A simple trogocytosis-based method to detect, quantify, characterize and purify antigen-specific live lymphocytes by flow cytometry, via their capture of membrane fragments from antigen-presenting cells

We have developed a method exploiting the phenomenon of trogocytosis to detect lymphocytes reacting specifically with target cells by flow cytometry. Trogocytosis is a process by which lymphocytes capture fragments of the plasma membrane from the antigen-presenting cells (APCs) expressing their cognate antigen. For this method, a label (such as a fluorescent lipid or biotin) is first incorporated in the membrane of APCs. These labeled cells are then co-cultured for a few hours with a population of cells containing the lymphocytes to be detected. After this period of stimulation, lymphocytes that have performed trogocytosis are identified by their acquisition of the label initially present on the APC membrane using flow cytometry. A major advantage of this method is its compatibility with the simultaneous detection of phenotypic and/or functional markers on the lymphocytes. Furthermore, cells can be recovered alive and active after detection of trogocytosis, and are therefore available for further characterization or even conceivably for therapeutic purposes.     

A novel validation and calibration method for motion capture systems based on micro-triangulation

Motion capture systems are widely used to measure human kinematics. Nevertheless, users must consider system errors when evaluating their results. Most validation techniques for these systems are based on relative distance and displacement measurements. In contrast, our study aimed to analyse the absolute volume accuracy of optical motion capture systems by means of engineering surveying reference measurement of the marker coordinates (uncertainty: 0.75 mm). The method is exemplified on an 18 camera OptiTrack Flex13 motion capture system. The absolute accuracy was defined by the root mean square error (RMSE) between the coordinates measured by the camera system and by engineering surveying (micro-triangulation). The original RMSE of 1.82 mm due to scaling error was managed to be reduced to 0.77 mm while the correlation of errors to their distance from the origin reduced from 0.855 to 0.209. A simply feasible but less accurate absolute accuracy compensation method using tape measure on large distances was also tested, which resulted in similar scaling compensation compared to the surveying method or direct wand size compensation by a high precision 3D scanner. The presented validation methods can be less precise in some respects as compared to previous techniques, but they address an error type, which has not been and cannot be studied with the previous validation methods.     

A new method to characterize saturation functions by their first four moments

The calculation of the first four moments of saturation functions is proposed as a method to describe the properties of enzymes or receptors models. The values of these moments in the case of the Langmuir or Michaelis-Menten equation and the Hill equation are reviewed. They have been calculated for the second degree Adair equation and in the case of binding site heterogeneity. A method for generalization to cases of greater complexity is also proposed. The advantage of this method over the classical ones—graphical representations and derivation of coefficients like n_H, $\text{[L]0.9}\text{[L]0.1}$…—is essentially that the moments are defined by one single value independently of any particular model for the whole of the saturation curve.     

A new method to characterize chemically and topographically nanopatterned surfaces

Surface chemistry of topographically patterned grooved samples with ridges of 150 nm width, adsorbed with self-assembled monolayers (SAMs) of alkanethiols on gold, have been characterized by near edge X-ray absorption fine structure (NEXAFS) spectroscopy. Analysis reveals that NEXAFS may discriminate between different chemistries adsorbed to the tops, sidewalls and grooves of the patterns.     

A method to characterize the passive elasticity in contracting muscle bundles

There is concern among researchers whether the passive muscle properties, characterized by purely passive material testing procedures, are an appropriate representation of the actual passive component of the muscle. This aspect is of particular importance in the biomechanical analysis of heart muscle response where it is generally agreed that the so-called parallel elasticity cannot be ignored as is done justifiably in the analysis of skeletal muscle response. In the present article, a method of quantifying the passive elasticity in contracting muscle bundles is presented. The method consists of imposing isometric transients (such as the quick-stretch or quick release) on a muscle bundle during the contraction phase and observing the differences in decayed force levels between a normal twitch and that of a perturbed twitch. The proposed method provides a means of obtaining useful passive properties from contracting muscle bundles and circumvents the difficulty of having to characterize muscle properties from separate experiments on quiescent muscle bundles.     

A novel antibody-cell conjugation method to enhance and characterize cytokine-induced killer cells

BackgroundCytokine-induced killer (CIK) cells are an ex vivo–expanded cellular therapy product with potent anti-tumor activity in a subset of patients with solid and hematologic malignancies. We hypothesize that directing CIK cells to a specific tumor antigen will enhance CIK cell anti-tumor cytotoxicity.MethodsWe present a newly developed method for affixing antibodies directly to cell surface proteins. First, we evaluated the anti-tumor potential of CIK cells after affixing tumor-antigen targeting monoclonal antibodies. Second, we evaluated whether this antibody-conjugation method can profile the surface proteome of CIK cells.ResultsWe demonstrated that affixing rituximab or daratumumab to CIK cells enhances cytotoxic killing of multiple lymphoma cell lines in vitro. These ‘armed’ CIK cells exhibited enhanced intracellular signaling after engaging tumor targets. Cell surface proteome profiling suggested mechanisms by which antibody-armed CIK cells concurrently activated multiple surface proteins, leading to enhanced cytolytic activity. Our surface proteome analysis indicated that CIK cells display enhanced protein signatures indicative of immune responses, cellular activation and leukocyte migration.ConclusionsHere, we characterize the cell surface proteome of CIK cells using a novel methodology that can be rapidly applied to other cell types. Our study also demonstrates that without genetic modification CIK cells can be rapidly armed with monoclonal antibodies, which endows them with high specificity to kill tumor targets.     

A new method to determine sex and gonad size in live fishes by using ultrasonography

A method to determine, non-invasively, sex and gonad size employing ultrasonography and using Atlantic salmon, Salmo salar Linnaeus, as an example, is described. Although gonads of immature males may be difficult to discern, gonads of females are always visible and hence sexing is possible by deduction. Gonad diameter can be estimated by ultrasonography; the correlation coefficients between ultrasonographic and ruler-derived measurements were 0.926 for females and 0.754 for males. The appearance of ultrasonographic images of the gonads during sexual maturation are described. The results are discussed and suggestions made to improve the quality of images and reliability of measurements.     

On the relationship between spontaneous mutation rates in vivo and in vitro.

Recent estimates of spontaneous mutation rates in man, in which previous sources of bias are corrected, indicate that the average is about 3 x 10(-7) per locus per generation, a much lower figure than is generally accepted. Assuming 100 to 1000 cell divisions between each gametic union, this information predicts that cellular mutation rats should be in the order of 10(-9) per locus per generation. Since none of the mutation rates measured in cultured cells are this low (average for seven characters equals 7 x 10(-7)), the size of mutation rates in cultured cells cannot be used to substantiate the claim of epigenetic inheritance. Furthermore, this information suggests that in multicellular organisms the germinal tissue is sequestered from mutagenic insult or subjected to selection against mutational damage so as to keep the genetic load of a species at a tolerable level. Alternatively, cell culture environments may present an extremely abnormal situation to somatic cells, thus elevating the mutation rate.     

A method for in vivo diffusion tracer studies combining perfusion fixation with intravenous tracer injection

Summary A method permitting exact time studies in diffusion tracer experiments in vivo is described. The method consist in application of perfusion fixation via the aorta abdominalis after the injection of the tracer substance. As the animal can be prepared for perfusion before the tracer is injected, time accuracy is in the order of a few seconds. Some applications of the method are demonstrated. A further advantage of the method is good tissue preservation. This perfusion technique is also recommended for study when exact timing is necessary, for example in autoradiography.Supported by grants from the Fonds national suisse de la Recherche scientifique, Berne, Switzerland. We wish to thank M. P. Detraz for technical assistance.     

A point cluster method for in vivo motion analysis: applied to a study of knee kinematics.

A new method for deriving limb segment motion from markers placed on the skin is described. The method provides a basis for determining the artifact associated with nonrigid body movement of points placed on the skin. The method is based on a cluster of points uniformly distributed on the limb segment. Each point is assigned an arbitrary mass. The center of mass and the inertia tensor of this cluster of points are calculated. The eigenvalues and eigenvectors of the inertia tensor are used to define a coordinate system in the cluster as well as to provide a basis for evaluating non-rigid body movement. The eigenvalues of the inertia tensor remain invariant if the segment is behaving as a rigid body, thereby providing a basis for determining variations for nonrigid body movement. The method was tested in a simulation model where systematic and random errors were introduced into a fixed cluster of points. The simulation demonstrated that the error due to nonrigid body movement could be substantially reduced. The method was also evaluated in a group of ten normal subjects during walking. The results for knee rotation and translation obtained from the point cluster method compared favorably to results previously obtained from normal subjects with intra-cortical pins placed into the femur and tibia. The resulting methodology described in this paper provides a unique approach to the measurement of in vivo motion using skin-based marker systems.     

Sensitive method to identify and characterize proteinases in situ after SDS-PAGE

Cells and body fluids contain numerous, different proteinases; to identify and characterize them are both important and difficult tasks. Especially difficult to identify and characterize are highly specific proteinases. Here, we present an extremely sensitive and quantitative method to characterize proteinases fractionated by SDS-PAGE that cleave specific rhodamine-based fluorogenic substrates. To test the sensitivity of the technique, we used trypsin as our model system. Filter paper impregnated with rhodamine-based fluorogenic substrates was placed on a gel, and bands of fluorescence originating from specific proteinases were visualized in real time. The method is very sensitive; picogram amounts of trypsin can be detected. The method should be very general, in that even proteinases whose substrates require amino acids C-terminal to the cleavage site may be identified and characterized. The results allow one to obtain not only information on the substrate specificity of a specific enzyme but also information about its molecular weight.     

Actin stress fibres and cell-cell adhesion molecules in tendons: organisation in vivo and response to mechanical loading of tendon cells in vitro.

Tendons consist of parallel longitudinal rows of cells separated by collagen fibres. The cells are in intimate contact longitudinally within rows, and laterally via sheet-like lateral cell processes between rows. At points of contact, they are linked by gap junctions. Since tendons stretch under load, such cell contacts require protection. Here we describe the organisation of the actin cytoskeleton and actin-based cell-cell interactions in vivo and examine the effect of cyclic tensile loading on tendon cells in vitro. Cells within longitudinal rows contained short longitudinally running actin stress fibres. Each fibre was aligned with similar fibres in the cells longitudinally on either side, and fibres appeared to be linked via adherens junctions. Overall, these formed long oriented rows of stress fibres running along the rows of tendon cells. In culture, junctional components n-cadherin and vinculin and the stress fibre component tropomyosin increased in strained cultures, whereas actin levels remained constant. These results suggest that: (1) cells are linked via actin-associated adherens junctions along the line of principal strain; and (2) under load, cells appear to attach themselves more strongly together, and assemble more of their cytoplasmic actin into stress fibres with tropomyosin. Taken together, this suggests that cell-cell contacts are protected during stretch, and also that the stress fibres, which are contractile, may provide an active mechanism for recovery from stretch. In addition, stress fibres are ideally oriented to monitor tensile load and thus may be important in mechanotransduction and the generation of signals passed via the gap junction network.     

Integrating modelling, motion capture and x-ray fluoroscopy to investigate patellofemoral function during dynamic activity

Accurate measurement of knee-joint kinematics is critical for understanding the biomechanical function of the knee in vivo. Measurements of the relative movements of the bones at the knee are often used in inverse dynamics analyses to estimate the net muscle torques exerted about the joint, and as inputs to finite-element models to accurately assess joint contact. The fine joint translations that contribute to patterns of joint stress are impossible to measure accurately using traditional video-based motion capture techniques. Sub-millimetre changes in joint translation can mean the difference between contact and no contact of the cartilage tissue, leading to incorrect predictions of joint loading. This paper describes the use of low-dose X-ray fluoroscopy, an in vivo dynamic imaging modality that is finding increasing application in human joint motion measurement. Specifically, we describe a framework that integrates traditional motion capture, X-ray fluoroscopy and anatomically-based finite-element modelling for the purpose of assessing joint function during dynamic activity. We illustrate our methodology by applying it to study patellofemoral joint function, wherein the relative movements of the patella are predicted and the corresponding joint-contact stresses are calculated for a step-up task.     

The relationships between cyclic fatigue loading, changes in initial mechanical properties, and the in vivo temporal mechanical response of the rat patellar tendon

Damage accumulation underlies tendinopathy. Animal models of overuse injuries do not typically control loads applied to the tendon. Our in vivo model in the rat patellar tendon allows direct control of the loading applied to the tendon. Despite this advantage, natural variation among tendons results in different amounts of damage induced by the same loading protocol. Our objectives were to (1) assess changes in the initial mechanical parameters (hysteresis, stiffness of the loading and unloading load-displacement curves, and elongation) after fatigue loading to identify parameters that are indicative of the induced damage, and (2) evaluate the relationships between these identified initial damage indices with the stiffness 7 day after loading. Left patellar tendons of adult, female retired breeder, Sprague-Dawley rats (n = 68) were fatigue loaded per our previously published in vivo fatigue loading protocol. To induce a range of damage, fatigue loading consisted of either 5, 100, 500 or 7200 cycles that ranged from 1 N to 40 N. Diagnostic tests were applied before and immediately after fatigue loading, and after 45 min of recovery to deduce recoverable and non-recoverable changes in initial damage indices. Relationships between these initial damage indices and the 7-day stiffness (at sacrifice) were determined. Day-0 hysteresis, loading and unloading stiffness exhibited cycle-dependent changes. Initial hysteresis loss correlated with the 7-day stiffness. k-means cluster analysis demonstrated a relationship between 7-day stiffness and day-0 hysteresis and unloading stiffness. This analysis also separated samples that exhibited low from high damage in response to both high or low number of cycles; a key delineation for interpretation of the biological response in future studies. Identifying initial parameters that reflect the induced damage is critical since the ability of the tendon to repair depends on the damage induced and the number of applied loading cycles.     

A new method for assessing relative dynamic motion of vertebral bodies during cyclic loading in vitro.

A new experimental technique for measuring generalized three-dimensional motion of vertebral bodies during cyclic loading in vitro is presented. The system consists of an orthogonal array of three lasers mounted rigidly to one vertebra, and a set of three mutually orthogonal charge-coupled devices mounted rigidly to an adjacent vertebra. Each laser strikes a corresponding charge-coupled device screen. The mathematical model of the system is reduced to a linear set of equations with consequent matrix algebra allowing fast real-time data reduction during cyclic movements of the spine. The range and accuracy of the system is well suited for studying thoracolumbar motion segments. Distinct advantages of the system include miniaturization of the components, the elimination of the need for mechanical linkages between the bodies, and a high degree of accuracy which is not dependent on viewing volume as found in photogrammetric systems. More generally, the spectrum of potential applications of systems of this type to the real-time measurement of the relative motion of two bodies is extremely broad.     

A method to identify and characterize Z-DNA binding proteins using a linear oligodeoxynucleotide.

An oligodeoxynucleotide that readily flips to the Z-DNA conformation in 10mM MgCl2 was produced by using Klenow enzyme to incorporate 5-bromodeoxycytosine and deoxyguanosine into a (dC-dG)22 template. During synthesis the oligomer can be labeled with 32P to high specific activity. The labeled oligodeoxynucleotide can be used in bandshift experiment to detect proteins that bind Z-DNA. This allows the binding specificity of such proteins to be determined with high reliability using unlabeled linear and supercoiled DNA competitors. In addition, because the radioactive oligodeoxynucleotide contains bromine atoms, DNA-protein complexes can be readily crosslinked using UV light. This allows an estimate to be made of the molecular weight of the proteins that bind to the radioactive probe. Both techniques are demonstrated using a goat polyclonal anti-Z-DNA antiserum.