The fluorolysis assay, a highly sensitive method for measuring the cytolytic activity of T cells at very low numbers

We have developed a highly sensitive cytolysis test, the fluorolysis assay, as a simple nonradioactive and inexpensive alternative to the standard Cr-51-release assay. P815 cells were stably transfected with a plasmid expressing the enhanced green fluorescent protein (EGFP) gene. These target cells were coated with or without cognate peptide or anti-CD3 Ab and then incubated with CD8(+) T cells to allow antigen-specific or nonspecific lysis. The degree of target cell lysis was measured using flow cytometry to count the percentage of viable propidium iodide(-) EGFP(+) cells, whose numbers were standardized to a reference number of fluorochrome-linked beads. By using small numbers of target cells (200-800 per reaction) and extended incubation times (up to 2 days), the antigen-specific cytolytic activity of one to two activated CD8(+) T cells of a CTL line could be detected. The redirected fluorolysis assay also measured the activity of very few ( greater than or equal to6) primary CD8(+) T cells following polyclonal activation. Importantly, antigen-specific lysis by small numbers ( greater than or equal to 25) of primary CD8(+) T cells could be directly measured ex vivo. This exquisite sensitivity of the fluorolysis assay, which was at least 8-33-folds higher than an optimized 51 Cr-release assay, allows in vitro and ex vivo studies of immune responses that would otherwise not be possible due to low CTL numbers or frequencies. (C) 2002 Elsevier Science B.V. All rights reserved.

Marx as a Critical Discourse Analyst: The genesis of a critical method and its relevance to the critique of global capital

In this paper we identify elements in Marx´s economic and political writings that are relevant to contemporary critical discourse analysis (CDA). We argue that Marx can be seen to be e n gaging in a form of discourse analysis. We identify the elements in Marx´s historical materialist method that support such a perspective, and exemplify these in a longitudinal comparison of Marx´s texts.

R-matrices and the tensor product graph method

A systematic method for constructing trigonometric R-matrices corresponding to the (multiplicity-free) tensor product of any two affinizable representations of a quantum algebra or superalgebra has been developed by the Brisbane group and its collaborators. This method has been referred to as the Tensor Product Graph Method. Here we describe applications of this method to untwisted and twisted quantum affine superalgebras.

Control of chaotic instabilities in a spinning spacecraft with dissipation using Lyapunov's method

Control of chaotic instability in a simplified model of a spinning spacecraft with dissipation is achieved using an algorithm derived using Lyapunov's second method. The control method is implemented on a realistic spacecraft parameter configuration which has been found to exhibit chaotic instability for a range of forcing amplitudes and frequencies when a sinusoidally varying torque is applied to the spacecraft. Such a torque, may arise in practice from an unbalanced rotor or from vibrations in appendages. Numerical simulations are performed and the results are studied by means of time history, phase space, Poincare map, Lyapunov characteristic exponents and bifurcation diagrams. (C) 2002 Elsevier Science Ltd. All rights reserved.

Development of a fully quantitative approach to the interpretation of stress echocardiography using radial and longitudinal myocardial velocities

Background: Tissue Doppler may be used to quantify regional left ventricular function but is limited by segmental variation of longitudinal velocity from base to apex and free to septal walls. We sought to overcome this by developing a composite of longitudinal and radial velocities. Methods and Results. We examined 82 unselected patients undergoing a standard dobutamine echocardiogram. Longitudinal velocity was obtained in the basal and mid segments of each wall using tissue Doppler in the apical views. Radial velocities were derived in the same segments using an automated border detection system and centerline method with regional chords grouped according to segment location and temporally averaged. In 25 patients at low probability of coronary disease, the pattern of regional variation in longitudinal velocity (higher in the septum) was the opposite of radial velocity (higher in the free wall) and the combination was homogenous. In 57 patients undergoing angiography, velocity in abnormal segments was less than normal segments using longitudinal (6.0 +/- 3.6 vs 9.0 +/- 2.2 cm/s, P = .01) and radial velocity (6.0 +/- 4.0 vs 8.0 +/- 3.9 cm/s, P = .02). However, the composite velocity permitted better separation of abnormal and normal segments (13.3 +/- 5.6 vs 17.5 +/- 4.2 cm/s, P = .001). There was no significant difference between the accuracy of this quantitative approach and expert visual wall motion analysis (81% vs 84%, P = .56). Conclusion: Regional variation of uni-dimensional myocardial velocities necessitates site-specific normal ranges, probably because of different fiber directions. Combined analysis of longitudinal and radial velocities allows the derivation of a composite velocity, which is homogenous in all segments and may allow better separation of normal and abnormal myocardium.

Automated regional myocardial displacement for facilitating the interpretation of dobutamine echocardiography

Quantification of stress echocardiography may overcome the training requirements and subjective nature of visual wall motion score (WMS) assessment, but quantitative approaches may be difficult to apply and require significant time for image processing. The integral of long-axis myocardial velocity is displacement, which may be represented as a color map over the left ventricular myocardium. This study was designed to explore the feasibility and accuracy of measuring long-axis myocardial displacement, derived from tissue Doppler, for the detection of coronary artery disease (CAD) during dobutamine stress echocardiography (DBE). One hundred thirty patients underwent standard DBE, including 30 patients at low risk of CAD, 30 patients with normal coronary angiography (both groups studied to define normal ranges of displacement), and 70 patients who underwent coronary angiography in whom the accuracy of normal ranges was tested. Regional myocardial displacement was obtained by analysis of color tissue Doppler apical images acquired at peak stress. Displacement was compared with WMS, and with the presence of CAD by angiography. The analysis time was 3.2 +/- 1.5 minutes per patient. Segmental displacement was correlated with wall motion (normal 7.4 +/- 3.2 mm, ischemia 5.8 +/- 4.2 mm, viability 4.6 +/- 3.0 mm, scar 4.5 +/- 3.5 mm, p <0.001). Reversal of normal base-apex displacement was an insensitive (19%) but specific (90%) marker of CAD. The sum of displacements within each vascular territory had a sensitivity and specificity of 89% and 79%, respectively, for prediction of significant CAD, compared with 86% and 78%, respectively, for WMS (p = NS). The displacements in the basal segments had a sensitivity and specificity of 83% and 78%, respectively (p = NS). Regional myocardial displacement during DBE is feasible and offers a fast and accurate method for the diagnosis of CAD. (C),2002 by Excerpta Medica, Inc.

A four-stage index 2 Diagonally Implicit Runge-Kutta method

High index Differential Algebraic Equations (DAEs) force standard numerical methods to lower order. Implicit Runge-Kutta methods such as RADAU5 handle high index problems but their fully implicit structure creates significant overhead costs for large problems. Singly Diagonally Implicit Runge-Kutta (SDIRK) methods offer lower costs for integration. This paper derives a four-stage, index 2 Explicit Singly Diagonally Implicit Runge-Kutta (ESDIRK) method. By introducing an explicit first stage, the method achieves second order stage calculations. After deriving and solving appropriate order conditions., numerical examples are used to test the proposed method using fixed and variable step size implementations. (C) 2001 IMACS. Published by Elsevier Science B.V. All rights reserved.

Application of a simplified method based on regular regime approach to determine the effective moisture diffusivity of mixture of low molecular weight sugars and maltodextrin during desorption

The binary diffusivities of water in low molecular weight sugars; fructose, sucrose and a high molecular weight carbohydrate; maltodextrin (DE 11) and the effective diffusivities of water in mixtures of these sugars (sucrose, glucose, fructose) and maltodextrin (DE 11) were determined using a simplified procedure based on the Regular Regime Approach. The effective diffusivity of these mixtures exhibited both the concentration and molecular weight dependence. Surface stickiness was observed in all samples during desorption, with fructose exhibiting the highest and maltodextrin the lowest. (C) 2002 Elsevier Science Ltd. All rights reserved.

Comparison of an ELISA and an LC/MS/MS method for measuring tacrolimus concentrations and making dosage decisions in transplant recipients

This study compared an enzyme-linked immunosorbent assay (ELISA) to a liquid chromatography-tandem mass spectrometry (LC/MS/MS) technique for measurement of tacrolimus concentrations in adult kidney and liver transplant recipients, and investigated how assay choice influenced pharmacokinetic parameter estimates and drug dosage decisions. Tacrolimus concentrations measured by both ELISA and LC/MS/MS from 29 kidney (n = 98 samples) and 27 liver (n = 97 samples) transplant recipients were used to evaluate the performance of these methods in the clinical setting. Tacrolimus concentrations measured by the two techniques were compared via regression analysis. Population pharmacokinetic models were developed independently using ELISA and LC/MS/MS data from 76 kidney recipients. Derived kinetic parameters were used to formulate typical dosing regimens for concentration targeting. Dosage recommendations for the two assays were compared. The relation between LC/MS/MS and ELISA measurements was best described by the regression equation ELISA = 1.02 . (LC/MS/MS) + 0.14 in kidney recipients, and ELISA = 1.12 . (LC/MS/MS) - 0.87 in liver recipients. ELISA displayed less accuracy than LC/MS/MS at lower tacrolimus concentrations. Population pharmacokinetic models based on ELISA and LC/MS/MS data were similar with residual random errors of 4.1 ng/mL and 3.7 ng/mL, respectively. Assay choice gave rise to dosage prediction differences ranging from 0% to 30%. ELISA measurements of tacrolimus are not automatically interchangeable with LC/MS/MS values. Assay differences were greatest in adult liver recipients, probably reflecting periods of liver dysfunction and impaired biliary secretion of metabolites. While the majority of data collected in this study suggested assay differences in adult kidney recipients were minimal, findings of ELISA dosage underpredictions of up to 25% in the long term must be investigated further.

Linguistic evaluation method for the environmental assessment of infrastructure projects

A soft linguistic evaluation method is proposed for the environmental assessment of physical infrastructure projects based on fuzzy relations. Infrastructure projects are characterized in terms of linguistic expressions of 'performance' with respect to factors or impacts and the 'importance' of those factors/impacts. A simple example is developed to illustrate the method in the context of three road infrastructure projects assessed against five factors/impacts. In addition, a means to include hard or crisp factors is presented and illustrated with respect to a sixth factor.

Assessing clinical reasoning: a method to monitor its development in a PBL curriculum

The aim of this study was to develop and trial a method to monitor the evolution of clinical reasoning in a PBL curriculum that is suitable for use in a large medical school. Termed Clinical Reasoning Problems (CRPs), it is based on the notion that clinical reasoning is dependent on the identification and correct interpretation of certain critical clinical features. Each problem consists of a clinical scenario comprising presentation, history and physical examination. Based on this information, subjects are asked to nominate the two most likely diagnoses and to list the clinical features that they considered in formulating their diagnoses, indicating whether these features supported or opposed the nominated diagnoses. Students at different levels of medical training completed a set of 10 CRPs as well as the Diagnostic Thinking Inventory, a self-reporting questionnaire designed to assess reasoning style. Responses were scored against those of a reference group of general practitioners. Results indicate that the CRPs are an easily administered, reliable and valid assessment of clinical reasoning, able to successfully monitor its development throughout medical training. Consequently, they can be employed to assess clinical reasoning skill in individual students and to evaluate the success of undergraduate medical schools in providing effective tuition in clinical reasoning.