Добірка наукової літератури з теми "In-Capillary labeling"

To assess the reliability of fluorescence methods for a quantitative staining of brain capillaries, three different immunohistochemical fluorescent markers were used in the rat brain. Staining of the basement membrane by antibodies directed against fibronectin was compared, in the same brain section, with simultaneous staining of the vascular endothelium constituents nonmuscle myosin or von Willebrand factor (factor VIII). These stainings all resulted in identical patterns, which demonstrates their suitability for capillary staining in the brain. It has been claimed that fixation of the tissue results in the appearance of spurious capillary spots. Such a fixation artifact could be excluded using nonmuscle myosin staining. These results validate the methods of quantitative fluorescent microscopical staining of capillary morphology in the brain and therefore support our concept of a continuous perfusion of all capillaries in the brains of conscious rats.

Aquaporin (AQP7) is expressed in proximal tubules and is involved in glycerol uptake. The cellular expression and physiological function in other organs remain largely undefined. AQP7 knockout (KO) mice were generated and used for immunohistochemical analyses to define the organ and cellular expression of AQP7. AQP7 labeling was found in kidney proximal tubule, heart, skeletal muscle, testis, epididymis, as well as in white and brown adipose tissue (WAT and BAT) of wild-type mice. Importantly, immunoreactivity was completely absent from these tissues in AQP7 KO mice. At the cellular level, the capillary endothelium WAT and BAT displayed prominent staining, whereas AQP7 labeling in adipocyte membranes was undetectable. Double-labeling confocal microscopy revealed coexpression of AQP7 with capillary AQP1 but not with adipocyte GLUT4. Moreover, immunoelectron microscopy and RT-PCR of isolated microvessels confirmed the vascular AQP7 expression. Distinct immunolabeling of the capillary endothelium was also observed in both skeletal and heart muscle with no apparent staining of skeletal or cardiac myocytes. As previously reported, specific immunolabeling was confined to brush border in segment 3 renal proximal tubules and to spermatids and spermatozoa in male reproductive tract. The expression of AQP7 was induced up to 2.2-fold in WAT of mice with streptozotocin-induced diabetes mellitus (S-DM) compared with controls and fasting for 72 h (but not 24 h) induced significant increase in AQP7 expression. In conclusion, AQP7 is expressed in capillary endothelia of adipose tissue (and cardiac and striated muscle) and is upregulated in WAT in response to S-DM supporting its role in glycerol metabolism.

We detected the hematopoietic stem and progenitor cell marker CD133 using immunogold labeling during angiogenesis in a three-dimensional collagen gel culture. CD133-positive cells were present in capillary tubes newly formed from aortic explants in vitro. The CD133-positive cell population had the capacity to form capillary tubes. Lovastatin strongly inhibited cell migration from aortic explants and caused the degradation of the capillary tubes. The present study provides insight into the function of CD133 during angiogenesis as well as an explanation for the antiangiogenic effect of statins.

Changes in distribution of intrarenal blood flow were studied in anesthetized rats during the acute phase of postischemic renal failure (1 h renal artery occlusion, 1 h reflow). Distribution of capillary plasma flow was determined by injecting fluorescein-isothiocyanate-globulin and lissamine-rhodamine-B200-globulin 1, 3, or 10 min prior to rapid freezing of the kidney. In histological sections it was possible to differentiate among the vessels perfused during the time of labeling because of their respective fluorescence. In these experiments all glomeruli became labeled within 1 min, although in contrast to the controls, the glomerular capillary network itself was not filled completely in the postocclusion organs. Incomplete labeling was far more pronounced, however, in the postglomerular network of the occlusion experiments. Due to this effect in the cortex and in the medulla, 11 and 58% of tissue, respectively, were found lying at a distance of more than 60 microns from the next vessel labeled after 1 min of dye circulation. In the control experiments there was no tissue within this distance. Prolonging the time of labeling up to 10 min caused little change in this pattern of distribution. In the occlusion experiments, the globulins were observed in nearly all Bowman spaces, but in less than half of the tubular lumina. The results strengthen the view that the ischemic insult leads primarily to disturbance of the postglomerular perfusion, which then results in trophic damage of the tubular system mainly within the renal medulla.

The pattern of capillary perfusion was studied in the brain of anesthetized rats. Two plasma labels were used to demonstrate the density of capillaries perfused during a 10-min period [fluorescein isothiocyanate (FITC) globulin], as well as during a 10-, 3-, or 1-s period [lissamine-rhodamine B 200 (RB200) globulin, infused into the left heart chamber], respectively. A special biopsy cutting-freezing system was used to withdraw brain tissue via a cranial window for histological analysis of dye distribution at the end of the infusion period. Complete labeling of all capillaries was already found after 10 s of dye circulation. However, intra-arterial dye infusion for 3 and 1 s led to reduced filling of capillaries: cortex 86.6 +/- 5.2 and 6.8 +/- 1.8%, hippocampus 95.0 +/- 1.6 and 9.9 +/- 2.1%, and thalamus 97.9 +/- 1.0 and 11.7 +/- 1.8%, respectively. The period of 1 s was found to be the circulation time from left heart chamber to brain capillaries. It can thus be concluded that in the studied brain areas greater than 85% of capillaries are reached by a plasma flow within 2 s and that the remaining small fraction completely fills within 10 s.

Frozen sections of chicken tissues containing aortic and capillary endothelial cells were immunolabeled with two mouse monoclonal antibodies directed to different epitopes of the chicken integrin beta-chain. Integrin is an integral membrane protein complex that is believed to mediate a transmembrane linkage between the extracellular matrix and the actin cytoskeleton. In immunofluorescence experiments with semi-thin frozen sections, the aortic endothelial cells were labeled for integrin all around their surfaces, whereas capillary endothelial cells of heart and kidney were labeled only on their basal surfaces. At the immunofluorescence level of resolution, the distribution of integrin appeared to be correlated with that of F-actin in double-labeling experiments with NBD-phallacidin. These different distributions of integrin on the two types of endothelial cells were definitively confirmed by immunoelectron microscopic labeling with the monoclonal antibodies on ultra-thin frozen sections. These results therefore indicate that the luminal surfaces, as well as the underlying cytoskeleton of capillary endothelial cells, are significantly different in structure from those of aortic endothelial cells. These differences may reflect the vastly different hemodynamic stress to which the two types of endothelial cells are subjected, and in addition may mediate different adhesion properties of the luminal surfaces of the two cell types.

Brain capillary endothelial cells (BCECs) express transferrin receptors. The uptake of a potential drug vector (OX26, or anti—transferrin receptor antibody IgG2a) conjugated to polyethyleneglycol-coated liposomes by BCECs was studied using in situ perfusion in 18-day-old rats in which the uptake of OX26 is almost twice as high as in the adult rat. Using radio-labeling, the uptake of OX26 by BCECs after 15-minute perfusion was approximately 16 times higher than that of nonimmune IgG2a (Ni-IgG2a). OX26 and OX26-conjugated liposomes selectively distributed to BCECs, leaving choroid plexus epithelium, neurons, and glia unlabeled. Ni-IgG2a and unconjugated liposomes did not reveal any labeling of BCECs. The labeling of BCECs by OX26 was profoundly higher than that of transferrin. Perfusion with albumin for 15 minutes did not reveal any labeling of neurons or glia, thus confirming the integrity of the blood—brain barrier. The failure to label neurons and glia shows that OX26 and OX26-conjugated liposomes did not pass through BCECs. The expression of transferrin receptors by endothelial cells selective to the brain qualifies OX26 as a candidate for blood-to-endothelium transport. A specifically designed formulation of liposomes may allow for their degradation within BCECs, leading to subsequent transport of liposomal cargo further into the brain.

La glycosylation est l'une des principales modifications post-traductionnelles des protéines. La détection de modifications mineures de la glycosylation des protéines peut aider à l’identification de nouveaux biomarqueurs diagnostiques ou au contrôle qualité des produits biothérapeutiques. La cartographie des glycanes est l'une des approches les plus efficaces mais cette stratégie reste encore limitée par les nombreuses et longues étapes de traitement d’échantillon. Ces étapes impliquent la libération, le marquage et l’analyse des glycanes par une technique de séparation. Ces opérations manuelles pouvant induire des interférences provoquent des problèmes de reproductibilité. La miniaturisation et l'automatisation des différentes étapes sont nécessaires mais restent difficiles. Les dispositifs mis au point pour la libération ou le marquage par fluorescence des glycanes concernent davantage les N- que les O-glycanes. A ce jour, aucun microréacteur avec des enzymes immobilisées (IMER) pour la libération de O-glycanes ou aucun marquage en capillaire de glycanes avec l'acide 8-aminopyrène-1,3,6-trisulfonique (APTS) n’a été reporté dans la littérature. Mon projet de thèse vise à contribuer au développement d'un microsystème intégrant la libération des glycanes des glycoprotéines, leur marquage fluorescent en ligne, et leur séparation par électrophorèse capillaire (EC) couplée à la détection par fluorescence induite par laser (LIF).La première partie expérimentale décrit le développement d'un IMER à base d'un monolithe photo-polymérisé pour libérer les O-glycanes des glycoprotéines. Un monolithe à base de poly(méthacrylate de glycidyl-co-poly(diacrylate d'éthylène glycol)) de 10 mm a été synthétisé dans un capillaire après optimisation des proportions monomère/réticulant et porogènes/monomères. Il présente une perméabilité relativement élevée et une bonne stabilité mécanique et chimique. Différentes chimies d'immobilisation, le temps d'immobilisation et la concentration en O-glycosidase ont également été testés. Les O-glycanes libérés de la fétuine ou l'asialofétuine ont ensuite été marqués avec l'APTS en tubes puis analysés par EC-LIF. Pour la première fois, une O-glycosidase active est immobilisée sur un support solide. La digestion in-IMER de l'asialofétuine a montré une efficacité similaire à celle de la digestion en solution (rendement de libération de 56%) mais avec une vitesse plus grande (temps de contact < 20 s). Dans la deuxième partie expérimentale, une stratégie de préconcentration électrocinétique a été étudiée pour augmenter la sensibilité de détection des N- et O-glycanes par EC-LIF. J'ai participé à une étude menée par un autre doctorant du laboratoire en appliquant ces optimisations aux O-glycanes. En utilisant de nouveaux électrolytes combinés à l’injection d’un grand volume d'échantillon par modulation du flux électroosmotique, un facteur d'enrichissement de ~ 200 a été obtenu pour différentes glycoprotéines (IgG, fétuine, érythropoïétine) et quel que soit le type (N-, O-) de glycanes.La troisième partie expérimentale présente le marquage fluorescent en ligne des N-glycanes avec l'APTS suivi de leur analyse par EC-LIF. Une stratégie de mélange basée sur la diffusion transversale des profils d'écoulement laminaire a été exploitée pour réaliser ce marquage à l'intérieur du capillaire de séparation. Après optimisation des paramètres de mélange, l'approche en capillaire a été appliquée avec succès au marquage et à la cartographie des N-glycanes issus de l'immunoglobuline G (IgG) humaine et de l'anticorps monoclonal Rituximab.En conclusion, ce travail de thèse a contribué à développer des méthodes innovantes pour la libération de O-glycanes en microréacteur, le marquage fluorescent en ligne de N-glycanes, leur séparation et leur préconcentration par EC. Toutes les étapes ont été réalisées au sein d'un capillaire en silice et sont donc intégrables pour développer un microsystème dédié à l'analyse des glycanes
Glycosylation is one of the most critical post-translational modifications of proteins. Detecting minor modifications of protein glycosylation can help identify new diagnosis biomarkers or control the quality of biotherapeutics. The glycan mapping is one of the most efficient approaches to detecting glycosylation modifications but the workflow is still limited by the high number and time-consuming steps required to achieve it. These steps entail the glycan release, labeling, and profiling by a separation technique. These manual operations can induce interferences, provoking reproducibility issues. Miniaturization and automation of the workflow are still needed but remain challenging. For instance, devices developed for glycan release or glycan labeling kits focus more on N- than on O-glycans. Neither the O-glycan release in an immobilized enzyme reactor (IMER) nor the in-capillary labeling of glycans with the 8-aminopyrene-1,3,6-trisulfonic acid (APTS) has been reported until now. My thesis project aims at contributing to the development of a microsystem, integrating the glycan release, their online fluorescent labeling, and their separation by capillary electrophoresis (CE) coupled with laser-induced fluorescence (LIF) detection.The first experimental part describes the development of an IMER based on a photo-polymerized monolith to release O-glycans from glycoproteins. A 10 mm poly (glycidyl methacrylate-co-poly (ethylene glycol) diacrylate) monolith was synthesized in a capillary after optimizing the monomer/crosslinker and porogens/monomers ratios. The optimized monolith showed a relatively high permeability and good mechanical and chemical stability. Different immobilization chemistries, immobilization time and O-glycosidase enzyme concentration were also tested. The O-glycans released from fetuin or asialofetuin were then offline fluorescently labeled with APTS and analyzed by CE-LIF. For the first time, an active O-glycosidase was immobilized on a solid support. The in-IMER digestion of asialofetuin provided a similar digestion efficiency to the in-solution one (releasing yield of 56%) and brought speed with a residence time for digestion of less than 20 s.In the second experimental part, an electrokinetic preconcentration strategy was investigated to increase the CE-LIF detection sensitivity of N- and O-glycans. I participated in the thesis study conducted by another PhD student in the lab by applying these optimizations to O-glycans. By using new background electrolytes combined with large volume sample injection via electroosmotic flow modulation, an enrichment factor of ~ 200 was obtained for different glycoproteins (IgG, fetuin, erythropoietin) whatever the type (N-, O-) of glycans.The third experimental part reports the online fluorescent labeling of N-glycans with APTS, followed by their subsequent online CE-LIF analysis. A mixing strategy based on the transverse diffusion of laminar flow profiles was exploited to achieve this labeling inside the separation capillary. After an in-depth optimization of the mixing parameters, the in-capillary approach was successfully applied to the labeling and mapping of N-glycans from human immunoglobulin G (IgG) and monoclonal antibody Rituximab.In conclusion, this thesis work contributed to developing innovative methods for O-glycan release, N-glycan online labeling, separation, and preconcentration by CE. All the steps were performed within a silica capillary and are therefore amenable to further integration for developing a microsystem dedicated to glycan analysis

Thesis (Ph. D.)--University of Illinois at Urbana-Champaign, 2006.
Source: Dissertation Abstracts International, Volume: 68-02, Section: B, page: 0941. Adviser: Jonathan V. Sweedler. Includes bibliographical references. Available on microfilm from Pro Quest Information and Learning.

Reduction in the capillary density in the nailfold region is frequently observed in patients suffering from Hypertension (Feng J, 2010). Loss of capillaries results in avascular regions which have been well characterized in many diseases (Mariusz, 2009). Nailfold capillary images need to be pre-processed so that noise can be removed, background can be separated and the useful parameters may be computed using image processing algorithms. Smoothing filters such as Gaussian, Median and Adaptive Median filters are compared using Mean Squared Error and Peak Signal-to-Noise Ratio. Otsu's thresholding is employed for segmentation. Connected Component Labeling algorithm is applied to calculate the number of capillaries per mm. This capillary density is used to identify rarefaction of capillaries and also the severity of rarefaction. Avascular region is detected by determining the distance between the peaks of the capillaries using Euclidian distance. Detection of rarefaction of capillaries and avascular regions can be used as a diagnostic tool for Hypertension and various other diseases.

Rapid, automated sample preparation of bacterial cells and spores is required for threat analysis by remotely deployed chemical and biological warning systems. Sandia is designing, building, and testing an automated front-end sample preparation system based on miniature and microfluidic components, with the goal of concentrating bacterial species collected from the air, harvesting and solubilizing proteins from them, and delivering them to Sandia’s MicroChemLab capillary gel electrophoresis system1,2 for analysis (Fig. 1). Miniature, motorized valves and pumps control flow between system components connected by fused silica capillaries (Fig. 4). Sample processing modules include concentration by dielectrophoresis in an array of insulating posts or by mechanical filtration; heat-activated chemical lysis; mechanical filtration; removal of chemical lysis agents by size exclusion chromatography (SEC); and in-capillary fluorescent labeling.

Abstract During miscible gas injection for enhanced oil recovery, the composition of the fluids can change throughout the reservoir as the oil and gas phases develop miscibility. Measuring and modeling relative permeability as compositional regions are traversed creates many challenges. In simulators, the association of each phase with a relative permeability curve sometimes creates discontinuities when phases disappear across miscibility boundaries. Some newer relative permeability models attempt to resolve these issues by changing the standard "oil" and "gas" method of phase labeling and instead labeling phases according to a physical property that is continuous and tied to composition, most notably the fluid density or Gibbs free energy (GFE). Ideally, a relative permeability model will be based on experimental measurements. A handful of all relative permeability experiments focus on studying changes in relative permeability brought about by changes in fluid composition with increasing capillary number. However, there is also evidence to suggest that composition can impact relative permeability even at capillary numbers well below the capillary desaturation threshold. In this research, two-phase gas/oil core flood experiments were performed with ethane as the gas phase and equilibrated octane as the oil phase. Pressure was varied so that the composition (density and GFE) of the gas and oil were changing. The capillary numbers were kept low and constant to prevent capillary desaturation of the oil phase. The experiments were then repeated with an added residual brine phase to test the effect of composition with a third phase present. The results show that changing the density and GFE of the oil and gas phases in either two-phase or three-phase flow had no impact on the relative permeability curves. However, significant changes were observed when comparing two-phase to three-phase oil and gas relative permeabilities. When only gas and oil were flowing in the core, the oil phase formed a continuous layer on the pore surfaces. The addition of residual brine caused the oil to form droplets, reducing the relative permeability of both the oil and gas phases in the absence of a continuous layer of oil. These findings verify previous history-matched relative permeabilities in literature and show that the oil phase connectivity is more important than compositional parameters.

Abstract Hysteresis of transport properties like relative permeability (Kr) can lead to computational problems and inaccuracies for various applications including CO2 sequestration and chemical enhanced oil recovery (EOR). Computational problems in multiphase numerical simulation include phase labeling issues and path dependencies that can create discontinuities. To mitigate hysteresis, modeling Kr as a state function that honors changes in physical parameters like wettability is a promising solution. In this research, we apply the state function concept to develop a physics-informed data-driven approach for predicting Kr in the space of its state parameters. We extend the development of the relative permeability equation-of-state (kr-EoS) to create a predictive physics-based model using Artificial Neural Networks (ANN). We predict kr as a function of phase saturation (S) and phase connectivity (χ^), as well as the specific S−χ^ path taken during the displacement, while maintaining other state parameters constant such as wettability, pore structure, and capillary number. We use numerical data generated from pore-network simulations (PNM) to test the predictive capability of the EoS. Physical limits within S−χ^ space are used to constrain the model and improve its predictability outside of the region of measured data. We find that the predicted relative permeabilities result in a smooth and physically consistent estimate. Our results show that ANN can more accurately estimate kr surface compared to using a high-order polynomial response surface. With only a limited amount of drainage and imbibition data with an initial phase saturation greater than 0.7, we provide a good prediction of kr from ANN for all other initial conditions, over the entire S−χ^ space. Finally, we show that we can predict the specific path taken in the S−χ^ space along with the corresponding kr for any initial condition and flow direction, which makes the approach practical when phase connectivity information is not available. This research demonstrates the first application of a physics-informed data-driven approach for prediction of relative permeability using ANN.

In this study we show that bovineadrenal capillary endothelial cells(BACE) contain heparin (HP); this HP has been found associated with the cell surface (i.e; trypsin-removable^and intracellularly. How-ever, experiments with [ sjsodium sulfate labelling, demonstrate that BACE cells donot synthesize HP de novo, but they uptake it from serum. We have studied binding, uptake, and metabolism odifferent molecular weight-HPs: 13 Kd-HP from bovine source, 14 Kd-HP from porcine source, 4.5 Kd, and 2.5-HP fragments. Comparison among different HPs, was carried out by calculating the IC from competition curves for [3HJ- HP. Binding of labelled-HP to BACE cells was specificand saturable. Dextran sulfate and glycosaminoglycans did not compete for binding; only heparan sulfate showed some competition. Binding of different HPs was strictly dependent on their molecular weight; 2.5 Kd- HP was unable to bind to cells, although sulfation degree of this fragment and of unfractionated HP was almost identical. Therefore, we assume that a specific oligosaccharide sequence could be responsible for HP binding to BACE cells; this hypothetical "binding sequence" could then be lost in very low molecular weight-HP fragments. BACE cells are also able to internalize HP, and they release its low molecular weight degradation products into culture medium. Thus we suggest that endothelial cells might represent a site for the metabolism of endogenous and exogenous HP in vivo.