Endothelial cell preservation at hypothermic to normothermic conditions using clinical and experimental organ preservation solutions

Introduction

Endothelial barrier function is pivotal for the outcome of organ transplantation. Since hypothermic preservation (gold standard) is associated with cold-induced endothelial damage, endothelial barrier function may benefit from organ preservation at warmer temperatures. We therefore assessed endothelial barrier integrity and viability as function of preservation temperature and perfusion solution, and hypothesized that endothelial cell preservation at subnormothermic conditions using metabolism-supporting solutions constitute optimal preservation conditions.Methods: Human umbilical vein endothelial cells (HUVEC) were preserved at 4–37 °C for up to 20 h using Ringer's lactate, histidine–tryptophan–ketoglutarate solution, University of Wisconsin (UW) solution, Polysol, or endothelial cell growth medium (ECGM). Following preservation, the monolayer integrity, metabolic capacity, and ATP content were determined as positive parameters of endothelial cell viability. As negative parameters, apoptosis, necrosis, and cell activation were assayed. A viability index was devised on the basis of these parameters.Results: HUVEC viability and barrier integrity was compromised at 4 °C regardless of the preservation solution. At temperatures above 20 °C, the cells' metabolic demands outweighed the preservation solutions' supporting capacity. Only UW maintained HUVEC viability up to 20 °C. Despite high intracellular ATP content, none of the solutions were capable of sufficiently preserving HUVEC above 20 °C except for ECGM.Conclusion: Optimal HUVEC preservation is achieved with UW up to 20 °C. Only ECGM maintains HUVEC viability at temperatures above 20 °C.     

Enzymatic, but not non-enzymatic, 1O2-mediated peroxidation of polyunsaturated fatty acids forms part of the EXECUTER1-dependent stress response program in the flu mutant of Arabidopsis thaliana

The conditional flu mutant of Arabidopsis accumulates excess amounts of protochlorophyllide within plastid membranes in the dark and generates singlet oxygen upon light exposure. By varying the length of the dark period, the level of the photosensitizer protochlorophyllide may be modulated, and conditions have been established that either endorse the cytotoxicity of (1)O(2) or reveal its signaling role. Two criteria have been used to distinguish between these two modes of activity of (1)O(2): the impact of the EXECUTER1 mutation and the prevalence of either non-enzymatic or enzymatic lipid peroxidation. During illumination of etiolated flu seedlings, toxic effects of (1)O(2) prevail and non-enzymatic lipid peroxidation proceeds rapidly. In contrast, in light-grown flu plants that were subjected to an 8 h dark/light shift, lipid peroxidation occurred almost exclusively enzymatically. The resulting oxidation product, 13-hydroperoxy octadecatrienoic acid (13-HPOT), serves as a substrate for synthesis of 12-oxo phytodienoic acid (OPDA) and jasmonic acid (JA), both of which are known to control various metabolic and developmental processes in plants. Inactivation of the EXECUTER1 protein abrogates not only (1)O(2)-mediated cell death and growth inhibition of flu plants, but also enzymatic lipid peroxidation. However, inactivation of jasmonate biosynthesis in the aos/flu double mutant does not affect (1)O(2)-mediated growth inhibition and cell death. Hence, JA and OPDA do not act as second messengers during (1)O(2) signaling, but form an integral part of a stress-related signaling cascade activated by (1)O(2) that encompasses several signaling pathways known to be activated by abiotic and biotic stressors.     

Benznidazole/Itraconazole Combination Treatment Enhances Anti-Trypanosoma cruzi Activity in Experimental Chagas Disease

The nitroheterocyclic drugs nifurtimox and benznidazole are first-line drugs available to treat Chagas disease; however, they have limitations, including long treatment courses and toxicity. Strategies to overcome these limitations include the identification of new drugs with specific target profiles, re-dosing regimens for the current drugs, drug repositioning and combination therapy. In this work, we evaluated combination therapy as an approach for optimization of the current therapeutic regimen for Chagas disease. The curative action of benznidazole/itraconazole combinations was explored in an established infection of the mice model with the T. cruzi Y strain. The activities of the benznidazole/itraconazole combinations were compared with the results from those receiving the same dosage of each individual drug. The administration of benznidazole/itraconazole in combination eliminated parasites from the blood more efficiently than each drug alone. Here, there was a significant reduction of the number of treatment days (number of doses) necessary to induce parasitemia suppression with the benznidazole/itraconazole combination, as compared to each compound administered alone. These results clearly indicate the enhanced effects of these drugs in combination, particularly at the dose of 75 mg/kg, as the effects observed with the drug combinations were four times more effective than those of each drug used alone. Moreover, benznidazole/itraconazole treatment was shown to prevent or decrease the typical lesions associated with chronic experimental Chagas disease, as illustrated by similar levels of inflammatory cells and fibrosis in the cardiac muscle tissue of healthy and treated mice. These results emphasize the importance of exploring the potential of combination treatments with currently available compounds to specifically treat Chagas disease.     

Routine cervical cerclage in higher order multiple gestation -- does it prolong the pregnancy?

Preterm birth following cervical dilatation is the greatest threat to infants of a multiple pregnancy. Lacking reliable data concerning the effect of prophylactic cerclage, we compared a study group to controls for maternal and perinatal outcome. Sixteen of 94 triplet-, 9 of 18 quadruplet/quintuplet-pregnancies, treated with prophylactic cerclage, were retrospectively compared to those without cervical cerclage respectively. Kruskal-Wallis test and Mann-Whitney-U test were performed as non-parametric one way analysis of variance. For the analysis of frequencies Chi Square test or Fisher's exact test were performed. Odds ratio with 95% confidence interval was used to compare the need for intravenous tocolysis as well as perinatal morbidity and mortality. Gestational age at delivery was not different from the controls in all studied groups. Birth weight revealed a 200 g dominance for the "no cerclage-triplets", while this significant difference was inverted for quadruplets/quintuplets (1245 g vs. 1069 g). With respect to gestational age at birth, need for hospitalisation or medical intervention no benefit was achieved. Moreover, perinatal outcome analysed by arterial pH, APGAR-Score and perinatal mortality was not altered by a prophylactic cerclage. Perinatal morbidity for quadruplets and quintuplets was even higher in cerclage pregnancies. Therefore, these retrospective results disclaim a positive impact of cervical cerclage on pregnancy management or perinatal outcome in multifetal pregnancies.     

HAT: Hypergeometric Analysis of Tiling-arrays with application to promoter-GeneChip data

Background  

Tiling-arrays are applicable to multiple types of biological research questions. Due to its advantages (high sensitivity, resolution, unbiased), the technology is often employed in genome-wide investigations. A major challenge in the analysis of tiling-array data is to define regions-of-interest, i.e., contiguous probes with increased signal intensity (as a result of hybridization of labeled DNA) in a region. Currently, no standard criteria are available to define these regions-of-interest as there is no single probe intensity cut-off level, different regions-of-interest can contain various numbers of probes, and can vary in genomic width. Furthermore, the chromosomal distance between neighboring probes can vary across the genome among different arrays.     

Fluoro-Jade B Staining Following Zymosan Microinjection into the Spinal Cord White Matter

1. The fluorescein derivate Fluoro-Jade B (FJB), which primarily labels dead or dying neurons, was used to study the acute focal inflammation in the spinal cord white matter. Inflammation was induced by microinjection of the yeast particulate zymosan to evaluate the biological effects of intraspinal macrophages activation without the confounding effects of physical trauma.2. A single bolus of zymosan (Sigma, 75 nL) was stereotaxically injected at the thoracic level into the lateral white matter of rat spinal cord. A standard Fluoro-Jade B staining protocol was applied to spinal cord sections at 6, 12, 24 h and 2, 4 days postinjection. Neutral Red, NADPH-diaphorase, Iba1-IR, and DAPI staining protocols accomplished examination of the cells participating in the acute inflammatory response.3. Zymosan caused formation of clearly delineated inflammation lesions localized in the lateral white matter of the spinal cord. Fluoro-Jade B stained cells in the area of inflammation were not observed at 12 h postinjection while mild FJB staining appeared at 24 h and intense staining was observed at 2 and 4 days postinjection.4. This study shows that the acute response to zymosan-induced inflammation in the rat spinal cord white matter causes a gradual appearance of phagocytic microglia/macrophages and delayed FJB staining of the inflammatory cells.5. FJB, a reliable marker of dying neurons, is a more universal agent than formerly believed. One possible explanation for the gradual appearance of FJB-stained cells in the area of inflammation is that specific time is required for sufficient levels of proteins and/or myelin debris of axonal origin to appear in the cytoplasm of phagocytic microglia/macrophages.     

Salivary melatonin rhythm as a marker of the circadian system in healthy children and those with attention-deficit/hyperactivity disorder

Attention-deficit/hyperactivity disorder (ADHD) is the most common neurobehavioral disorder of childhood. Problems with sleep structure, efficiency, and timing have been reported in some, but not all, studies on ADHD children. As the sleep-wake cycle belongs to circadian rhythms, the timekeeping circadian system might be involved in ADHD. To assess whether the circadian system of ADHD children differs from that of controls, the rhythm of the pineal hormone melatonin was used as a reliable marker of the system. Saliva from 34 ADHD and 43 control 6- to 12-yr-old children was sampled at 2-h intervals throughout the entire 24-h cycle, and the melatonin profiles of the ADHD and control children were compared. The nocturnal melatonin peaks of the ADHD and control group did not differ significantly. The high nocturnal interindividual variability of the peaks seen in adulthood was present already in the studied children. The 24-h melatonin profiles of all the ADHD subjects did not differ significantly from those of the control subjects. Categorization of subjects according to age, into groups of 6- to 7-yr-old (9 ADHD, 5 control), 8- to 9-yr-old (16 ADHD, 26 control), and 10- to 12-yr-old (9 ADHD, 12 control) children, revealed significant differences between the ADHD and control group in the melatonin rhythm waveform, but not in nocturnal melatonin peaks; the peaks were about the same in both groups and did not change significantly with increasing age. In the oldest, but not in the younger, children, the melatonin signal duration in the ADHD group was shorter than in the control group. The difference might be due to the fact that whereas in the control group both the evening melatonin onset and the morning offset phase delayed in the oldest children relative to those in the youngest children, in the ADHD group only the onset, but not the offset, phase delayed with increasing age. The data may indicate subtle differences between the circadian system of ADHD and control children during development.