Granulocyte-colony stimulating factor (G-CSF) to treat acute-on-chronic liver failure: A multicenter randomized trial (GRAFT study).

BACKGROUND & AIMS: Based on positive results from small single center studies, granulocyte-colony stimulating factor (G-CSF) is being widely used for the treatment of patients with acute-on-chronic liver failure (ACLF). Herein, we aimed to evaluate the safety and efficacy of G-CSF in patients with ACLF. METHODS: In this multicenter, prospective, controlled, open-label phase II study, 176 patients with ACLF (EASL-CLIF criteria) were randomized to receive G-CSF (5 µg/kg daily for the first 5 days and every third day thereafter until day 26) plus standard medical therapy (SMT) (n = 88) or SMT alone. The primary efficacy endpoint was 90-day transplant-free survival analyzed by Cox regression modeling. The key secondary endpoints were overall and transplant-free survival after 360 days, the development of ACLF-related complications, and the course of liver function scores during the entire observation period. RESULTS: Patients treated with G-CSF had a 90-day transplant-free survival rate of 34.1% compared to 37.5% in the SMT group (hazard ratio [HR] 1.05; 95% CI 0.711-1.551; p = 0.805). Transplant-free and overall survival at 360 days did not differ between the 2 arms (HR 0.998; 95% CI 0.697-1.430; p = 0.992 and HR 1.058; 95% CI 0.727-1.548; p = 0.768, respectively). G-CSF did not improve liver function scores, the occurrence of infections, or survival in subgroups of patients without infections, with alcohol-related ACLF, or with ACLF defined by the APASL criteria. Sixty-one serious adverse events were reported in the G-CSF+SMT group and 57 were reported in the SMT group. In total, 7 drug-related serious adverse reactions occurred in the G-CSF group. The study was prematurely terminated due to futility after conditional power calculation. CONCLUSIONS: In contrast to previous findings, G-CSF had no significant beneficial effect on patients with ACLF in this multicenter controlled trial, which suggests that it should not be used as a standard treatment for ACLF. CLINICALTRIALS. GOV NUMBER: NCT02669680 LAY SUMMARY: Granulocyte-colony stimulating factor was considered as a novel treatment for acute-on-chronic liver failure (ACLF). We performed the first randomized, multicenter, controlled phase II trial, which showed that G-CSF did not improve survival or other clinical endpoints in patients with ACLF. Therefore, G-CSF should not be used to treat liver disease outside clinical studies.

Ecto-Nucleotide Triphosphate Diphosphohydrolase-2 (NTPDase2) Deletion Increases Acetaminophen-Induced Hepatotoxicity.

Ecto-nucleotidase triphosphate diphosphohydrolase-2 (NTPDase2) is an ecto-enzyme that is expressed on portal fibroblasts in the liver that modulates P2 receptor signaling by regulating local concentrations of extracellular ATP and ADP. NTPDase2 has protective properties in liver fibrosis and may impact bile duct epithelial turnover. Here, we study the role of NTPDase2 in acute liver injury using an experimental model of acetaminophen (APAP) intoxication in mice with global deletion of NTPDase2. Acute liver toxicity was caused by administration of acetaminophen in wild type (WT) and NTPDase2-deficient (Entpd2 null) mice. The extent of liver injury was compared by histology and serum alanine transaminase (ALT). Markers of inflammation, regeneration and fibrosis were determined by qPCR). We found that Entpd2 expression is significantly upregulated after acetaminophen-induced hepatotoxicity. Entpd2 null mice showed significantly more necrosis and higher serum ALT compared to WT. Hepatic expression of IL-6 and PDGF-B are higher in Entpd2 null mice. Our data suggest inducible and protective roles of portal fibroblast-expressed NTPDase2 in acute necrotizing liver injury. Further studies should investigate the relevance of these purinergic pathways in hepatic periportal and sinusoidal biology as such advances in understanding might provide possible therapeutic targets.

Human Stem Cells Promote Liver Regeneration After Partial Hepatectomy in BALB/C Nude Mice.

BACKGROUND: Mesenchymal stem cells (MSCs) have been suggested to augment liver regeneration after surgically and pharmacologically induced liver failure. To further investigate this we processed human bone marrow-derived MSC according to good manufacturing practice (GMP) and tested those cells for their modulatory capacities of metabolic alterations and liver regeneration after partial hepatectomy in BALB/c nude mice. METHODS: Human MSCs were obtained by bone marrow aspiration of healthy donors as in a previously described GMP process. Transgenic GFP-MSCs were administered i.p. 24 h after 70% hepatectomy in BALB/c nude mice, whereas control mice received phosphate-buffered saline. Mice were sacrificed 2, 3, and 5 d after partial hepatectomy. Blood and organs were harvested and metabolic alterations as well as liver regeneration subsequently assessed by liver function tests, multianalyte profiling immunoassays, histology, and immunostaining. RESULTS: Hepatocyte and sinusoidal endothelial cell proliferation were significantly increased after partial hepatectomy in mice receiving MSC compared to control mice (Hepatocyte postoperative day 3, P < 0.01; endothelial cell postoperative day 5, P < 0.05). Hepatocyte fat accumulation correlated inversely with hepatocyte proliferation (r2 = 0.4064, P < 0.01) 2 d after partial hepatectomy, with mice receiving MSC being protected from severe fat accumulation. No GFP-positive cells could be detected in the samples. Serum levels of IL-6, HGF, and IL-10 were significantly decreased at day 3 in mice receiving MSC when compared to control mice (P < 0.05). Relative body weight loss was significantly attenuated after partial hepatectomy in mice receiving MSC (2 d and 3 d, both P < 0.001) with a trend toward a faster relative restoration of liver weight, when compared to control mice. CONCLUSIONS: Human bone marrow-derived MSC attenuate metabolic alterations and improve liver regeneration after partial hepatectomy in BALB/c nude mice. Obtained results using GMP-processed human MSC suggest functional links between fat accumulation and hepatocyte proliferation, without any evidence for cellular homing. This study using GMP-proceeded MSC has important regulatory implications for an urgently needed translation into a clinical trial.

Mononuclear-cell-derived microparticles attenuate endothelial inflammation by transfer of miR-142-3p in a CD39 dependent manner.

Plasma microparticles (MP) bear functional active ectonucleotidases of the CD39 family with implications in vascular inflammation. MP appear to be able to fuse with cells and transfer genetic information. Here, we tested whether levels of different immunomodulatory microRNAs (miRs) in plasma MP are modulated by CD39 after experimental hepatectomy. We further investigated whether horizontal transfer of miR-142-3p between mononuclear (MNC) and endothelial cells via MP is regulated by purinergic signaling. Partial hepatectomy was performed in C57BL/6 wild type and Cd39 null mice. MP were collected via ultracentrifugation. MNC were stimulated with nucleotides and nucleosides, in vitro, and tested for miR-142-3p levels. Fusion of MNC-derived MP and endothelial cells with subsequent transfer of miR-142-3p was imaged by flow cytometry and confocal microscopy. Endothelial inflammation and apoptosis were quantified after transfection with miR-142-3p. Significantly lower miR-142-3p levels were observed in plasma MP of Cd39 null mice after partial hepatectomy, when compared to C57BL/6 wild types (p < 0.05). In contrast to extracellular nucleotides, anti-inflammatory adenosine significantly increased miR-142-3p levels in MNC-derived MP, in vitro (p < 0.05). MNC-derived MP are able to transfer miR-142-3p to endothelial cells by fusion. Transfection of endothelial cells with miR-142-3p decreased TNF-α levels (p < 0.05) and endothelial apoptosis (p < 0.05). MiR-142-3p levels in MNC-derived MP are modulated by nucleoside signaling and might reflect compensatory responses in vascular inflammation. Our data suggest the transfer of genetic information via shed MP as a putative mechanism of intercellular communication-with implications in organ regeneration.

The value of microparticles in detecting acute rejection episodes after liver transplantation.

CONTEXT: Non-invasive markers for diagnosis of acute rejection (AR) following liver transplantation have not been developed, yet. OBJECTIVE: We analyzed the correlation of plasma microparticle levels (MP) with AR. MATERIALS AND METHODS: MP (CD4, CD8, CD25, CD31, MHC) of 11 AR patients and 11 controls were analyzed within the first week after transplantation. RESULTS: CD4, CD8 and CD31 positive MP were higher in the AR, whereas overall MP count, CD25 and MHCI positive MP proportions did not differ between both groups. DISCUSSION AND CONCLUSION: MP dynamics within the first period of transplantation could help to clarify on-going mechanisms of immunomodulation.

Absolute quantification of microparticles by flow cytometry in ascites of patients with decompensated cirrhosis: a cohort study.

BACKGROUND: Microparticles (MPs) are small (<1 µm) cell membrane-derived vesicles that are formed in response to cellular activation or early stages of apoptosis. Increased plasma MP levels have been associated with liver disease severity. Here we investigated the clinical impact of ascites MPs in patients with decompensated liver cirrhosis. METHODS: Ascites and blood samples of 163 patients with cirrhosis (ascites n = 163, blood n = 31) were collected between February 2011 and December 2012. MPs were obtained from ascites and from blood by two-step ultracentrifugation and quantified by flow cytometry. Quantitative absolute MP levels were correlated with clinical and laboratory baseline parameters as well as patient outcomes. Ascites microparticles were stained with antibodies against CD66b (neutrophils) and CD3 (lymphocytes) in a subgroup of 60 matched patients. RESULTS: MPs were detected in all ascites and blood samples. Absolute ascites MP levels correlated with blood levels (r = 0.444, p = 0.011). Low ascites MP levels (<488.4 MP/µL) were associated with a poor 30-day survival probability (<488.4 MP/µL 71.1% vs. >488.4 MP/µL 94.7%, log rank p = 0.001) and such patients had a higher relative amount of ascites microparticles derived from neutrophils and lymphocytes. Low levels of ascites MPs, high MELD score and antibiotic treatment were independent risk factors for death within 30 days. CONCLUSIONS: Ascites MP levels predict short-term survival along with the liver function in patients with decompensated cirrhosis. Further studies which evaluate ascites MPs as disease specific biomarker with a validation cohort and which investigate its underlying mechanisms are needed. Neutrophils and lymphocytes contributed more frequently to the release of microparticles in patients with low ascites levels, possibly indicating an immune activation in this cohort.

Correlation of cell-free DNA plasma concentration with severity of non-alcoholic fatty liver disease.

BACKGROUND: The assessment of fibrosis and inflammatory activity is essential to identify patients with non-alcoholic fatty liver disease (NAFLD) at risk for progressive disease. Serum markers and ultrasound-based methods can replace liver biopsy for fibrosis staging, whereas non-invasive characterization of inflammatory activity remains a clinical challenge. Cell-free DNA (cfDNA) is a novel non-invasive biomarker for assessing cellular inflammation and cell death, which has not been evaluated in NAFLD. METHODS: Patients and healthy controls from two previous studies were included. NAFLD disease activity and severity were non-invasively characterized by liver stiffness measurement (transient elastography, TE) including steatosis assessment with controlled attenuation parameter (CAP), single-proton magnetic resonance spectroscopy (1H-MRS) for determination of hepatic fat fraction, aminotransferases and serum ferritin. cfDNA levels (90 and 222 bp fragments) were analyzed using quantitative real-time PCR. RESULTS: Fifty-eight NAFLD patients (age 62 ± 11 years, BMI 28.2 ± 3.5 kg/m2) and 13 healthy controls (age 38 ± 12 years, BMI 22.4 ± 2.1 kg/m2) were included. 90 bp cfDNA levels were significantly higher in NAFLD patients compared to healthy controls: 3.7 (1.3-23.1) vs. 2.9 (1.4-4.1) ng/mL (p = 0.014). In the NAFLD cohort, circulating cfDNA correlated significantly with disease activity and severity, especially in patients with elevated liver stiffness (n = 13, 22%) compared to cases with TE values ≤7 kPa: cf90 bp 6.05 (2.41-23.13) vs. 3.16 (1.29-7.31) ng/mL (p < 0.001), and cf222 bp 14.41 (9.27-22.90) vs. 11.32 (6.05-18.28) ng/mL (p = 0.0041). CONCLUSIONS: Cell-free DNA plasma concentration correlates with established non-invasive markers of NAFLD activity and severity. Therefore, cfDNA should be further evaluated as biomarker for identifying patients at risk for progressive NAFLD.

Biological characterization of novel nitroimidazole-peptide conjugates in vitro and in vivo.

Recently, we reported on the design of a multimodal peptide conjugate useful as delivery platform for targeting hypoxic cells. A nitroimidazole (2-(2-nitroimidazol-1-yl)acetic acid, NIA) moiety, which is selectively entrapped in hypoxic cells, was coupled to a cell-penetrating peptide serving as the transporter. Furthermore, attachment of a bifunctional linker allowed the introduction of a diagnostic or therapeutic radiometal. However, although selective tumor accumulation could be detected in vivo, a fast renal clearance of the compound was observed. The present study aims to improve the system by using the more proteolytically stable all-d version of the peptide carrier (DsC18), by attaching two NIA moieties instead of one (DsC18(NIA)2 ) to enhance the tumor uptake, and by incorporating the bifunctional chelator NODAGA instead of DOTA (NODAGA-DsC18(NIA)2 ) to optimize labeling chemistry. First, we characterized in vitro the novel all-d peptide compared with its parent l-version. Then, in order to investigate and compare the pharmacological profiles of the peptides, these were radiolabeled with 64 CuII and 68 GaIII , and the biodistribution and kinetics were evaluated in vivo. Our results show the versatility of the d-peptide as cell-penetrating peptide and transporter. However, attaching two NIA groups modified the system in such a way that no selective tumor uptake could be observed compared with the peptide without NIA moieties. Still, this work highlights new pharmacokinetic data on the biodistribution of such compounds in vivo. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.

Influence of the metal center and linker on the intracellular distribution and biological activity of organometal-peptide conjugates.

Organometallic complexes conjugated to cell-penetrating peptides (CPPs) are promising systems for diagnostic imaging and therapeutic applications in human medicine. Recently, we reported on the synthesis of cymantrene(CpMn(CO)(3))-CPP conjugates with biological activity on different cancer cell lines. However, the precise mechanism of cytotoxicity remained elusive in these studies. To investigate the role of the metal center and the linker between the CpM(CO)(3) moiety and the peptide, a number of derivatives with manganese replaced by rhenium and the keto linker originally used substituted by a methylene group were prepared and fully characterized by (1)H NMR spectroscopy, infrared spectroscopy, electrospray ionization mass spectrometry, and elemental analysis as well as X-ray structure determination. The organometal-peptide conjugates as well as carboxyfluorescein-labeled derivatives thereof were prepared by solid-phase peptide synthesis, purified by high-performance liquid chromatography, and analyzed by mass spectrometry. Fluorescence microscopy studies of MCF-7 human breast cancer cells revealed an efficient cellular uptake and pronounced nuclear localization of the bioconjugates with the methylene linker compared with systems with the keto group. In addition, the latter also showed a higher cytotoxicity. In contrast, the variation of the metal center from manganese to rhenium had a negligible effect. The structure-activity relationships determined in the present work will aid in the further tuning of the biological activity of organometal-peptide conjugates.

Cellular impact and selectivity of half-sandwich organorhodium(III) anticancer complexes and their organoiridium(III) and trichloridorhodium(III) counterparts.

Half-sandwich organorhodium(III) complexes and their trichloridorhodium(III) counterparts are potent anticancer agents that enhance the formation of reactive oxygen species and invoke a strong induction of apoptosis in leukemia cells. The antiproliferative activity towards human MCF-7 and HT-29 adenocarcinoma cells of novel nonintercalating complexes containing the 5-substituted phenanthroline ligands 5,6-dimethylphenanthroline, 5-chlorophenanthroline, and 5-nitrophenanthroline (phen*) increases dramatically in the order [(η(5)-C(5)Me(5))IrCl(phen*)](CF(3)SO(3)) < [(η(5)-C(5)Me(5))RhCl(phen*)](CF(3)SO(3)) < mer-[RhCl(3)(DMSO)(phen*)] (DMSO is dimethyl sulfoxide). Improved activity was also achieved by attaching a cell-penetrating peptide to the dipyrido[3,2-a:2',3'-c]phenazine (dppz) ligand of an organorhodium(III) complex. Whereas 5-substitution led to significant improvements in the activity of the organoiridium(III) and trichloridorhodium(III) compounds in comparison with the parent phenanthroline complex, the IC(50) values of their organorhodium(III) counterparts remained effectively invariable. The high activities of the trichloridorhodium(III) complexes (IC(50) = 0.06-0.13 µM) were accompanied by pronounced selectivity towards human cancer cells in comparison with immortalized HEK-293 cells. In contrast, [(η(5)-C(5)Me(5))RhCl(5,6-Me(2)phen)](CF(3)SO(3)) (phen is phenanthroline) was markedly more active towards BJAB lymphoma cells than ex vivo healthy leukocytes and caused an immediate decrease in cellular adhesion possibly associated with interactions with membrane proteins. Its dppz analogue invoked an initial increase in glycolysis to compensate for reduced respiration before inducing a delayed onset of cell death. Strong antimitochondrial activity with respiration impairment and release of cytochrome c was established for both complexes.

Antimicrobial peptides with cell-penetrating peptide properties and vice versa.

Antimicrobial peptides (AMPs) are a group of peptides that are active against a diverse spectrum of microorganisms. Due to their mode of action, AMPs are a promising class of molecules that could overcome the problems of increasing resistance of bacteria to conventional antibiotics. Furthermore, AMPs are strongly membrane-active and some are able to translocate into cells without the necessity for permanent membrane permeabilization. This feature has brought them into focus for use as transport vectors in the context of drug delivery. Since the plasma membrane restricts transport of bioactive substances into cells, great research interest lies in the development of innovative ways to overcome this barrier and to increase bioavailability. In this context, peptide-based transport systems, such as cell-penetrating peptides (CPPs), have come into focus, and their efficiency has been demonstrated in many different applications. However, more recently, also some AMPs have been used as efficient vectors for intracellular translocation of various active molecules. This review summarizes recent efforts in this interesting field of drug delivery. Moreover, some examples of the application of CPPs as efficient antimicrobial substances will be discussed.

Extended liver resection in mice: state of the art and pitfalls-a systematic review.

BACKGROUND: Rodent models of liver resection have been used to investigate and evaluate the liver's complex physiology and pathology since 1931. First documented by Higgins and Anderson, such models were created to understand liver regeneration mechanisms to improve outcomes in patients undergoing extensive liver resection for liver cancer or other underlying liver diseases. METHODS: A systematic search was conducted using Pubmed, gathering publications up to January 2019, which engaged with the mouse model of extended liver resection as a method itself. The results of this search were filtered according to their language, novelty, and relevancy. RESULTS: The Boolean search found 3741 articles on Pubmed, with 3130 publications remaining when filtered by language and the presence of a full text. In total, 21 of these publications examined the key themes of the animal model described. The mortality varied from 0 to 50% depending on the surgeon's experience and the resection method. The liver resection was mainly performed with classic sutures (14 out of 21 publications) and isoflurane was used for anaesthesia (10 out of 21 publications) in combination with analgesics (buprenorphine or ketamine/xylazine). The most used mouse strain was C57BL/6 (7 of 21 publications) which was on average 11 weeks old with a weight of 23 g. CONCLUSION: Through the overview, laid out in the selected publications, this paper reviews the shift of the extended liver resection model from rat to the mouse, describes the state of the art in the experimental setting, and discusses the possible limitations and pitfalls. Clearly, the extended liver resection in mice is a reproducible, practical and easy to learn method.

Sensing Acute Cellular Rejection in Liver Transplant Patients Using Liver-Derived Extracellular Particles: A Prospective, Observational Study.

Acute cellular rejection (ACR) after liver transplantation (LT) goes along with allograft dysfunction, which is diagnosed by liver biopsy and concomitant histological analysis, representing the gold standard in clinical practice. Yet, liver biopsies are invasive, costly, time-intensive and require expert knowledge. Herein we present substantial evidence that blood plasma residing peripheral liver-derived extracellular particles (EP) could be employed to diagnose ACR non-invasively. In vitro experiments showed organ-specific EP release from primary human hepatocytes under immunological stress. Secondly, analysis of consecutive LT patients (n=11) revealed significant heightened EP concentrations days before ACR. By conducting a diagnostic accuracy study (n = 69, DRKS00011631), we explored the viability of using EP as a liquid biopsy for diagnosing ACR following LT. Consequently, novel EP populations in samples were identified using visualization of t-distributed stochastic neighbor embedding (viSNE) and self-organizing maps (FlowSOM) algorithms. As a result, the ASGR1+CD130+Annexin V+ EP subpopulation exhibited the highest accuracy for predicting ACR (area under the curve: 0.80, 95% confidence interval [CI], 0.70-0.90), with diagnostic sensitivity and specificity of 100% (95% CI, 81.67-100.0%) and 68.5% (95% CI, 55.3-79.3%), respectively. In summary, this new EP subpopulation presented the highest diagnostic accuracy for detecting ACR in LT patients.

Protease-activatable organometal-Peptide bioconjugates with enhanced cytotoxicity on cancer cells.

Over the past years, numerous promising new metalorganic lead structures have been developed exhibiting highly active cytostatic properties. However, the efficiency of such chemotherapeutics in the treatment of tumors is often limited by their low therapeutic index due to their short half-life, lack of tumor selectivity, and associated side effects. Furthermore, the membrane barrier often restricts their cellular uptake by passive diffusion. In this contribution, we describe the synthesis, cellular uptake, and biologic activity of a series of cymantrene-peptide conjugates. Cymantrene CpMn(CO)(3) is a robust organometallic group, which is stable in air and water and easy to functionalize. In this work, some new cymantrene derivatives with different linkers between the half-sandwich complex and the carboxylate group were attached to the cell-penetrating peptide sC18 that should act as a transporter for the metal moiety. All conjugates were characterized for their cytotoxic activity on human breast adenocarcinoma cells (MCF-7) and human colon carcinoma cells (HT-29). We found that bioconjugates bearing two cymantrene groups were more active than the monofunctionalized ones. By the introduction of a cathepsin B cleavage site next to the organometallic group, the biologic activity could be in increased even further. Fluorescence microscopy studies and apoptosis assays gave preliminary hints on the mode of action of these systems.

Influence of the new standardized clinical cryopreservation/slow thawing protocol on immunogenicity of arterial allografts in rats.

OBJECTIVES AND DESIGN: At the present time there are two waiting list for patients with vascular prosthetic infection indicated for arterial transplantation in the Czech Republic. The inclusion of each patient for cold-stored or cryopreserved arterial transplantation is the preference of indicating surgeon. In this experimental work we studied the immunogenicity of rat aortal allografts treated by our new clinical cryopreservation/slow thawing protocol. MATERIAL AND METHODS: Brown-Norway (BN) (N = 6, 203-217 g) or Lewis (LEW) (N = 6, 248-254 g) abdominal aortal grafts treated in accordance with our new clinical cryopreservation/slow thawing protocol were orthotopically transplanted to Lewis recipients (N = 12, 191-245 g). Aortal wall histology and infiltration by recipient immune cells, as well as donor specific anti MHC class I and II antibodies in recipient serum were studied in both isografts and allografts on day 30 postransplant. Core data of cryopreserved allografts were compared to our previous data of cold-stored aortal allografts treated in accordance with our clinical cold-storage protocol. RESULTS: Cryopreserved allografts showed regular morphology of aortal wall with clear differentiation of all three basic anatomical layers on day 30 postransplant. Intimal layer showed no hyperplasia, luminal surface was covered by endothelial cells. No statistical difference was observed in tunica media thickness between isografts and allografts. The medial layer showed no necrosis, shrinkage or immunoglobuline G deposition in any experimental group. The adventitial infiltration by immune cells was significantly higher (P<0.05) in allografts. Cryopreserved allografts showed significant lower activation of both cell- and antibody mediated immunity compared to historical data of cold-stored allografts. CONCLUSION: Aortal wall histology of rat allografts treated by our new standardized clinical cryopreservation/slow thawing protocol was comparable to that of the cryopreserved isografts on day 30 posttranspant. The immunogenicity of cryopreserved aortal allografts was significantly lower compared to that of cold-stored aortal allografts.

Increased Cell-Free DNA Plasma Concentration Following Liver Transplantation Is Linked to Portal Hepatitis and Inferior Survival.

Donor organ quality is crucial for transplant survival and long-term survival of patients after liver transplantation. Besides bacterial and viral infections, endogenous damage-associated molecular patterns (DAMPs) can stimulate immune responses. Cell-free DNA (cfDNA) is one such DAMP that exhibits highly proinflammatory effects via DNA sensors. Herein, we measured cfDNA after liver transplantation and found elevated levels when organs from resuscitated donors were transplanted. High levels of cfDNA were associated with high C-reactive protein, leukocytosis as well as granulocytosis in the recipient. In addition to increased systemic immune responses, portal hepatitis was observed, which was associated with increased interface activity and a higher numbers of infiltrating neutrophils and eosinophils in the graft. In fact, the cfDNA was an independent significant factor in multivariate analysis and increased concentration of cfDNA was associated with inferior 1-year survival. Moreover, cfDNA levels were found to be decreased significantly during the postoperative course when patients underwent continuous veno-venous haemofiltration. In conclusion, patients receiving livers from resuscitated donors were characterised by high postoperative cfDNA levels. Those patients showed pronounced portal hepatitis and systemic inflammatory responses in the short term leading to a high mortality. Further studies are needed to evaluate the clinical relevance of cfDNA clearance by haemoadsorption and haemofiltration in vitro and in vivo.

Diagnostic Biomarkers to Diagnose Acute Allograft Rejection After Liver Transplantation: Systematic Review and Meta-Analysis of Diagnostic Accuracy Studies.

Objective: A systematic review and meta-analysis of diagnostic biomarkers for noninvasive diagnosis of acute allograft rejection following liver transplantation. Background: Noninvasive blood and urine markers have been widely explored in recent decades for diagnosing acute rejection after liver transplantation. However, none have been translated into routine clinical use so far due to uncertain diagnostic accuracy, and liver biopsy remains the gold standard. Methods: Systematic literature searches of Medline, Cochrane and Embase were conducted up to February 2019 to identify studies evaluating the use of noninvasive markers in diagnosing allograft rejection following liver transplantation. Meta-analysis was performed using a random effects model with DerSimonian-Laird weighting and the hierarchical summary receiver operating curve. Results: Of 560 identified studies, 15 studies (1,445 patients) met the inclusion criteria. The following markers were tested: acid labile nitroso-compounds (NOx), serum amyloid A protein, procalcitonin, peripheral blood eosinophil count, peripheral blood T-cell activation and interleukin 2 (IL-2) receptor, guanylate-binding protein-2 mRNA, graft-derived cell-free DNA, pi-glutathione S-transferase, alpha-glutathione S-transferase and serum HLA class I soluble antigens. Only eosinophil count was tested in multiple studies, and they demonstrated high heterogeneity (I2 = 72% [95% CI: 0.5-0.99]). IL-2 receptor demonstrated the highest sensitivity (89% [95% CI: 0.78-0.96]) and specificity (81% [95% CI: 0.69-0.89]). Conclusion: IL-2 receptor expression demonstrated the highest diagnostic accuracy, while the peripheral eosinophil count was the only marker tested in more than one study. Presently, liver biopsy remains superior to noninvasive diagnostic biomarkers as most studies exhibited inferior designs, hindering possible translation into clinical application.

Cymantrene conjugation modulates the intracellular distribution and induces high cytotoxicity of a cell-penetrating peptide.

The conjugation of cymantrene CpMn(CO)(3) to cell-penetrating peptide hCT(18-32)-k7 alters the intracellular distribution in MCF-7 cells compared to the unmodified peptide, as visualized by fluorescence microscopy, and leads to an increased nuclear accumulation; the peptide and cymantrene compound themselves are not toxic, but the bioconjugate shows a significant cytotoxicity with an IC(50) value of 36 micromol l(-1).

The nanomolar sensing of nicotinamide adenine dinucleotide in human plasma using a cycling assay in albumin modified simulated body fluids.

Nicotinamide adenine dinucleotide (NAD), a prominent member of the pyridine nucleotide family, plays a pivotal role in cell-oxidation protection, DNA repair, cell signalling and central metabolic pathways, such as beta oxidation, glycolysis and the citric acid cycle. In particular, extracellular NAD+ has recently been demonstrated to moderate pathogenesis of multiple systemic diseases as well as aging. Herein we present an assaying method, that serves to quantify extracellular NAD+ in human heparinised plasma and exhibits a sensitivity ranging from the low micromolar into the low nanomolar domain. The assay achieves the quantification of extracellular NAD+ by means of a two-step enzymatic cycling reaction, based on alcohol dehydrogenase. An albumin modified revised simulated body fluid was employed as standard matrix in order to optimise enzymatic activity and enhance the linear behaviour and sensitivity of the method. In addition, we evaluated assay linearity, reproducibility and confirmed long-term storage stability of extracellular NAD+ in frozen human heparinised plasma. In summary, our findings pose a novel standardised method suitable for high throughput screenings of extracellular NAD+ levels in human heparinised plasma, paving the way for new clinical discovery studies.

Angiogenic miRNAs, the angiopoietin axis and related TIE2-expressing monocytes affect outcomes in cholangiocarcinoma.

BACKGROUND: Tumour angiogenesis is modulated on both an epigenetic and protein level and has potential implications for immune cell responses. However, the importance of related angiogenic biomarkers in cholangiocarcinoma (CCA) is unknown. This study assessed human CCA samples for the expression of angiogenesis-associated microRNAs, angiopoietins (Angs) and monocytes expressing the Ang-receptor, TIE2, with regards to prognostic significance after liver resection. METHODS: Angiogenic miRNAs were analysed in frozen samples of intrahepatic CCA (iCC; n = 43) and hilar CCA (HC; n = 45). Ang-1 and Ang-2, as well as TIE2-expressing monocytes (TEMs), were detected in paraffin-embedded iCC sections (n = 88). MiRNA expression and the abundance of TEMs and Angs were correlated with clinicopathological characteristics and survival. RESULTS: MiR-126 was downregulated in 76.7% of all CCA samples, with high relative expression associated with smaller tumours and reduced lymph node metastasis. High Ang-1 expression was associated with less lymphangiosis carcinomatosa and better histological grading (all p < 0.05). The absence of TEMs in iCC correlated with elevated CA19-9 levels. High relative miR-126 and low miR-128 levels were associated with improved survival in iCC and HC, respectively (all p < 0.05). High miR-126, low miR-128 and TEMs were independent prognostic factors for recurrence-free and overall survival (all p < 0.05). CONCLUSIONS: These results suggest that angiogenic miRNAs, Angs and TEMs are of prognostic value in CCA. In addition to the possible functional links between angiogenic miRNA expression profiles, Angs and immune-cell responses by TEMs, these data have clinical implications as novel diagnostic tools.