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31.
Blockade of Thrombopoietin Reduces Organ Damage in Experimental Endotoxemia and Polymicrobial Sepsis
Alessandra Cuccurullo Elisabetta Greco Enrico Lupia Paolo De Giuli Ornella Bosco Erica Martin-Conte Tiziana Spatola Emilia Turco Giuseppe Montrucchio 《PloS one》2016,11(3)
Background and Purpose
Thrombopoietin (TPO), a growth factor primarily involved in thrombopoiesis may also have a role in the pathophysiology of sepsis. In patients with sepsis, indeed, TPO levels are markedly increased, with disease severity being the major independent determinant of TPO concentrations. Moreover, TPO increases and correlates with ex vivo indices of platelet activation in patients with burn injury upon sepsis development, and may contribute to depress cardiac contractility in septic shock. Still, the role of TPO in sepsis pathophysiology remains controversial, given the protective role of TPO in other experimental disease models, for instance in doxorubicin-induced cardiotoxicity and myocardial ischemia/reperfusion injury. The aim of our study was to define the contribution of TPO in the development of organ damage induced by endotoxemia or sepsis, and to investigate the effects of inhibiting TPO in these conditions.Methods
We synthesized a chimeric protein able to inhibit TPO, mTPOR-MBP, and studied its effect in two murine experimental models, acute endotoxemia and cecal ligation and puncture (CLP) model.Results
In both models, TPO levels markedly increased, from 289.80±27.87 pg/mL to 465.60±45.92 pg/mL at 3 hours in the LPS model (P<0.01), and from 265.00±26.02 pg/mL to 373.70±26.20 pg/mL in the CLP model (P<0.05), respectively. Paralleling TPO levels, also platelet-monocyte aggregates increased, from 32.86±2.48% to 46.13±1.39% at 3 hours in the LPS model (P<0.01), and from 43.68±1.69% to 56.52±4.66% in the CLP model (P<0.05). Blockade of TPO by mTPOR-MBP administration reduced histological damage in target organs, namely lung, liver, and gut. In particular, neutrophil infiltration and lung septal thickening were reduced from a score of 1.86±0.34 to 0.60±0.27 (P<0.01) and from 1.43±0.37 to 0.40±0.16 (P<0.05), respectively, in the LPS model at 3 hours, and from a score of 1.75±0.37 to 0.38±0.18 (P<0.01) and from 1.25±0.31 to 0.13±0.13 (P<0.001), respectively, in the CLP model. Similarly, the number of hepatic microabscesses was decreased from 14.14±1.41 to 3.64±0.56 in the LPS model at 3 hours (P<0.001), and from 1.71±0.29 to 0.13±0.13 in the CLP model (P<0.001). Finally, the diameter of intestinal villi decreased from 90.69±3.95 μm to 70.74±3.60 μm in the LPS model at 3 hours (P<0.01), and from 74.29±4.29 μm to 57.50±1.89 μm in the CLP model (P<0.01). This protective effect was associated with the blunting of the increase in platelet-monocyte adhesion, and, on the contrary, with increased platelet-neutrophil aggregates in the circulation, which may be related to decreased neutrophil sequestration into the inflamed tissues. Conversely, circulating cytokine levels were not significantly changed, in both models, by mTPOR-MBP administration.Conclusion
Our results demonstrate that TPO participates in the development of organ damage induced by experimental endotoxemia or polymicrobial sepsis via a mechanism involving increased platelet-leukocyte adhesion, but not cytokine release, and suggest that blocking TPO may be useful in preventing organ damage in patients affected by systemic inflammatory response or sepsis. 相似文献32.
33.
Luca Goldoni Tiziana Beringhelli Walter Rocchia Natalia Realini Daniele Piomelli 《Analytical biochemistry》2016
Absolute analyte quantification by nuclear magnetic resonance (NMR) spectroscopy is rarely pursued in metabolomics, even though this would allow researchers to compare results obtained using different techniques. Here we report on a new protocol that permits, after pH-controlled serum protein removal, the sensitive quantification (limit of detection [LOD] = 5−25 μM) of hydrophilic nutrients and metabolites in the extracellular medium of cells in cultures. The method does not require the use of databases and uses PULCON (pulse length-based concentration determination) quantitative NMR to obtain results that are significantly more accurate and reproducible than those obtained by CPMG (Carr–Purcell–Meiboom–Gill) sequence or post-processing filtering approaches. Three practical applications of the method highlight its flexibility under different cell culture conditions. We identified and quantified (i) metabolic differences between genetically engineered human cell lines, (ii) alterations in cellular metabolism induced by differentiation of mouse myoblasts into myotubes, and (iii) metabolic changes caused by activation of neurotransmitter receptors in mouse myoblasts. Thus, the new protocol offers an easily implementable, efficient, and versatile tool for the investigation of cellular metabolism and signal transduction. 相似文献
34.
The introduction of integration tests on a pre-entry or post-arrival basis has drawn attention to changing national integration policies. Yet, at the local level, language and civic culture courses have often been implemented before the development of national programmes. The way these policies came into place and the extent to which they responded to similar challenges has often been overlooked. This article explores the development of these measures in four cities – Stuttgart, Vienna, Barcelona and Turin – to find out which mechanisms account for convergence in policymaking. Integration courses present traits of similarity in their contents and target groups; however, this does not imply a consistent process towards convergence. Local introduction initiatives are the product of local policy arenas, which display different policy frames of migrant integration. On the other hand, new legislation and changes in the available financial resources for integration appear to be crucial factors that trigger divergence. 相似文献
35.
Marcello Locatelli Roberta Cifelli Giuseppe Carlucci Annalisa Romagnoli 《Journal of enzyme inhibition and medicinal chemistry》2016,31(1):106-111
A new and specific HPLC–DAD method for the direct determination of Prulifloxacin and its active metabolite, Ulifloxacin, in human plasma has been developed. Plasma samples were analysed after a simple solid phase extraction (SPE) clean-up using a new HILIC stationary phase based high-performance liquid chromatography (HPLC) column and an ammonium acetate buffer (5?mM, pH 5.8)/acetonitrile (both with 1% Et3N, v/v) mobile phase in isocratic elution mode, with Danofloxacin as the internal standard. Detection was performed using DAD from 200 to 500?nm and quantitative analyses were carried out at 278?nm. The LOQ of the method was 1?μg/mL of the cited analytes and the calibration curve showed a good linearity up to 25?μg/mL. For both analytes the precision (RSD%) and the trueness (bias%) of the method fulfil with International Guidelines. The method was applied for stability studies, at three QC concentration levels, in human plasma samples stored at different temperature of?+?25,?+?4 and ?20?°C in order to evaluate plasma stability profiles. 相似文献
36.
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38.
Bettendorff L Wirtzfeld B Makarchikov AF Mazzucchelli G Frédérich M Gigliobianco T Gangolf M De Pauw E Angenot L Wins P 《Nature chemical biology》2007,3(4):211-212
Several important cofactors are adenine nucleotides with a vitamin as the catalytic moiety. Here, we report the discovery of the first adenine nucleotide containing vitamin B1: adenosine thiamine triphosphate (AThTP, 1), or thiaminylated ATP. We discovered AThTP in Escherichia coli and found that it accumulates specifically in response to carbon starvation, thereby acting as a signal rather than a cofactor. We detected smaller amounts in yeast and in plant and animal tissues. 相似文献
39.
Taking tissue slices of the embryonic and newborn pancreas is a novel approach for the study of the perinatal development of this gland. The aim of this study was to describe the morphology and physiology of in vivo and in vitro developing -cells. In addition, we wanted to lay a foundation for the functional analysis of other pancreatic cells, either alone or as part of an integrative pancreatic physiology approach. We used cytochemistry and light microscopy to detect specific markers and the whole-cell patch-clamp to assess the function of single -cells. The insulin signal in the embryonic -cells was condensed to a subcellular compartment and redistributed throughout the cytosol during the first 2 days after birth. The hormone distribution correlated well with the development of membrane excitability and hormone release competence in -cells. Endocrine cells survived in the organotypic tissue culture and maintained their physiological properties for weeks. We conclude that our preparation fulfills the criteria for a method of choice to characterize the function of developing pancreas in wild-type and genetically modified mice that die at birth. We suggest organotypic culture for in vitro studies of the development and regeneration of -cells.This work was supported by the European Commission (grant QLG1-CT-2001-02233 to TMR, AR and MR), the DFG Research Center for Molecular Physiology of the Brain (CMPB) and the Max-Planck Society (MR) 相似文献
40.
Palomba L Bianchi M Persichini T Magnani M Colasanti M Cantoni O 《Free radical biology & medicine》2004,36(3):319-329
Exposure of PC12 cells to A23187 or thapsigargin caused a concentration-dependent release of arachidonic acid (AA) mediated by cytosolic phospholipase A2 (PLA2). Under the same conditions, however, analysis of nitric oxide (NO) formation revealed that activation of NO synthase (NOS) is best described by a bell-shaped curve. Reduced detection of NO observed at increasing A23187 or thapsigargin concentrations was not due to formation of peroxynitrite or to activation of NO-consuming processes, but rather to AA-dependent inhibition of NOS activity. Furthermore, NO formation observed under optimal conditions for NOS activity was suppressed by AA as well as by the PLA2 activator melittin. Finally, the effects of AA were not the consequence of direct enzyme inhibition, because this lipid messenger failed to inhibit formation of NO by purified neuronal NOS, but were mediated by an AA-dependent signaling and not by downstream products of the cyclooxygenase and lipoxygenase pathways. In conclusion, the present study underscores a novel mechanism whereby endogenous, or exogenous, AA promotes inhibition of NOS activity. Because AA is generated in response to various agonists acting on membrane receptors and extensively released in inflammatory conditions, these findings have important physiopathological implications. 相似文献