Intravital FRAP imaging using an E-cadherin-GFP mouse reveals disease- and drug-dependent dynamic regulation of cell-cell junctions in live tissue

E-cadherin-mediated cell-cell junctions play a prominent role in maintaining the epithelial architecture. The disruption or deregulation of these adhesions in cancer can lead to the collapse of tumor epithelia that precedes invasion and subsequent metastasis. Here we generated an E-cadherin-GFP mouse that enables intravital photobleaching and quantification of E-cadherin mobility in live tissue without affecting normal biology. We demonstrate the broad applications of this mouse by examining E-cadherin regulation in multiple tissues, including mammary, brain, liver, and kidney tissue, while specifically monitoring E-cadherin mobility during disease progression in the pancreas. We assess E-cadherin stability in native pancreatic tissue upon genetic manipulation involving Kras and p53 or in response to anti-invasive drug treatment and gain insights into the dynamic remodeling of E-cadherin during in situ cancer progression. FRAP in the E-cadherin-GFP mouse, therefore, promises to be a valuable tool to fundamentally expand our understanding of E-cadherin-mediated events in native microenvironments

Test 1 analyser for determination of ESR. 1. Practical evaluation and comparison with the Westergren technique

various modifications of the Erythrocyte Sedimentation Rate (ESR) determination have been suggested since the original Westergren procedure that has been adopted as the gold standard by the International Council for Standardization in Haematology (ICSH). Recently, an automated method, (Alifax Test 1), based on a technique completely different from Westergren, has been introduced. In this comparative study, ESR of blood from 680 patients with various rheumatic diseases was determined on both Test 1 and the StaRRsed automated ESR analyser which performs measurements in accordance with ICSH specifications. Furthermore the robustness of the new technique was evaluated. Direct correlation of Test 1 and StaRRsed measurements confirmed the results of previous studies: an overall correlation coefficient of R = 0.90. However, further statistical analysis showed that, depending on the instrument that was used, in 78 samples (i.e. 11.5%) the results could lead to different treatment suggestions. Furthermore it appeared that several procedural factors could influence the final Test 1 outcome. Due to its sensitivity for procedural variations, Test 1 measurements should be carried out under strictly standardized conditions. Especially at the higher ESR levels the Test 1 technique is, however, not a reliable alternative for the ICSH approved 'Westergren' metho

Photometric measurements of red blood cell aggregation: light transmission versus light reflectance

Red blood cell (RBC) aggregation is the reversible and regular clumping in the presence of certain macromolecules. This is a clinically important phenomenon, being significantly enhanced in the presence of acute phase reactants (e.g., fibrinogen). Both light reflection (LR) and light transmission (LT) from or through thin layers of RBC suspensions during the process of aggregation are accepted to reflect the time course of aggregation. It has been recognized that the time courses of LR and LT might be different from each other. We aim to compare the RBC aggregation measurements based on simultaneous recordings of LR and LT. The results indicate that LR during RBC aggregation is characterized by a faster time course compared to simultaneously recorded LT. This difference in time course of LR and LT is reflected in the calculated parameters reflecting the overall extent and kinetics of RBC aggregation. Additionally, the power of parameters calculated using LR and LT time courses in detecting a given difference in aggregation are significantly different from each other. These differences should be taken into account in selecting the appropriate calculated parameters for analyzing LR or LT time courses for the assessment of RBC aggregatio

Laser-assisted optical rotational cell analyzer measurements reveal early changes in human RBC deformability induced by photodynamic treatment

BACKGROUND: The ability to deform is important for circulating RBCs in vivo, and earlier studies showed that this property can objectively be measured in vitro by the LORCA. In this study it was investigated whether photodynamic treatment of human RBCs (meant to inactivate contaminating pathogens) affects deformability STUDY DESIGN AND METHODS: WBC-reduced RBC suspensions (30% Hct) were treated with 1,9-dimethylmethylene blue (DMMB) and red light. Changes in deformability were analyzed by LORCA measurements, in which elongation of the cells is measured at increasing shear stress. The effect of DMMB concentration and light dose was determined as well as the interfering effect of two scavengers of reactive oxygen species, that is, dipyridamole and Trolox. RESULTS: Photodynamic treatment with DMMB resulted in clear changes in RBC deformability. Deformability changes occurred before onset of hemolysis. Under relatively mild treatment conditions, especially deformability at low shear stress was decreased, whereas deformability changes at high shear stress only occurred under harsher treatment conditions. Inclusion of dipyridamole and/or Trolox primarily prevented deformability changes at high shear stress. CONCLUSION: LORCA measurements can effectively be used to detect changes in deformability that are induced by photodynamic treatment of human RBCs. A change in deformability represents an early marker of RBC damage under these condition

Small artery remodeling and erythrocyte deformability in L-NAME-induced hypertension: Role of transglutaminases

Background: Hypertension is associated with inward remodeling of small arteries and decreased erythrocyte deformability, both impairing proper tissue perfusion. We hypothesized that these alterations depend on transglutaminases, cross-linking enzymes present in the vascular wall, monocytes/macrophages and erythrocytes. Methods and Results: Wild-type (WT) mice and tissue-type transglutaminase (tTG) knockout (KO) mice received the nitric oxide inhibitor N omega- nitro-L-arginine methyl ester hydrochloride (L-NAME) to induce hypertension. After 1 week, mesenteric arteries from hypertensive WT mice showed a smaller lumen diameter (-6.9 +/- 2.0%, p = 0.024) and a larger wall-to-lumen ratio (11.8 +/- 3.5%, p = 0.012) than controls, whereas inward remodeling was absent in hypertensive tTG KO mice. After 3 weeks, the wall-to-lumen ratio was increased in WT (20.8 +/- 4.8%, p = 0.005) but less so in tTG KO mice (11.7 +/- 4.6%, p = 0.026), and wall stress was normalized in WT but not in tTG KO mice. L-NAME did not influence expression of tTG or an alternative transglutaminase, coagulation factor XIII (FXIII). Suppression of FXIII by macrophage depletion was associated with increased tTG in the presence of L-NAME. L-NAME treatment decreased erythrocyte deformability in the WT mice (-15.3% at 30 dynes/cm(2), p = 0.014) but not in the tTG KO mice. Conclusion: Transglutaminases are involved in small artery inward remodeling and erythrocyte stiffening associated with nitric oxide inhibition-related hypertensio

Parameterization of red blood cell elongation index--shear stress curves obtained by ektacytometry

Measurement of red blood cell (RBC) deformability by ektacytometry yields a set of elongation indexes (EI) measured at various shear stresses (SS) presented as SS-EI curves, or tabulated data. These are useful for detailed analysis, but may not be appropriate when a simple comparison of a global parameter between groups is required. Based on the characteristic shape of SS-EI curves, two approaches have been proposed to calculate the maximal RBC elongation index (EI(max)) and the shear stress required for one-half of this maximal deformation (SS(1/2)): (i) linear Lineweaver-Burke (LB) model; (ii) Streekstra-Bronkhorst (SB) model. Both approaches have specific assumptions and thus may be subject to the measurement conditions. Using RBC treated with various concentrations of glutaraldehyde (GA) and data obtained by ektacytometry, the two approaches have been compared for nine different ranges of SS between 0.6-75 Pa. Our results indicate that: (i) the sensitivity of both models can be affected by the SS range and limits employed; (ii) over the entire range of SS-data, a non-linear curve fitting approach to the LB model gave more consistent results than a linear approach; (iii) the LB method is better for detecting SS(1/2) differences between RBC treated with 0.001-0.005% glutaraldehyde (GA) and for a 40% mixture of rigid cells but is equally sensitive to SB for 10% rigid cells; and (iv) the LB and SB methods for EI(max) are equivalent for 0.001% and 0.003% GA and 40% rigid, with the SB better for 0.005% GA and the LB better for 10% rigi

Comparison of three commercially available ektacytometers with different shearing geometries

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