Contribution of perfusion techniques to the evaluation of the hemostatic effectiveness of platelet concentrates.

Perfusion systems allowing the morphometric analysis of platelet interactions with vessel subendothelium under flow conditions have been applied to evaluate the quality and function of stored platelets. Studies performed in vitro indicate that despite the existence of storage lesions, platelets in concentrates stored for up to 5 days retain their ability to interact with the subendothelium. Perfusion studies ex vivo with nonanticoagulated blood from anemic-thrombocytopenic patients have shown the critical hemorrheological role of red blood cells facilitating platelet interactions with subendothelium. Similar studies performed on severely thrombocytopenic patients who received transfusions of platelets stored at 4 degrees C indicate that incompletely viable platelets can contribute to primary hemostasis through procoagulant mechanisms. The latter results suggest that storage lesions which contribute to impairment of platelet function may result in enhancement of platelet procoagulant activities. Perfusion techniques have contributed to the evaluation of the hemostatic effectiveness of platelet concentrates. These techniques will provide a useful model to test the impact of new storage technologies on platelet hemostatic function.     

Differential protein expression in endothelial cells exposed to serum from patients with acute graft-vs-host disease,depending on steroid response

Graft-versus-host disease (GVHD) is a complication of allogeneic haematopoietic cell transplantation. Endothelial injury is crucial as pathophysiological substrate for GVHD. GVHD first-line treatment is high-dose corticosteroids, although some patients are steroid-refractory. Through the present study, we compared the endothelial proteomic profiles in response to serum from steroid-refractory acute GVHD (SR-aGVHD) and steroid-sensitive acute GVHD (SS-aGVHD) patients. Blood samples from SR-aGVHD (n = 4) and SS-aGVHD (n = 8) patients were collected at aGVHD diagnosis. Endothelial cell cultures were exposed (48 h) to patients' serum. Protein extraction and proteomic analysis were performed. Differences were statistically evaluated by multivariate analysis. Forty-four proteins contributed to separate all samples into the two study groups, among which 15 participated significantly (p < 0.05), 10 exhibiting a fold change >1.2. Differentially expressed proteins were mainly associated with oxidative phosphorylation (Cytochrome C oxidase subunit 6B1, CX6B1), inflammation and angiogenesis (Apolipoprotein D, APOD), cell survival (Rapamycin-insensitive companion of mTOR, RICTR), and oxidative stress (Riboflavin kinase, RIFK). This pilot study used a novel approach to distinguish the aGVHD response to steroid treatment. The proteins differentially expressed could constitute potential biomarkers for steroid-treatment response. These findings signify a step forward to identify the mechanisms of response to steroids, of high clinical relevance considering the SR-aGVHD elevated mortality.     

The development of wingless,a homeotic mutation of Drosophila

The mutation wingless produces a homeotic transformation in which the distal structures (appendages) of both the wing and haltere discs are replaced by a duplication of the proximal structures (thorax). However, not all of the mutant discs show mutant phenotype; some of them differentiate normal appendages. Gynandromorph and clonal analyses suggest that the phenotype does not result from massive cell death followed by regeneration and/or duplication. We conjecture that the mutant phenotype is caused by a specific failure in the process of compartmentalization. In contrast to other homeotic mutants, wingless is not cell autonomous; that is, mutant clones show wildtype phenotype when produced in wildtype wings.     

Pharmacological dose of melatonin reduces cytosolic calcium load in response to cholecystokinin in mouse pancreatic acinar cells

Intracellular Ca²⁺ overload has been considered a common pathological precursor of pancreatic injury. In this study, the effects of melatonin on Ca²⁺ mobilization induced by cholecystokinin octapeptide (CCK-8) in freshly isolated mouse pancreatic acinar cells have been examined. Changes in intracellular free Ca²⁺ concentration were followed by single cell fluorimetry. For this purpose, cells were loaded with the Ca²⁺-sensitive fluorescent dye fura-2-acetoxymethyl ester. In order to evaluate the contribution of Ca²⁺ transport at the plasma membrane, at the endoplasmic reticulum (ER) or at the mitochondria, cells were incubated with CCK-8 alone or in combination with LaCl₃, thapsigargin (Tps), or FCCP to, respectively, uncouple Ca²⁺ transport at these localizations. The experiments were performed in the absence or in the presence of melatonin in combination with the stimuli mentioned. Our results show that the total Ca²⁺ mobilization evoked by CCK-8 was attenuated by a 30 % in the presence of 100 µM melatonin compared with the responses induced by CCK-8 alone. Upon inhibition of Ca²⁺ transport into the ER by Tps, Ca²⁺ mobilization was also reduced in the presence of melatonin. In the presence of LaCl₃ plus melatonin, the total Ca²⁺ mobilization induced by CCK-8 was significantly decreased, compared with the response obtained without melatonin but in the presence of LaCl₃. No major differences were found when the cells were incubated with CCK-8 or Tps alone or in combination with LaCl₃ plus melatonin and FCCP, compared with the responses obtained in the absence of FCCP. The initial Ca²⁺ release from intracellular stores evoked by CCK-8 or Tps was not significantly reduced in the presence of melatonin. The effect of melatonin could be explained on the basis of a stimulated Ca²⁺ transport out of the cell through the plasma membrane and by a stimulation of Ca²⁺ reuptake into the ER. Accumulation of Ca²⁺ into mitochondria might not be a major mechanism stimulated by melatonin. We conclude that melatonin alleviates intracellular Ca²⁺ accumulation, a situation potentially leading to cell damage in the exocrine pancreas.     

Differential localization of von Willebrand factor,fibronectin and 13-HODE in human endothelial cell cultures

Summary Von Willebrand factor (vWF), fibronectin (FN) and 13-hydroxy-octadecadienoic acid (13-HODE) are known to influence the regulation of the adhesive properties of vascular surfaces. In the present study vWF, FN and 13-HODE were comparatively localized in endothelial cells (EC) and in the extracellular matrix (ECM) produced by EC. An indirect immunofluorescent technique was applied to coverslips containing human EC cultures previously fixed and permeabilized following different procedures: A. Alcohol/acetone; B. Paraformaldehyde alone and C. Paraformaldehyde followed by Triton X-100. vWF was observed inside EC (A), on the ECM produced by EC (B) or in EC and ECM (C) depending on the fixation procedures used. FN was mainly localized in the ECM despite the fixation procedures employed. FN was only seen in relation to cell bodies after strong permeabilization (A). Under our experimental conditions 13-HODE was never found in ECM. This latter antigen was observed randomly dispersed in those preparations fixed with alcohol/acetone, indicating that it is probably extracted by this fixative. 13-HODE was detected in granular shaped structures in EC after permeabilization with detergent (C). These results suggest that the cellular localization of vWF and FN is compatible with an adhesive role related to the abluminal side of ECs. 13-HODE was readily observed after mild permeabilization. This finding would be morphologically consistent with its contribution to the regulation of the vessel wall thromboresistance.     

Glucanase gene diversity in prokaryotic and eukaryotic organisms

A number of bacteria and eukaryotes produce extracellular enzymes that degrade various types of polysaccharides including the glucans starch, cellulose and hemicellulose (xylan). The similarities in the modes of expression and specificity of enzyme classes, such as amylase, cellulose and xylanase, suggest common genetic origins for particular activities. Our determination of the extent of similarity between these glucanases suggests that such data may be of very limited use in describing the early evolution of these proteins. The great diversity of these proteins does allow identification of their most highly conserved (and presumably functionally important) regions.