Ischaemiás-reperfusiós károsodások funkcionális és strukturális változásainak kivédése különböző (máj, vékonybél, szabad lebeny, amputatum) experimentalis sebészi modelleken = Protection of functional and structural changes of ischemia-reperfusion injuries on different experimental surgical models (liver, small bowel, free flap, limb)

A kutatás fő célkitűzései különböző szerveken végzett sebészi beavatkozások során az érleszorítások ischaemia-reperfusió (I/R) okozta károsodásainak vizsgálata volt megfelelő protektív módszerek kidolgozásához az esetleges klinikai szövődmények felismeréséhez nyolc kísérleti modellben. Főbb eredményeink: 1. Felhívtuk a figyelmet máj, vékonybél, musculus latissimus dorsi lebeny, amputatum és arterio-venosus (A-V) shunt modellekben arra, hogy a lokális és szisztémás keringés haemorheologiai paramétereinek -vvs deformálhatóság és vvs aggregatio- változásai az I/R károsodások korai jelzői lehetnek, melyeket szövettani, immunhisztokémiai és rutin kémiai vizsgálatok eredményei is alátámasztottak. 2. Máj modellnél megerősítettük a Báron manőver -a lig. hepatoduodenale leszorítás- jogosultságát, az egy vagy kétszeri alkalmazásának preventív hatását, mivel a további leszorítások a korai postoperatív szövődmények kockázatát növelik. 3. Az A-V shunt modell eredmények a haemodialysisre kerülő betegek hosszabb ideig biztosítható ""nyitva tartásához"", a kezelés folytatásához adtak biztató adatokat. 4. A musculus latissimus dorsi lebeny eredményei a szabadlebeny átültetések biztonságát növelheti a korai postoperatív időszakban, a plasztikai/rekonstrukciós sebészetben. | The main objectives of the research were in eight experimental models to investigate the ischemia-reperfusion (I/R) injuries due to vascular clamping during surgical interventions on various organs in order to develop proper protecting methods for recognizing the possible clinical complications. Our main findings are the followings: 1. We attracted attention in liver, small bowel, latissimus dorsi muscle (LDM) flap, amputate and arterio-venosus (A-V) shunt models that the changes of the systemic and local hemorheological parameters -RBC deformability and RBC aggregation- can be the early signs of I/R injuries, which was also supported by histological, immunohistochemical and routine chemical test results. 2. In case of the liver model the utility of the Báron’s manoeuvre -clamping of the hepatoduodenal ligament- was strengthened, with its preventive effect as applied one or two times, since further clamping periods raise the risk of early postoperative complications. 3. The results of the A-V shunt model supplied reassuring data for the management of longer lifetime shunts and for the treatment continuance in patients undergoing hemodialysis. 4. The results of the LDM flap model can increase the safety of free-flap transplantations in the early postoperative period in the plastic/reconstructive surgery

Isolation of Exosomes from Blood Plasma: Qualitative and Quantitative Comparison of Ultracentrifugation and Size Exclusion Chromatography Methods

BACKGROUND: Exosomes are emerging targets for biomedical research. However, suitable methods for the isolation of blood plasma-derived exosomes without impurities have not yet been described. AIM: Therefore, we investigated the efficiency and purity of exosomes isolated with potentially suitable methods; differential ultracentrifugation (UC) and size exclusion chromatography (SEC). METHODS AND RESULTS: Exosomes were isolated from rat and human blood plasma by various UC and SEC conditions. Efficiency was investigated at serial UC of the supernatant, while in case of SEC by comparing the content of exosomal markers of various fractions. Purity was assessed based on the presence of albumin. We found that the diameter of the majority of isolated particles fell into the size range of exosomes, however, albumin was also present in the preparations, when 1h UC at 4 degrees C was applied. Furthermore, with this method only a minor fraction of total exosomes could be isolated from blood as deduced from the constant amount of exosomal markers CD63 and TSG101 detected after serial UC of rat blood plasma samples. By using UC for longer time or with shorter sedimentation distance at 4 degrees C, or UC performed at 37 degrees C, exosomal yield increased, but albumin impurity was still observed in the isolates, as assessed by transmission electron microscopy, dynamic light scattering and immunoblotting against CD63, TSG101 and albumin. Efficiency and purity were not different in case of using further diluted samples. By using SEC with different columns, we have found that although a minor fraction of exosomes can be isolated without significant albumin content on Sepharose CL-4B or Sephacryl S-400 columns, but not on Sepharose 2B columns, the majority of exosomes co-eluted with albumin. CONCLUSION: Here we show that it is feasible to isolate exosomes from blood plasma by SEC without significant albumin contamination albeit with low vesicle yield

Differential detergent sensitivity of extracellular vesicle subpopulations

This work shows for the first time that exosomes are more resistant to detergents than microvesicles and apoptotic bodies.</p

Tubulin Binds to the Cytoplasmic Loop of TRESK Background K+ Channel In Vitro.

The cytoplasmic loop between the second and third transmembrane segments is pivotal in the regulation of TRESK (TWIK-related spinal cord K+ channel, K2P18.1, KCNK18). Calcineurin binds to this region and activates the channel by dephosphorylation in response to the calcium signal. Phosphorylation-dependent anchorage of 14-3-3 adaptor protein also modulates TRESK at this location. In the present study, we identified molecular interacting partners of the intracellular loop. By an affinity chromatography approach using the cytoplasmic loop as bait, we have verified the specific association of calcineurin and 14-3-3 to the channel. In addition to these known interacting proteins, we observed substantial binding of tubulin to the intracellular loop. Successive truncation of the polypeptide and pull-down experiments from mouse brain cytosol narrowed down the region sufficient for the binding of tubulin to a 16 amino acid sequence: LVLGRLSYSIISNLDE. The first six residues of this sequence are similar to the previously reported tubulin-binding region of P2X2 purinergic receptor. The tubulin-binding site of TRESK is located close to the protein kinase A (PKA)-dependent 14-3-3-docking motif of the channel. We provide experimental evidence suggesting that 14-3-3 competes with tubulin for the binding to the cytoplasmic loop of TRESK. It is intriguing that the 16 amino acid tubulin-binding sequence includes the serines, which were previously shown to be phosphorylated by microtubule-affinity regulating kinases (MARK kinases) and contribute to channel inhibition. Although tubulin binds to TRESK in vitro, it remains to be established whether the two proteins also interact in the living cell

Systematic transcriptomic and phenotypic characterization of human and murine cardiac myocyte cell lines and primary cardiomyocytes reveals serious limitations and low resemblances to adult cardiac phenotype

Background Cardiac cell lines and primary cells are widely used in cardiovascular research. Despite increasing number of publications using these models, comparative characterization of these cell lines has not been performed, therefore, their limitations are undetermined. We aimed to compare cardiac cell lines to primary cardiomyocytes and to mature cardiac tissues in a systematic manner. Methods and results Cardiac cell lines (H9C2, AC16, HL-1) were differentiated with widely used protocols. Left ventricular tissue, neonatal primary cardiomyocytes, and human induced pluripotent stem cell-derived cardiomyocytes served as reference tissue or cells. RNA expression of cardiac markers (e.g. Tnnt2, Ryr2) was markedly lower in cell lines compared to references. Differentiation induced increase in cardiac- and decrease in embryonic markers however, the overall transcriptomic profile and annotation to relevant biological processes showed consistently less pronounced cardiac phenotype in all cell lines in comparison to the corresponding references. Immunocytochemistry confirmed low expressions of structural protein sarcomeric alpha-actinin, troponin I and caveolin-3 in cell lines. Susceptibility of cell lines to sI/R injury in terms of viability as well as mitochondrial polarization differed from the primary cells irrespective of their degree of differentiation. Conclusion Expression patterns of cardiomyocyte markers and whole transcriptomic profile, as well as response to sI/R, and to hypertrophic stimuli indicate low-to-moderate similarity of cell lines to primary cells/cardiac tissues regardless their differentiation. Low resemblance of cell lines to mature adult cardiac tissue limits their potential use. Low translational value should be taken into account while choosing a particular cell line to model cardiomyocytes

Validation of the United Kingdom copy-number alteration classifier in 3239 children with B-cell precursor ALL

Genetic abnormalities provide vital diagnostic and prognostic information in pediatric acute lymphoblastic leukemia (ALL) and are increasingly used to assign patients to risk groups. We recently proposed a novel classifier based on the copy-number alteration (CNA) profile of the 8 most commonly deleted genes in B-cell precursor ALL. This classifier defined 3 CNA subgroups in consecutive UK trials and was able to discriminate patients with intermediate-risk cytogenetics. In this study, we sought to validate the United Kingdom ALL (UKALL)-CNA classifier and reevaluate the interaction with cytogenetic risk groups using individual patient data from 3239 cases collected from 12 groups within the International BFM Study Group. The classifier was validated and defined 3 risk groups with distinct event-free survival (EFS) rates: good (88%), intermediate (76%), and poor (68%) (P < .001). There was no evidence of heterogeneity, even within trials that used minimal residual disease to guide therapy. By integrating CNA and cytogenetic data, we replicated our original key observation that patients with intermediate-risk cytogenetics can be stratified into 2 prognostic subgroups. Group A had an EFS rate of 86% (similar to patients with good-risk cytogenetics), while group B patients had a significantly inferior rate (73%, P < .001). Finally, we revised the overall genetic classification by defining 4 risk groups with distinct EFS rates: very good (91%), good (81%), intermediate (73%), and poor (54%), P < .001. In conclusion, the UKALL-CNA classifier is a robust prognostic tool that can be deployed in different trial settings and used to refine established cytogenetic risk groups

Mast cell secretome: Soluble and vesicular components

Mast cells are multifunctional master cells implicated in both innate and adaptive immune responses. Their role has been best characterized in allergy and anaphylaxis; however, emerging evidences support their contribution to a wide variety of human diseases. Mast cells, being capable of both degranulation and subsequent recovery, have recently attracted substantial attention as also being rich sources of secreted extracellular vesicles (including exosomes and microvesicles). Along with secreted de novo synthesized soluble molecules and secreted preformed granules, the membrane-enclosed extracellular vesicles represent a previously unexplored part of the mast cell secretome. In this review article we summarize available data regarding the different soluble molecules and membrane-enclosed structures secreted by mast cells. Furthermore, we provide an overview of the release mechanisms including degranulation, piecemeal degranulation, transgranulation, and secretion of different types of extracellular vesicles. Finally, we aim to give a summary of the known biological functions associated with the different mast cell-derived secretion products. The increasingly recognized complexity of mast cell secretome may provide important novel clues to processes by which mast cells contribute to the development of different pathologies and are capable of orchestrating immune responses both in health and disease