity profiles become blunted from the parabolic as in Poiseuille flow, as pseudo-shear rate (= mean fluid velocity/vessel diameter) is decreased from approximately 100 s(-1) to 5 s(-1). At control flow rates, the short distance between venular junctions does not appear to permit significant axial migration and red cell depletion of the peripheral fluid layer before additional red cells and aggregates are infused from a feeding tributary. Formation of a cell-free plasma layer at the vessel wall and sedimentation in vivo are evident only at very low pseudo-shear rates (<5 s(-1)). These findings may explain in large part observations in whole organs of increased venous resistance with reduction of blood flow.

Blood Flow Velocity↗

PC12 cell aggregation and dopamine production on EHS-derived extracellular matrix.

PC12 cells on plastic grow as a single-layered lawn of cells which synthesize, store and secrete dopamine. In contrast, PC12 cells cultured on Englebreth-Holm-Swarm (EHS) tumor-derived extracellular matrix grow into multicellular aggregates. Matrix dissolution and cell migration appear to follow aggregate formation. PC12 cell plating efficiency is decreased on EHS-matrix but the doubling time of cells on EHS-matrix is comparable to plastic. Dopamine secretion and cellular content determined with a radioenzymatic assay as well as dopamine synthesis determined with cation-exchange chromatography are similar on a per cell basis in cultures of PC12 cells on plastic and EHS-matrix.

Animals↗