Prognostic value of circulating tumour cells in limited-stage small-cell lung cancer: analysis of the concurrent once-daily versus twice-daily radiotherapy (CONVERT) randomised controlled trial.

BACKGROUND: The clinical significance of circulating tumour cells (CTCs) in limited-stage small-cell lung cancer (LS-SCLC) is not well defined. We report a planned exploratory analysis of the prevalence and prognostic value of CTCs in LS-SCLC patients enrolled within the phase III randomised CONVERT (concurrent once-daily versus twice-daily chemoradiotherapy) trial. PATIENTS AND METHODS: Baseline blood samples were enumerated for CTCs using CellSearch in 75 patients with LS-SCLC who were enrolled in the CONVERT trial and randomised between twice- and once-daily concurrent chemoradiation. Standard statistical methods were used for correlations of CTCs with clinical factors. Log-rank test and Cox regression analyses were applied to establish the associations of 2, 15 and 50 CTC thresholds with progression-free survival (PFS) and overall survival (OS). An optimal CTC count threshold for LS-SCLC was established. RESULTS: CTCs were detected in 60% (45/75) of patients (range 0-3750). CTC count thresholds of 2, 15 and 50 CTCs all significantly correlate with PFS and OS. An optimal CTC count threshold in LS-SCLC was established at 15 CTCs, defining 'favourable' and 'unfavourable' prognostic risk groups. The median OS in <15 versus ≥15 CTCs was 26.7 versus 5.9 m (P = 0.001). The presence of ≥15 CTCs at baseline independently predicted ≤1 year survival in 70% and ≤2 years survival in 100% of patients. CONCLUSION: We report the prognostic value of baseline CTC count in an exclusive LS-SCLC population at thresholds of 2, 15 and 50 CTCs. Specific to LS-SCLC, ≥15 CTCs was associated with worse PFS and OS independent of all other factors and predicted ≤2 years survival. These results may improve disease stratification in future clinical trial designs and aid clinical decision making. TRIAL REGISTRATION: ClinicalTrials.gov identifier: NCT00433563.

Hazard perception skills of young drivers with Attention Deficit Hyperactivity Disorder (ADHD) can be improved with computer based driver training: An exploratory randomised controlled trial.

BACKGROUND: Young drivers with Attention Deficit Hyperactivity Disorder (ADHD) are at higher risk of road traffic injuries than their peers. Increased risk correlates with poor hazard perception skill. Few studies have investigated hazard perception training using computer technology with this group of drivers. OBJECTIVES: *Determine the presence and magnitude of the between-group and within- subject change in hazard perception skills in young drivers with ADHD who receive Drive Smart training. *Determine whether training-facilitated change in hazard perception is maintained over time. METHODS: This was a feasibility study, randomised control trial conducted in Australia. The design included a delayed treatment for the control group. Twenty-five drivers with a diagnosis of ADHD were randomised to the Immediate Intervention or Delayed Intervention group.The Immediate Intervention group received a training session using a computer application entitled Drive Smart. The Delayed Intervention group watched a documentary video initially (control condition), followed by the Drive Smart computer training session. The participant's hazard perception skill was measured using the Hazard Perception Test (HPT). FINDINGS: After adjusting for baseline scores, there was a significant betweengroup difference in post-intervention HPT change scores in favour of the Immediate Intervention group. The magnitude of the effect was large. There was no significant within-group delayed intervention effect. A significant maintenance effect was found at 6-week follow-up for the Immediate Intervention group. CONCLUSIONS: The hazard perception skills of participants improved following training with large effect size and some maintenance of gain. A multimodal approach to training is indicated to facilitate maintenance. A full-scale trial is feasible.

Fluid flow structure in arterial bypass anastomosis.

The fluid flow through a stenosed artery and its bypass graft in an anastomosis can substantially influence the outcome of bypass surgery. To help improve our understanding of this and related issues, the steady Navier-Stokes flows are computed in an idealized arterial bypass system with partially occluded host artery. Both the residual flow issued from the stenosis--which is potentially important at an earlier stage after grafting--and the complex flow structure induced by the bypass graft are investigated. Seven geometric models, including symmetric and asymmetric stenoses in the host artery, and two major aspects of the bypass system, namely, the effects of area reduction and stenosis asymmetry, are considered. By analyzing the flow characteristics in these configurations, it is found that (1) substantial area reduction leads to flow recirculation in both upstream and downstream of the stenosis and in the host artery near the toe, while diminishes the recirculation zone in the bypass graft near the bifurcation junction, (2) the asymmetry and position of the stenosis can affect the location and size of these recirculation zones, and (3) the curvature of the bypass graft can modify the fluid flow structure in the entire bypass system.

Examination of synovial fluid and serum following intravenous injections of hyaluronan for the treatment of osteoarthritis in dogs.

A randomized, blinded, prospective clinical trial was performed to determine the effects of intravenous (i.v.) administration of hyaluronan sodium (HA) on serum glycosaminoglycans (GAG) concentrations, synovial fluid (SF) hyaluronan concentrations and viscosity in dogs treated for unilateral rupture of the cranial cruciate ligament. Twenty-two dogs undergoing tibial plateau leveling osteotomy were used in this study. Synovial fluid from both stifles and serum were collected prior to surgery and at 2, 4, and 8 weeks following surgery. Dogs received either 1.0 ml (10 mg) of sodium hyaluronate (treatment group 1; n = 10) or equal volume of 0.9% NaCl (treatment group 2; n = 12), i.v. immediately, 2 and 4 weeks following surgery. Synovial fluid viscosity was evaluated using a magnetically driven, acoustically tracked, translating-ball rheometer. Synovial fluid HA disaccharide content was measured by fluorophore-assisted carbohydrate electrophoresis. Serum GAG concentrations were measured by alcian blue spectrophotometric assay. Data were analyzed using a Wilcoxon sign rank test (p < 0.05). Mean +/- SD viscosity (cP) was significantly higher (p = 0.011) in SF obtained from the intact stifle (450 +/- 604.1) than injured (54.8 +/- 60.8) prior to surgery. Mean +/- SD HA concentrations (ug/ml) were significantly higher (p = 0.02) in synovial fluid obtained from the injured stifles (281.4 +/- 145.9) than intact stifles (141.6 +/- 132.5). No significant difference was noted within or between treatment groups in SF viscosity, HA concentrations, or serum GAG concentrations at any time following surgery. Stifles with cranial cruciate ligament insufficiency had significant alterations in SF viscosity and HA concentrations.

Altered rheology of lymphocytes in the diabetic mouse.

AIMS/HYPOTHESIS: Clinical complications associated with diabetes may be related to altered physical properties of leucocytes. We used micropipette techniques to examine leucocyte rheology (specifically lymphocyte rheology) in the non-obese diabetic (NOD) mouse model of diabetes mellitus. We hypothesised that diabetes affects lymphocyte rheology, and specifically that lymphocyte membranes from diabetic mammals have a higher cortical tension than those from non-diabetic mammals. METHODS: Lymphocytes were isolated from diabetic and control mice. Lymphocyte deformation and activation were assessed with a micropipette apparatus. Cellular activation was assessed visually. Projection length into the micropipette during aspiration was used to calculate the viscosity of the cell. Recovery length following expulsion from the micropipette was used to derive the recovery time constant, which is the ratio of cortical tension : viscosity (T(o)/mu) for each cell. The cell cortical/surface tension was calculated from this ratio. RESULTS: Of 692 control lymphocytes, 29% were spontaneously activated compared with 39% of 624 diabetic cells (p<0.06) and 31.5% of 315 non-diabetic NOD cells (p=0.14). Viscosity values for diabetic lymphocytes were equivalent to those for control cells (1345.12+/-1420.97 Pa.s vs 996.84+/-585.07 Pa.s, p=0.13). The average T(o)/micro value for diabetic lymphocytes (35.4+/-16.5x10(-6) cm/s) was significantly higher than that for control cells (24.8+/-11.3x10(-6) cm/s, p<0.03) and cells from non-diabetic NOD mice (26.3+/-9.0x10(-6) cm/s, p<0.005). The mean cortical tension values for diabetic and control cells were 4.7+/-2.3x10(-4) N/m and 2.8+/-0.7x10(-4) N/m respectively (p<0.003). CONCLUSIONS/INTERPRETATION: Lymphocytes from diabetic mice tend to spontaneously activate. They have an equivalent cytoplasmic viscosity but a larger recovery time constant compared with cells from control mice. The results suggest that diabetic lymphocytes are stiffer than control cells.

Computational modeling of cell adhesion and movement using a continuum-kinetics approach.

Adhesion of leukocytes to substrate involves the coupling of disparate length and timescales between molecular mechanics and macroscopic transport, and existing models of cell adhesion do not use full cellular information. To address these challenges, a multiscale computational approach for studying the adhesion of a cell on a substrate is developed and assessed. The cellular level model consists of a continuum representation of the field equations and a moving boundary tracking capability to allow the cell to change its shape continuously. At the receptor-ligand level, a bond molecule is mechanically represented by a spring. Communication between the macro/micro- and nanoscale models is facilitated interactively during the computation. The computational model is assessed using an adherent cell, rolling and deforming along the vessel wall under imposed shear flows. Using this approach, we first confirm existing numerical and experimental results. In this study, the intracellular viscosity and interfacial tension are found to directly affect the rolling of a cell. Our results also show that the presence of a nucleus increases the bond lifetime, and decreases the cell rolling velocity. Furthermore, it is found that a cell with a larger diameter rolls faster, and decreases the bond lifetime. This study shows that cell rheological properties have significant effects on the adhesion process contrary to what has been hypothesized in most literature.

Fatal crashes involving young male drivers: a continuous time Poisson change-point analysis.

BACKGROUND: In an effort to reduce road trauma, the New Zealand government implemented a series of intervention programs over the last decade, with young male drivers as the main target audience. Previous research, however, found little or no evidence that these programs had any impact on this group of drivers despite an apparent decrease in their crash involvement. OBJECTIVE: To determine the approximate time when the decrease in the number of fatal crashes involving young male drivers occurred. METHOD: A Poisson change-point estimator was used to locate the most likely point where a decrease in the average number of monthly crashes had occurred. RESULTS: The most likely time of change was found to coincide with the time when the Transport Act 1992 was debated and passed in Parliament. The publicity given to the issue and the Government's signal of impending actions were sufficient to induce a significant change in the behaviours of young male drivers. IMPLICATIONS: This result can partially reconcile the difference between the apparent reduction in the number of fatal crashes involving young male drivers and the inability of previous studies to find a significant impact of the various intervention programs since the change occurred prior to the actual implementation of the programs. Nevertheless, it was argued that the implementation of the programs subsequently was necessary in sustaining the reduction.

Effects of nucleus on leukocyte recovery.

The rheological properties of a leukocyte significantly affect its biological and mechanical characteristics. To date, existing physical models of leukocyte are not capable of quantitatively explaining the wide range of deformation and recovery behaviors observed in experiment. However, a compound drop model has gained some success. In the present work, we investigate the effect of nucleus size and position, and the relative rheological properties of cytoplasm and nucleus, on cell recovery dynamics. Two nucleus sizes corresponding to that of neutrophil and lymphocyte are considered. Direct comparison between numerical simulations and experimental observation is made. Results indicate that the time scale ratio between the nucleus and cytoplasm plays an important role in cell recovery characteristics. Comparable time scales between the two cell components yield favorable agreement in recovery rates between numerical and experimental observations; disparate time scales, on the other hand, result in recovery behavior and cell shapes inconsistent with experiments. Furthermore, it is found that the nucleus eccentricity exhibits minimum influence on all major aspects of the cell recovery characteristics. The present work offers additional evidence in support of the compound cell model for predicting the rheological behavior of leukocytes.

Numerical analysis of the deformation of an adherent drop under shear flow.

The adhesion of leukocytes to substrates is an important biomedical problem and has drawn extensive research. In this study, employing both single and compound drop models, we investigate how hydrodynamics interacts with an adherent liquid drop in a shear flow. These liquid drop models have recently been used to describe the rheological behavior of leukocytes. Numerical simulation confirms that the drop becomes more elongated when either capillary number or initial contact angle increases. Our results show that there exists a thin region between the drop and the wall as the drop undergoes large stretching, which allows high pressure to build up and provides a lift force. In the literature, existing models regard the leukocyte as a rigid body to calculate the force and torque acting on the drop in order to characterize the binding between cell receptors and endothelial ligands. The present study indicates that such a rigid body model is inadequate and the force magnitude obtained from it is less than half of that obtained using the deformable drop models. Furthermore, because of its much higher viscosity, the cell nucleus introduces a hydrodynamic time scale orders of magnitude slower than the cytoplasm. Hence the single and compound drops experience different dynamics during stretching, but exhibit very comparable steady-state shapes. The present work offers a framework to facilitate the development of a comprehensive dynamic model for blood cells.

Rheological modelling of leukocytes.

A three-layer Newtonian model is investigated using a combined Eulerian-Lagrangian computational method to describe the dynamic behaviour of leukocytes. The model, composed of a cell membrane (outer layer), cytoplasm (middle layer) and nucleus (inner layer), can better describe the recovery characteristics because large viscosity and capillarity differences between layers are considered, and both Newtonian and seemingly non-Newtonian behaviours reported in the literature can be reproduced. It is found that, to describe adequately the various rheological characteristics of leukocytes, the presence of the highly viscous nucleus and its deformation/recovery, as well as the surface energy stored in the fluid interfaces, are critical. Photographs from pipette experiments using a fluorescent technique confirm the theoretical finding of the important role played by the nucleus in cell deformation.

Is there a need to treat hypokalaemia associated with intravenous salbutamol infusion?

OBJECTIVES: To investigate the evolution of metabolic effects associated with intravenous salbutamol infusion given together with 2 doses of intramuscular steroids in the treatment of preterm labour. METHODS: Preterm labour was inhibited with an intravenous infusion of salbutamol in 8 women between 26 and 32(+6) weeks with normal singleton pregnancies. Serum glucose concentration, serum potassium, sodium and insulin concentrations, and total white cell count both during the infusion as well as post-therapy, were plotted against time. RESULTS: Intravenous salbutamol infusion administered at a rate required to inhibit uterine contractions in preterm labour causes a rise in serum glucose and plasma insulin concentrations. The serum glucose and plasma insulin levels peaked soon after cessation of therapy and took 2-3 hours to come to pre-infusion levels. The decline in serum potassium concentration was gradual and plateaued after 2 hours. Once the salbutamol infusion was stopped the potassium levels were back to normal by 2 hours. There is an increase in total white cell count within an hour of the initiation of therapy. CONCLUSION: There is no need to administer insulin for hyperglycaemia and/or potassium for hypokalaemia unless the patient is a known diabetic or when a patient needs immediate surgery.

Numerical simulation of the flow of highly viscous drops down a tapered tube.

The flow of a highly viscous drop surrounded by an inviscid fluid inside a tapered tube is analyzed according to a Newtonian, liquid-drop model in which a variational method is used to simultaneously solve the hydrodynamic equations for low Reynolds-number flow and the equations for membrane equilibrium with a constant membrane tension. It is found that the flow in the end caps is plug and radial in the conical section of the drop. The results are compared to a simplified analytical theory that makes these assumptions. Very good agreement is found between the two approaches. Both approaches are used to analyze existing experimental results of passive neutrophils flowing down a tapered tube. The theoretical models give a good fit to published experimental data by Bagge et al. (1977) at driving pressures of 20 and 40 mm H2O for a membrane cortical tension of 0.024 dyn/cm and an apparent cytoplasmic viscosity of about 2400 and 1400 poise, respectively.

Viscosity of passive human neutrophils undergoing small deformations.

At issue is the type of constitutive equation that can be used to describe all possible types of deformation of the neutrophil. Here a neutrophil undergoing small deformations is studied by aspirating it into a glass pipet with a diameter that is only slightly smaller than the diameter of the spherically shaped cell. After being held in the pipet for at least seven seconds, the cell is rapidly expelled and allowed to recover its undeformed, spherical shape. The recovery takes approximately 15 s. An analysis of the recovery process that treats the cell as a simple Newtonian liquid drop with a constant cortical (surface) tension gives a value of 3.3 x 10(-5) cm/s for the ratio of the cortical tension to cytoplasmic viscosity. This value is about twice as large as a previously published value obtained with the same model from studies of large deformations of neutrophils. This discrepancy indicates that the cytoplasmic viscosity decreases as the amount of deformation decreases. An extrapolated value for the cytoplasmic viscosity at zero deformation is approximately 600 poise when a value for the cortical tension of 0.024 dyn/cm is assumed. Clearly the neutrophil does not behave like a simple Newtonian liquid drop in that small deformations are inherently different from large deformations. More complex models consisting either of two or more fluids or multiple shells must be developed. The complex structure inside the neutrophil is shown in scanning electron micrographs of osmotically burst cells and cells whose membrane has been dissolved away.

Relationship of clinical severity to packed cell rheology in sickle cell anemia.

There is wide variation in the clinical manifestations of sickle cell disease (SCD) from one affected individual to another. Many investigators have sought to discern parameters that would explain this variability. In the present studies we have attempted to correlate the frequency of painful events and the extent of end organ failure in SCD with rheologic properties of packed suspensions of sickle cells, using a magneto-acoustic ball microrheometer developed in our laboratory. Using this device we have measured the steady-state viscosity, and the viscous and elastic moduli of cell suspensions in 16 individuals with hemoglobin SS disease who were untransfused and in their steady state. The rheologic parameters were then correlated with clinical parameters. The clinical parameters measured were emergency department visits, hospitalizations, hemoglobin, reticulocyte count, age, and end organ failure (nephropathy, avascular necrosis of bone, stroke, retinopathy, resting hypoxemia after acute chest syndrome(s), leg ulcer, and priapism with impotence). The P value for the correlation between the steady state viscosity and end organ failure was .001 with a correlation coefficient (R value) of .73. The P value for the correlation between the viscous modulus of viscosity and end organ failure was .00006 with an R value of .83. The P value for the correlation between the elastic modulus of viscosity and end organ failure was .0006 with an R value of .76. However, there was no significant correlation between any component of packed cell rheology and emergency department visits or hospitalizations for pain.

A physical characterization of GAP A3 hybridoma cells: morphology, geometry, and mechanical properties.

Morphological, geometrical, and rheological properties of the GAP A3 hybridoma cell line have been evaluated as a function of the cell cycle. Interference contrast video microscopy and scanning electron microscopy (SEM) showed that a sample of cells taken from the middle of the exponential growth phase displayed a range of cell morphologies, consistent with a heterogeneous growing culture. Micropipet manipulation was used to measure the geometrical (cell volume) and mechanical (cortical tension and apparent cell viscosity) properties of single cells selected at random from a sample in the middle of the exponential growth phase. Consistent with the range of morphologies, cell volumes (1400 to 5700 microm(3)) and apparent viscosities (430 to 1.2 x 10(4) P) showed a wide range of values at 37 degrees C, demonstrating that a hybridoma cell line cannot be characterized by a single value for any one property, and that properties must be related to their cycle dependence when considering proliferating cells. Direct, video-microscopic observation of synchronized cells, and of individual cells that were followed throughout their cell cycle, allowed us to correlated distinct morphologies with phases of the cell cycle. As the cell cycle progresses, an increase in cell volume by a factor of 3 to 4 is accompanied by an overall increase in apparent cell viscosity by approximately the same ratio, consistent with an accumulation of more cytoplasmic material in the older cells. Also, a decrease in average apparent viscosity by a factor of 10. These results are important in order to evaluate the possible role of certain structural, cell-cycle dependent features in shear and abrasion sensitivity. This is a problem of current concern in the bioreactor culture of mammalian cells.

Time-dependent recovery of passive neutrophils after large deformation.

Experiments are performed in which a passive human neutrophil is deformed into an elongated "sausage" shape by aspirating it into a small glass pipette. When expelled from the pipette the neutrophil recovers its natural spherical shape in approximately 1 minute. This recovery process is analyzed according to a Newtonian, liquid-drop model in which a variational method is used to simultaneously solve the hydrodynamic equations for low Reynolds-number flow and the equations for membrane equilibrium with a constant membrane tension. The theoretical model gives a good fit to the experimental data for a ratio of membrane cortical tension to cytoplasmic viscosity of approximately 1.7 x 10(-5) cm/s (0.17 micron/s). However, when the cell is held in the pipette for only a short time period of 5 s or less, and then expelled, the cell undergoes an initial, rapid elastic rebound suggesting that the cell behaves in this instance as a Maxwell viscoelastic liquid rather than a Newtonian liquid with constant cortical tension.

Effects of cell lysis on the rheological behavior of red blood cell suspensions.

Rheological studies of lysed cell suspensions are performed with a magneto acoustic ball microrheometer. Two methods for lysing the cells are developed in order to provide cell volume concentrations identical to control intact cell suspensions. The first uses a freeze-thaw technique and the second uses sonication. It is found that cell suspensions disrupted by sonication have a lower viscosity than intact suspensions, whereas cell suspensions lysed by the freeze-thaw method exhibit a higher viscosity. Sonication is discovered to have a detrimental impact on the cell membrane, and to cause complete destruction of the cell membrane structure. Measurements of the steady state viscosity show that indeed the presence of the membrane is not detected, and that what is measured is mainly the viscosity of the hemoglobin solution. On the other hand, freeze-thaw results indicate that at least two phenomena occur. The first phenomenon, occurring during the first freeze-thaw cycle, produces an increase in viscosity and in viscoelasticity. The second one, taking place after subsequent freeze-thaw cycles, induces a decrease in the bulk rheological properties. Several possible mechanisms are presented to explain the observed phenomena.

A rheological study of packed red blood cell suspensions with an oscillating ball microrheometer.

Rheological properties of concentrated red blood cell suspensions are studied with a magneto acoustic microrheometer in which a ball is suspended in a vertically oriented cylindrical tube. The rheometer uses a conventional falling ball technique to measure steady state viscosity and a vertically oscillating, magnetically driven ball for viscoelastic measurements. The motion of the ball is tracked by ultrasound echo location in which sound waves are transmitted and received by an ultrasound transducer mounted at the base of the tube. The compact size of the rheometer allows rheological studies to be made with microliter quantities of opaque suspensions and permits sudden and accurate changes in temperature. Also, values for the adiabatic compressibility are evaluated from measurements of the speed of sound.

Osteoblasts increase their rate of division and align in response to cyclic, mechanical tension in vitro.

Bone adapts to physical deformation in vivo, yet the mechanism of the adaptive process remains unknown. One reason for this perplexity has been the difficulty in examining the effects of a well-defined deformation regimen on individual bone cells. With the utilization of novel, flexible-bottomed cell culture plates, one can study the effects of cyclic strain on the morphologic and biochemical adaptations of individual osteoblasts in vitro. Avian, calvarial osteoblast-like cells, from passes 2-5, responded to cyclic strain, by increasing their rates of DNA synthesis and cell division during the first 72 h after initiation of a continuous deformation regimen comprised of 3 cycles per min of 0-24% elongation. In addition, within hours after initiation of the deformation regimen, cells oriented 90 degrees to the applied strain field at the periphery of the culture plate in the region of maximum strain and elongation.