Hyaluronate levels in donor organ washout effluents: a simple and predictive parameter of graft viability

The principal cause of primary non-function in orthotopic liver transplantation is thought to be preservation injury to the microvasculature. We, therefore, evaluated if effluent levels of hyaluronate, whose uptake is an endothelial cell marker, could predict early graft function and ultimate graft outcome in orthotopic liver transplantation. A total of 102 cases were studied in two phases. In the first phase, we attempted to determine if a correlation existed between effluent hyaluronate levels, early graft function and ultimate graft outcome. This phase of the study was also used to determine hypothetical cut-off values for hyaluronate which could discriminate between good and bad livers. Thirty-two livers orthotopically transplanted to randomly selected primary recipients were studied. After varying periods of static cold storage (4°C) in University of Wisconsin solution, the livers were reinfused with cold (4°C) lactated Ringer’s solution. The first 50 ml of the reperfusion effluent was collected from the infrahepatic vena cava. Effluent samples were analyzed for hyaluronate. Linear regression analysis demonstrated a significant correlation between effluent hyaluronate levels and post-operative aspartate and alanine aminotransferase levels (p<0.001 for both). Logistic regression demonstrated a highly significant correlation (p = 0.0056) between effluent hyaluronate levels and ultimate graft outcome. Generation of Receiver Characteristics Curves indicated that a level between 400 and 430 μg·l(−1) could possibly discriminate between good livers and those at risk of early graft failure. The authenticity of this hyaluronate cut-off level was further confirmed in the second phase of the study where 70 consecutive primary crossmatch-negative transplants were performed. A highly significant difference was observed in peak aspartate and alanine aminotransferase levels in the first week (p<0.0006 and p<0.0005, respectively) between livers with effluent hyaluronate levels≤400 μg·l(−1) and livers with hyaluronate levels higher than 400 μg·l(−1) Logistic regression revealed a highly significant correlation between effluent hyaluronate levels and graft success (p=0.0001). Since hyaluronate uptake by the microvascular endothelial cell is significantly greater than production, high hyaluronate effluent levels in failed livers would be due to decreased hyaluronate uptake by the injured microvascular endothelial cell. We therefore conclude that effluent hyaluronate levels may prove to be a reliable preoperative test to assess early graft function and outcome in clinical orthotopic liver transplantation

Lipopolysaccharide-induced liver apoptosis is increased in interleukin-10 knockout mice

AbstractAlthough IL-10 down-regulates pro-inflammatory cytokine secretion by hepatic Kupffer cells, the mechanisms underlying its hepatoprotective effects are not fully clear. This study tested the hypothesis that IL-10 protects the liver against pro-inflammatory cytokines by counteracting their pro-apoptotic effects. Wild type and IL-10 knockout mice were treated with bacterial lipopolysaccharide and sacrificed 1, 4, 8, and 12 h later. Plasma ALT activity was measured as a marker of liver injury. Liver pathology and TUNEL response were assessed by histology. Plasma levels and whole liver mRNA levels were measured for TNF-α, IL-1β, TGF-β1, IL-10, and their respective receptors. Hepatic mRNA levels were measured for several pro-apoptotic adaptors/regulators, including FasL, Fas receptor, FADD, TRADD, Bad, Bak, Bax, and Bcl-XS, and anti-apoptotic regulators, including Bcl-w, Bcl-XL, Bcl-2, and Bfl-1. Caspase-3 activity in the liver was determined as well as immunohistochemistry for IL-1RII, TGF-βRII and Fas receptor. At all time points the livers from IL-10 knockout mice displayed a significantly increased number of apoptotic nuclei compared to wild type mice. Changes in plasma cytokine levels and their liver mRNA levels were consistent with suppression by IL-10 of pro-inflammatory cytokine secretion. In addition, pro-inflammatory cytokine receptor mRNA levels (TNF-α, TGF-β, and IL-1β) were markedly up-regulated by LPS at all time points in IL-10 knockout mice as compared to wild type mice. Expression of the pro-inflammatory cytokine receptor IL-1RII was similarly increased as shown by immunostaining. The mRNA levels of a typical pro-apoptotic cytokine, TRAIL, were increased and LPS also up-regulated the mRNA expression of other apoptotic factors to a larger extent in IL-10 knockout mice than in their wild type counterparts, suggestive of an IL-10 anti-apoptotic effect. In the livers of knockout mice, markedly increased caspase-3 activity was already evident at the 1-h time point following LPS administration, while in the wild type animals this increase was delayed. Immunostaining also indicated that LPS increased hepatic expression of the pro-apoptotic receptors Fas and TGF-βRII in IL-10 knockout mice. The data presented in this study show that: (i) IL-10 modulates not only the secretion of pro-inflammatory cytokines, but also the receptors of these cytokines, and ii) IL-10 protects the liver against LPS-induced injury at least in part by counteracting pro-inflammatory cytokine-induced liver apoptosis

Tobacco\u27s Minor Alkaloids: Effects on Place Conditioning and Nucleus Accumbens Dopamine Release in Adult and Adolescent Rats

Tobacco products are some of the most commonly used psychoactive drugs worldwide. Besides nicotine, alkaloids in tobacco include cotinine, myosmine, and anatabine. Scientific investigation of these constituents and their contribution to tobacco dependence is less well developed than for nicotine. The present study evaluated the nucleus accumbens dopamine-releasing properties and rewarding and/or aversive properties of nicotine (0.2-0.8 mg/kg), cotinine (0.5-5.0 mg/kg), anatabine (0.5-5.0 mg/kg), and myosmine (5.0-20.0 mg/kg) through in vivo microdialysis and place conditioning, respectively, in adult and adolescent male rats. Nicotine increased dopamine release at both ages, and anatabine and myosmine increased dopamine release in adults, but not adolescents. The dopamine release results were not related to place conditioning, as nicotine and cotinine had no effect on place conditioning, whereas anatabine and myosmine produced aversion in both ages. While the nucleus accumbens shell is hypothesized to play a role in strengthening drug-context associations following initiation of drug use, it may have little involvement in the motivational effects of tobacco constituents once these associations have been acquired. Effects of myosmine and anatabine on dopamine release may require a fully developed dopamine system, since no effects of these tobacco alkaloids were observed during adolescence. In summary, while anatabine and myosmine-induced dopamine release in nucleus accumbens may play a role in tobacco dependence in adults, the nature of that role remains to be elucidated

Semisynthetic Aurones Inhibit Tubulin Polymerization at the Colchicine-Binding Site and Repress PC-3 Tumor Xenografts in Nude Mice and Myc-Induced T-ALL in Zebrafish

Structure-activity relationships (SAR) in the aurone pharmacophore identified heterocyclic variants of the (Z)-2-benzylidene-6-hydroxybenzofuran-3(2H)-one scaffold that possessed low nanomolar in vitro potency in cell proliferation assays using various cancer cell lines, in vivo potency in prostate cancer PC-3 xenograft and zebrafish models, selectivity for the colchicine-binding site on tubulin, and absence of appreciable toxicity. Among the leading, biologically active analogs were (Z)-2-((2-((1-ethyl-5-methoxy-1H-indol-3-yl)methylene)-3-oxo-2,3-dihydrobenzofuran-6-yl)oxy)acetonitrile (5a) and (Z)-6-((2,6-dichlorobenzyl)oxy)-2-(pyridin-4-ylmethylene)benzofuran-3(2H)-one (5b) that inhibited in vitro PC-3 prostate cancer cell proliferation with IC50 values below 100 nM. A xenograft study in nude mice using 10 mg/kg of 5a had no effect on mice weight, and aurone 5a did not inhibit, as desired, the human ether-à-go-go-related (hERG) potassium channel. Cell cycle arrest data, comparisons of the inhibition of cancer cell proliferation by aurones and known antineoplastic agents, and in vitro inhibition of tubulin polymerization indicated that aurone 5a disrupted tubulin dynamics. Based on molecular docking and confirmed by liquid chromatography-electrospray ionization-tandem mass spectrometry studies, aurone 5a targets the colchicine-binding site on tubulin. In addition to solid tumors, aurones 5a and 5b strongly inhibited in vitro a panel of human leukemia cancer cell lines and the in vivo myc-induced T cell acute lymphoblastic leukemia (T-ALL) in a zebrafish model

Transcriptomic Analyses of Sexual Dimorphism of the Zebrafish Liver and the Effect of Sex Hormones

10.1371/journal.pone.0053562PLoS ONE81