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Background: Cardiovascular disease (CVD) is the leading cause of death after renal transplantation (RT). Sex-specific differences in CVD in the general population are well known. The aim of this study was to evaluate sex-specific differences in prevalence and course of subclinical cardiovascular (CV) organ damage in RT recipients during the first year after RT. Methods: In a prospective longitudinal study, we enrolled 121 patients (male 64%, age 51 ± 15 years). CV risk factors, left ventricular mass index (LVMI), and pulse wave velocity (PWV) were assessed at time of RT and 1 year later. Results: Women showed less prediagnosed CVD and better blood pressure (BP) control, and were less likely to be treated with calcium channel blockers (CCBs). Despite similar transplant function, LVMI increased in women and decreased in men ( p  = 0.027). In multivariable analysis, changes in LVMI were independently associated with female sex and systolic BP. Importantly, women receiving CCBs showed a decrease in LVMI and PWV. Conclusions: Our findings indicate a sex-specific association between RT and changes in LVMI. CCBs seem to have a positive impact on CV risk after RT, especially in women. Further studies on the effect of sex and CCB use in RT recipients are warranted.

Regulatory T cells (Tregs) are crucial in controlling allospecific immune responses. However, studies in human kidney recipients regarding the contribution of polyspecific Tregs have provided differing results and studies on alloreactive Tregs are missing completely. In this retrospective study, we specifically analyzed activated CD4 + CD25 high FOXP3 + GARP + Tregs in 17 patients of a living donor kidney transplantation cohort longitudinally over 24 months by flow cytometry (FOXP3: forkhead box protein 3, GARP: glycoprotein A repetitions predominant). We could demonstrate that Tregs of patients with end-stage renal disease (ESRD) are already pre-activated when compared to healthy controls. Furthermore, even though total CD4 + CD25 high FOXP3 + Treg numbers decreased in the first three months after transplantation, frequency of activated Tregs increased significantly representing up to 40% of all peripheral Tregs. In a cohort of living donor kidney transplantation recipients with stable graft function, frequencies of activated Tregs did not correlate with the occurrence of acute cellular rejection or chronic graft dysfunction. Our results will be important for clinical trials using adoptive Treg therapy after kidney transplantation. Adoptively transferred Tregs could be important to compensate the Treg loss at month 3, while they have to compete within the Treg niche with a large number of activated Tregs.

Hepatitis C virus (HCV) and the malaria parasite Plasmodium use the membrane protein CD81 to invade human liver cells. Here we mapped 33 host protein interactions of CD81 in primary human liver and hepatoma cells using high-resolution quantitative proteomics. In the CD81 protein network, we identified five proteins which are HCV entry factors or facilitators including epidermal growth factor receptor (EGFR). Notably, we discovered calpain-5 (CAPN5) and the ubiquitin ligase Casitas B-lineage lymphoma proto-oncogene B (CBLB) to form a complex with CD81 and support HCV entry. CAPN5 and CBLB were required for a post-binding and pre-replication step in the HCV life cycle. Knockout of CAPN5 and CBLB reduced susceptibility to all tested HCV genotypes, but not to other enveloped viruses such as vesicular stomatitis virus and human coronavirus. Furthermore, Plasmodium sporozoites relied on a distinct set of CD81 interaction partners for liver cell entry. Our findings reveal a comprehensive CD81 network in human liver cells and show that HCV and Plasmodium highjack selective CD81 interactions, including CAPN5 and CBLB for HCV, to invade cells., Competing Interests: The authors declare no conflict of interest.

With a single exception, all isolates of hepatitis C virus (HCV) require adaptive mutations to replicate efficiently in cell culture. Here, we show that a major class of adaptive mutations regulates the activity of a cellular lipid kinase, phosphatidylinositol 4-kinase IIIα (PI4KA). HCV needs to stimulate PI4KA to create a permissive phosphatidylinositol 4-phosphate-enriched membrane microenvironment in the liver and in primary human hepatocytes (PHHs). In contrast, in Huh7 hepatoma cells, the virus must acquire loss-of-function mutations that prevent PI4KA overactivation. This adaptive mechanism is necessitated by increased PI4KA levels in Huh7 cells compared with PHHs, and is conserved across HCV genotypes. PI4KA-specific inhibitors promote replication of unadapted viral isolates and allow efficient replication of patient-derived virus in cell culture. In summary, this study has uncovered a long-sought mechanism of HCV cell-culture adaptation and demonstrates how a virus can adapt to changes in a cellular environment associated with tumorigenesis.

Objective: Chronically HCV-infected orthotopic liver transplantation (OLT) recipients appear to have improved outcomes when their immunosuppressive regimen includes a mammalian target of rapamycin (mTOR) inhibitor. The mechanism underlying this observation is unknown., Design: We used virological assays to investigate mTOR signalling on the HCV replication cycle. Furthermore, we analysed HCV RNA levels of 42 HCV-positive transplanted patients treated with an mTOR inhibitor as part of their immunosuppressive regimen., Results: The mTOR inhibitor rapamycin was found to be a potent inhibitor for HCV RNA replication in Huh-7.5 cells as well as primary human hepatocytes. Half-maximal inhibition was observed at 0.01 µg/mL, a concentration that is in the range of serum levels seen in transplant recipients and does not affect cell proliferation. Early replication cycle steps such as cell entry and RNA translation were not affected. Knockdown of raptor, an essential component of mTORC1, but not rictor, an essential component of mTORC2, inhibited viral RNA replication. In addition, overexpression of raptor led to higher viral RNA replication, demonstrating that mTORC1, but not mTORC2, is required for HCV RNA replication. In 42 HCV-infected liver-transplanted or kidney-transplanted patients who were switched to an mTOR inhibitor, we could verify that mTOR inhibition decreased HCV RNA levels in vivo., Conclusions: Our data identify mTORC1 as a novel HCV replication factor. These findings suggest an underlying mechanism for the observed benefits of mTOR inhibition in HCV-positive OLT recipients and potentiate further investigation of mTOR-containing regimens in HCV-positive recipients of solid organ transplants., Competing Interests: Conflicts of Interest: None declared., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/.)

Hepatitis C virus (HCV) and human immunodeficiency virus (HIV-1) transmissions among people who inject drugs (PWID) continue to pose a challenging global health problem. Here, we aimed to analyse a universally applicable inactivation procedure, namely microwave irradiation, as a safe and effective method to reduce the risk of viral transmission. The exposure of HCV from different genotypes to microwave irradiation resulted in a significant reduction of viral infectivity. Furthermore, microwave irradiation reduced viral infectivity of HIV-1 and of HCV/HIV-1 suspensions indicating that this inactivation may be effective at preventing co-infections. To translate microwave irradiation as prevention method to used drug preparation equipment, we could further show that HCV as well as HIV-1 infectivity could be abrogated in syringes and filters. This study demonstrates the power of microwave irradiation for the reduction of viral transmission and establishment of this safety strategy could help reduce the transmission of blood-borne viruses.

Background: Hepatobiliary surgery with biliodigestive anastomosis (BDA) results in a loss of the sphincter of Oddi with consecutive ascension of bacteria into the bile system which may cause cholangitis in the postoperative course., Methods: Patients who received reconstruction with a BDA after hepatobiliary surgery were analyzed retrospectively for their postoperative course of disease depending on intraoperatively obtained bile cultures and antibiotic prophylaxis., Results: Two hundred forty-three patients were included in the analysis, 49.4 % of whom had received endoscopic stenting before the operation. Stenting was significantly associated with the presence of drug-resistant bacteria in the intraoperatively obtained bile sample (p < 0.001, OR = 4.09). Of all patients, 14.4 % developed postoperative cholangitis. This was significantly associated with the postoperative length of stay in the intensive care unit (p = 0.002, OR = 1.035). The highest incidence of postoperative cholangitis was found in patients with cholangiocellular carcinoma (n = 12, p = 0.046, OR = 2.178). Patients were more likely to harbor strains with resistance against the antibiotic that was given intraoperatively., Conclusion: The risk for the presence of drug-resistant bacteria is increased by preoperative stenting of the common bile duct. Bile culture by intraoperative swabs can be altered by the perioperative antibiotic prophylaxis as it induces microbiological selection in the common bile duct.

Objective: In HCV infected individuals graft infection occurs shortly after orthotopic liver transplantation (OLT). We aimed to describe the composition of the inflammatory response at this time, how it affects the HCV replication cycle and identify novel proviral and antiviral factors., Design: We used a Luminex assay to quantify 50 inflammatory mediators in sera before and shortly after OLT. In vitro grown HCV based on the JFH-1 isolate were used to characterise the effects of patient sera and individual mediators on HCV., Results: Although the mediator composition is highly variable between individuals, sera drawn immediately post-OLT significantly enhance HCV infectivity compared with control sera from before OLT in about half of the cases. Among 27 non-interferon inflammatory mediators fibroblast growth factor (FGF)-2 stood out as it enhanced HCV RNA replication and release of infectious particles. The effect was concentration-dependent and detectable in dividing and non-dividing cells. Moreover, pharmacological inhibition of FGF-2 receptor signalling abrogated the enhancing effect of FGF-2 and inhibited HCV replication in the absence of serum FGF-2 suggesting that HCV replication is dependent on basal activation of the FGF-2 triggered signalling pathway. Finally, in individuals with chronic HCV infection with high viral load, serum FGF-2 was significantly higher compared with those with low viral load., Conclusions: Although no single mediator may account for this effect, serum shortly post-OLT enhances HCV infection. FGF-2 is a novel endogenous driver of HCV replication and a potential therapeutic target., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/)

Objective: A monoclonal antibody (mAb) against the leukocyte common antigen CD45 (RT7 in rats) could facilitate bone marrow transplantation (BMT). This study in rats evaluates a depletive rat anti-RT7a mAb as isolated tool for BMT conditioning without using irradiation or any chemotherapeutic / immunosuppressive agent., Methods: The model used a CD45 di-allelic polymorphism (RT7a/RT7b). The anti-RT7a mAb was intravenously administered to LEW.1W rats (RT1uRT7a) at 5, 10 and 15 mg/kg. 1x10(8) BM cells of MHC syngeneic (RT1u), MHC disparate (RT1l) or MHC haploidentical (RT1u/l) donors were transplanted. All BM donor strains carried the RT7b allele so that their CD45+ cells were not affected by the anti-RT7a mAb. Recipients were monitored for reconstitution and donor-chimerism in blood leukocytes., Results: mAb dosages of 5 or 10 mg/kg were myelosuppressive, whereas 15 mg/kg was myeloablative. Multi-lineage donor-chimerism at day 100 indicated engraftment of MHC syngeneic BM after any used mAb dosage (5 mg/kg: 46+/-7%; 10 mg/kg: 62+/-5%; 15 mg/kg: 80+/-4%). MHC disparate BM resulted in autologous reconstitution after conditioning by 10 mg/kg of the mAb and caused transient chimerism ending up in death associated with aplasia after conditioning by 15 mg/kg of the mAb. MHC haploidentical BM (F1 to parental) engrafted only after conditioning by 15 mg/kg (chimerism at day 100: 78+/-7%). Abandonment of α/β TCR+ cell depletion from BM grafts impaired the engraftment process after conditioning using 15 mg/kg of the mAb in the MHC syngeneic setting (2 of 6 recipients failed to engraft) and the MHC haploidentical setting (3 of 6 recipients failed)., Conclusion: This depletive anti-RT7a mAb is myelosuppressive and conditions for engraftment of MHC syngeneic BM. The mAb also facilitates engraftment of MHC haploidentical BM, if a myeloablative dose is used. RT7b expressing, BM-seeded α/β TCR+ cells seem to impair the engraftment process after myeloablative mAb conditioning.

Hepatitis C virus (HCV) particles closely mimic human very-low-density lipoproteins (VLDL) to evade humoral immunity and to facilitate cell entry. However, the principles that govern HCV association with VLDL components are poorly defined. Using an siRNA screen, we identified ABHD5 (α/β hydrolase domain containing protein 5, also known as CGI-58) as a new host factor promoting both virus assembly and release. ABHD5 associated with lipid droplets and triggered their hydrolysis. Importantly, ABHD5 Chanarin-Dorfman syndrome mutants responsible for a rare lipid storage disorder in humans were mislocalised, and unable to consume lipid droplets or support HCV production. Additional ABHD5 mutagenesis revealed a novel tribasic motif that does not influence subcellular localization but determines both ABHD5 lipolytic and proviral properties. These results indicate that HCV taps into the lipid droplet triglyceride reservoir usurping ABHD5 lipase cofactor function. They also suggest that the resulting lipid flux, normally devoted to VLDL synthesis, also participates in the assembly and release of the HCV lipo-viro-particle. Altogether, our study provides the first association between the Chanarin-Dorfman syndrome protein and an infectious disease and sheds light on the hepatic manifestations of this rare genetic disorder as well as on HCV morphogenesis.

Within the field of regenerative medicine, the liver is of major interest for adoption of regenerative strategies due to its well-known and unique regenerative capacity. Whereas therapeutic strategies such as liver resection and orthotopic liver transplantation (OLT) can be considered standards of care for the treatment of a variety of liver diseases, the concept of liver cell transplantation (LCTx) still awaits clinical breakthrough. Success of LCTx is hampered by insufficient engraftment/long-term acceptance of cellular allografts mainly due to rejection of transplanted cells. This is in contrast to the results achieved for OLT where long-term graft survival is observed on a regular basis and, hence, the liver has been deemed an immune-privileged organ. Immune responses induced by isolated hepatocytes apparently differ considerably from those observed following transplantation of solid organs and, thus, LCTx requires refined immunological strategies to improve its clinical outcome. In addition, clinical usage of LCTx but also related basic research efforts are hindered by the limited availability of high quality liver cells, strongly emphasizing the need for alternative cell sources. This review focuses on the various immunological aspects of LCTx summarizing data available not only for hepatocyte transplantation but also for transplantation of non-parenchymal liver cells and liver stem cells.

Background: This study strives to define prognostic models for outcome after surgery for malignant pancreatic neuroendocrine tumors., Methods: Forty-one patients were included. Prognostic models for mortality and disease recurrence were developed with multivariate binary logistic regression., Results: The proposed prognostic model for tumor recurrence risk after surgery in percentage (AUROC = 0.774, 95%CI = 0.611-0.937) is: Risk in % = Exp(Y)/(1 + Exp(Y)), with Y = -4.360 + (0.015 × tumor diameter in cm) + (0.010 × preoperative platelet count in thousand/μl) + (1.077 × distant metastases, if yes = 1; if no = 0) + (-0.026 × Ki-67-positive cells in %) + (-1.086 × upper abdominal pain, if yes = 1; if no = 0). The proposed prognostic model for observed 3-year survival probability after surgery in % (AUROC = 0.932, 95%CI = 0.857-0.999) is: Survival probability in % = Exp(Y)/(1 + Exp(Y)), with Y = -12.492 + (0.054 × preoperative platelet count in thousand/μl) + (0.112 × minimal distance of the resection margin from the tumor in mm) + (-1.574 × number of positive lymph nodes) + (2.292 × histological tumor infiltration, if yes = 1; if no = 0) CONCLUSIONS: The platelet count was identified as a relevant risk factor. Proposed prognostic models with good model-fit display properties that indicate potential clinical usefulness., (© 2015 Wiley Periodicals, Inc.)

Unlabelled: To explore mechanisms of hepatitis C viral (HCV) replication we screened a compound library including licensed drugs. Flunarizine, a diphenylmethylpiperazine used to treat migraine, inhibited HCV cell entry in vitro and in vivo in a genotype-dependent fashion. Analysis of mosaic viruses between susceptible and resistant strains revealed that E1 and E2 glycoproteins confer susceptibility to flunarizine. Time of addition experiments and single particle tracking of HCV demonstrated that flunarizine specifically prevents membrane fusion. Related phenothiazines and pimozide also inhibited HCV infection and preferentially targeted HCV genotype 2 viruses. However, phenothiazines and pimozide exhibited improved genotype coverage including the difficult to treat genotype 3. Flunarizine-resistant HCV carried mutations within the alleged fusion peptide and displayed cross-resistance to these compounds, indicating that these drugs have a common mode of action., Conclusion: These observations reveal novel details about HCV membrane fusion; moreover, flunarizine and related compounds represent first-in-class HCV fusion inhibitors that merit consideration for repurposing as a cost-effective component of HCV combination therapies., (© 2015 by the American Association for the Study of Liver Diseases.)

Background. Despite aggressive intensive medical management acute liver failure (ALF) may require high-urgency liver transplantation (LTx). Available prognostic scores do not apply for all patients; reliable tools to identify individuals in need of LTx are highly required. The liver maximum function capacity test (LiMAx) might represent an appropriate option. Referring to a case of ALF after Amanita phalloides-intoxication the potential of the LiMAx-test in this setting is discussed. Presentation of Case. LiMAx was performed in a 27-year-old patient prior to and after high-urgency LTx. In accordance with clinical appearance of hepatic encephalopathy, coagulopathy, and acute kidney failure, the LiMAx-test constituted a fulminant course of ALF with hardly any detectable metabolic activity. Following LTx with a marginal donor organ (95% hepatosteatosis), uptake of liver function was demonstrated by postoperative increase of the LiMAx-value. The patient was discharged from hospital on postoperative day 26. Discussion. ALF often is associated with a critical state of the patient that requires almost immediate decision-making regarding further therapy. Application of a noninvasive liver function test might help to determine the prognosis of ALF and support decision-making for or against LTx as well as acceptance of a critical donor organ in case of a critically ill patient.

Background & Aims: Soraphen A (SorA) is a myxobacterial metabolite that inhibits the acetyl-CoA carboxylase, a key enzyme in lipid biosynthesis. We have previously identified SorA to efficiently inhibit the human immunodeficiency virus (HIV). The aim of the present study was to evaluate the capacity of SorA and analogues to inhibit hepatitis C virus (HCV) infection., Methods: SorA inhibition capacity was evaluated in vitro using cell culture derived HCV, HCV pseudoparticles and subgenomic replicons. Infection studies were performed in the hepatoma cell line HuH7/Scr and in primary human hepatocytes. The effects of SorA on membranous web formation were analysed by electron microscopy., Results: SorA potently inhibits HCV infection at nanomolar concentrations. Obtained EC50 values were 0.70 nM with a HCV reporter genome, 2.30 nM with wild-type HCV and 2.52 nM with subgenomic HCV replicons. SorA neither inhibited HCV RNA translation nor HCV entry, as demonstrated with subgenomic HCV replicons and HCV pseudoparticles, suggesting an effect on HCV replication. Consistent with this, evidence was obtained that SorA interferes with formation of the membranous web, the site of HCV replication. Finally, a series of natural and synthetic SorA analogues helped to establish a first structure-activity relationship., Conclusions: SorA has a very potent anti-HCV activity. Since it also interferes with the membranous web formation, SorA is an excellent tool to unravel the mechanism of HCV replication., (Copyright © 2015 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

The IFNL4 gene is negatively associated with spontaneous and treatment-induced clearance of hepatitis C virus infection. The activity of IFNλ4 has an important causal role in the pathogenesis, but the molecular details are not fully understood. One possible reason for the detrimental effect of IFNλ4 could be a tissue-specific regulation of an unknown subset of genes. To address both tissue and subtype specificity in the interferon response, we treated primary human hepatocytes and airway epithelial cells with IFNα, IFNλ3 or IFNλ4 and assessed interferon mediated gene regulation using transcriptome sequencing. Our data show a surprisingly similar response to all three subtypes of interferon. We also addressed the tissue specificity of the response, and identified a subset of tissue-specific genes. However, the interferon response is robust in both tissues with the majority of the identified genes being regulated in hepatocytes as well as airway epithelial cells. Thus we provide an in-depth analysis of the liver interferon response seen over an array of interferon subtypes and compare it to the response in the lung epithelium.

Unlabelled: Hepatitis C virus (HCV) is a positive-strand RNA virus that primarily infects human hepatocytes. Infections with HCV constitute a global health problem, with 180 million people currently chronically infected. Recent studies have reported that cholesterol 25-hydroxylase (CH25H) is expressed as an interferon-stimulated gene and mediates antiviral activities against different enveloped viruses through the production of 25-hydroxycholesterol (25HC). However, the intrinsic regulation of human CH25H (hCH25H) expression within the liver as well as its mechanistic effects on HCV infectivity remain elusive. In this study, we characterized the expression of hCH25H using liver biopsies and primary human hepatocytes. In addition, the antiviral properties of this protein and its enzymatic product, 25HC, were further characterized against HCV in tissue culture. Levels of hCH25H messenger RNA were significantly up-regulated both in HCV-positive liver biopsies and in HCV-infected primary human hepatocytes. The expression of hCH25H in primary human hepatocytes was primarily and transiently induced by type I interferon. Transient expression of hCH25H in human hepatoma cells restricted HCV infection in a genotype-independent manner. This inhibition required the enzymatic activity of CH25H. We observed an inhibition of viral membrane fusion during the entry process by 25HC, which was not due to a virucidal effect. Yet the primary effect by 25HC on HCV was at the level of RNA replication, which was observed using subgenomic replicons of two different genotypes. Further analysis using electron microscopy revealed that 25HC inhibited formation of the membranous web, the HCV replication factory, independent of RNA replication., Conclusion: Infection with HCV causes up-regulation of interferon-inducible CH25H in vivo, and its product, 25HC, restricts HCV primarily at the level of RNA replication by preventing formation of the viral replication factory., (© 2015 by the American Association for the Study of Liver Diseases.)

Hepatitis C virus (HCV) enters human hepatocytes through a multistep mechanism involving, among other host proteins, the virus receptor CD81. How CD81 governs HCV entry is poorly characterized, and CD81 protein interactions after virus binding remain elusive. We have developed a quantitative proteomics protocol to identify HCV-triggered CD81 interactions and found 26 dynamic binding partners. At least six of these proteins promote HCV infection, as indicated by RNAi. We further characterized serum response factor binding protein 1 (SRFBP1), which is recruited to CD81 during HCV uptake and supports HCV infection in hepatoma cells and primary human hepatocytes. SRFBP1 facilitates host cell penetration by all seven HCV genotypes, but not of vesicular stomatitis virus and human coronavirus. Thus, SRFBP1 is an HCV-specific, pan-genotypic host entry factor. These results demonstrate the use of quantitative proteomics to elucidate pathogen entry and underscore the importance of host protein-protein interactions during HCV invasion., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

Apolipoprotein E (ApoE), an exchangeable apolipoprotein, is necessary for production of infectious Hepatitis C virus (HCV) particles. However, ApoE is not the only liver-expressed apolipoprotein and the role of other apolipoproteins for production of infectious HCV progeny is incompletely defined. Therefore, we quantified mRNA expression of human apolipoproteins in primary human hepatocytes. Subsequently, cDNAs encoding apolipoproteins were expressed in 293T/miR-122 cells to explore if they complement HCV virus production in cells that are non-permissive due to limiting endogenous levels of human apolipoproteins. Primary human hepatocytes expressed high mRNA levels of ApoA1, A2, C1, C3, E, and H. ApoA4, A5, B, D, F, J, L1, L2, L3, L4, L6, M, and O were expressed at intermediate levels, and C2, C4, and L5 were not detected. All members of the ApoA and ApoC family of lipoproteins complemented HCV virus production in HCV transfected 293T/miR-122 cells, albeit with significantly lower efficacy compared with ApoE. In contrast, ApoD expression did not support production of infectious HCV. Specific infectivity of released particles complemented with ApoA family members was significantly lower compared with ApoE. Moreover, the ratio of extracellular to intracellular infectious virus was significantly higher for ApoE compared to ApoA2 and ApoC3. Since apolipoproteins complementing HCV virus production share amphipathic alpha helices as common structural features we altered the two alpha helices of ApoC1. Helix breaking mutations in both ApoC1 helices impaired virus assembly highlighting a critical role of alpha helices in apolipoproteins supporting HCV assembly. In summary, various liver expressed apolipoproteins with amphipathic alpha helices complement HCV virus production in human non liver cells. Differences in the efficiency of virus assembly, the specific infectivity of released particles, and the ratio between extracellular and intracellular infectivity point to distinct characteristics of these apolipoproteins that influence HCV assembly and cell entry. This will guide future research to precisely pinpoint how apolipoproteins function during virus assembly and cell entry.

Background: Following pig-to-primate kidney transplantation, endothelial cell activation and xenogenic activation of the recipient's coagulation eventually leading to organ dysfunction and microthrombosis can be observed. In this study, we examined the effect of a TNF-receptor fusion protein (TNF-RFP) on endothelial cell activation and coagulopathy utilizing an appropriate ex vivo perfusion system., Methods: Using an ex vivo perfusion circuit based on C1-Inhibitor (C1-Inh) and low-dose heparin administration, we have analyzed consumptive coagulopathy following contact of human blood with porcine endothelium. Porcine kidneys were recovered following in situ cold perfusion with Histidine-tryptophan-ketoglutarate (HTK) organ preservation solution and were immediately connected to a perfusion circuit utilizing freshly drawn pooled porcine or human AB blood. The experiments were performed in three individual groups: autologous perfusion (n = 5), xenogenic perfusion without any further pharmacological intervention (n = 10), or with addition of TNF-RFP (n = 5). After perfusion, tissue samples were obtained for real-time PCR and immunohistological analyses. Endothelial cell activation was assessed by measuring the expression levels of E-selectin, ICAM-1, and VCAM-1., Results: Kidney survival during organ perfusion with human blood, C1-Inh, and heparin, but without any further pharmacological intervention was 126 ± 78 min. Coagulopathy was observed with significantly elevated concentrations of D-dimer and thrombin-antithrombin complex (TAT), resulting in the formation of multiple microthrombi. Endothelial cell activation was pronounced, as shown by increased expression of E-selectin and VCAM-1. In contrast, pharmacological intervention with TNF-RFP prolonged organ survival to 240 ± 0 min (max. perfusion time; no difference to autologous control). Formation of microthrombi was slightly reduced, although not significantly, if compared to the xenogenic control. D-dimer and TAT were elevated at similar levels to the xenogenic control experiments. In contrast, endothelial cell activation, as shown by real-time PCR, was significantly reduced in the TNF-RFP group., Conclusion: We conclude that although coagulopathy was not affected, TNF-RFP is able to suppress inflammation occurring after xenoperfusion in this ex vivo perfusion model., (© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Background: Application of bone marrow cells (BMC) is a promising strategy for tolerance induction, but usually requires strong depletion of the host immune system. This study evaluates the ability of immunosuppressants to evolve tolerogenic properties of BMC in view of residual alloreactivity., Methods: The rat model used a major histocompatibility complex (MHC) class II disparate bone marrow transplantation (BMT) setting (LEW.1AR1 (RT1auu) → LEW.1AR2 (RT1aau)). Heart grafts (LEW.1WR1 (RT1uua)) were disparate for the complete MHC to recipients and for MHC class I to BMC donors. Limited conditioning was performed by total body irradiation of 6 Gy. Cyclosporine (CsA) or Sirolimus (Srl) were administered for 14 or 28 days. Transplantation of heart grafts (HTx) was performed at day 16 or at day 100 after BMT. Chimerism and changes in the T cell pool were detected by flow cytometry., Results: Mixed chimeras accepted HTx indefinitely, although the composition of the regenerated T cell pool was not changed to a basically donor MHC class II haplotype. Non-chimeric animals rejected HTx spontaneously. BMC recipients, who received HTx during T cell recovery at day 16, accepted HTx only after pre-treatment with Srl, although chimerism was lost. CsA pre-treatment led to accelerated HTx rejection as did isolated application of BMC., Conclusion: Srl evolves tolerogenic properties of allogeneic BMC to achieve indefinite acceptance of partly MHC disparate HTx despite residual alloreactivity and in particular loss of chimerism.

Matrine is a bioactive component of the traditional Chinese medical herb Sophora flavescens that has been used in China to treat various kinds of diseases including virus hepatitis. However, the molecular mechanisms underlying its hepatoprotective effects remains elusive. In the present study, primary human hepatocytes were employed to elucidate the protective effects and molecular mechanisms of matrine. We observed that low concentrations of matrine had no significant impact on albumin secretion, but high concentrations (>140 mg/L) of matrine decreased the albumin secretion in hepatocytes. Western blot data indicated that matrine at 140 mg/L at 72 h induced protein expression of CYP2A6, CYP2B6 and CYP3A4. Furthermore, high concentrations of matrine reduced LDH and AST levels and were cytotoxic to hepatocytes, leading to a decreased cell viability and total protein amount. Moreover, low concentrations of matrine, enhanced the ECOD activity and decreased the level of NO2 (-) induced by cytokines in human hepatocytes. Taken together, the present study sheds novel light on the molecular mechanisms of matrine and potential application of matrine in hepatic diseases.

Intrahepatic cholangiocarcinoma is the second most common primary liver tumor. The aim of this study was to analyze retrospectively the outcome of surgical treatment and prognostic factors. Clinical, histopathological and treatment data of 221 patients treated from 1995 to 2010 at our institution were investigated. Univariate and multivariate analysis of the patient's data was performed. Patients after R0 and R1 resection presented an overall survival of 67% and 54.5% after 1 year and 40% and 36.4% after 3 years, respectively. The survival of patients without resection of the tumor was dismal with 26% and 3.4% after 1 and 3 years, respectively. Survival after resection was not statistically different in cases with R0 versus R1 resection (P = 0.639, log rank). Univariate Cox regression revealed that higher T stages are a significant hazard for survival (P = 0.048, hazard ratio (HR): 1.211, 95% confidence interval (CI): 1.002-2.465). Patients with tumor recurrence had a significantly inferior long-term survival when compared to patients without recurrence (P < 0.001, log rank). Presence of lymph node metastasis (N1) was an independent prognostic factor for survival after resection in risk-adjusted multivariate Cox regression (P < 0.001, HR: 2.577, 95% CI: 1.742-3.813). Adjuvant chemotherapy did not improve patient survival significantly (P = 0.550, log rank). Surgical resection is still the best treatment option for intrahepatic cholangiocarcinoma regarding the patient's long-term survival. R0 and R1 resection enable both better survival rates when compared to surgical exploration without resection. T status, N status, and tumor recurrence seem to be the most important prognostic factors after resection., (© 2014 Japanese Society of Hepato-Biliary-Pancreatic Surgery.)

Background: After introduction of MELD-based allocation in Germany, decreased waiting list mortality and increased mortality after transplantation have been reported., Material and Methods: This study compares relevant outcome parameters in patients with high MELD ≥30 versus lower MELD scores in a retrospective analysis including 454 consecutively performed liver transplantations in adults (age >16 years) at Hannover Medical School between 01/01/2007 and 31/12/2012 and a follow-up until 31/12/2013. Multivariable risk-adjusted models were applied to identify independent risk factors for 90-day and long-term mortality., Results: MELD score ≥30 (n=117; 26.1%) was an independent risk factor for 90-day mortality (p=0.004, odds ratio: 3.045, 95% CI 1.439-6.498) and long-term mortality (p=0.016, hazard ratio: 1.620, 95% CI 1.095-2.396) and was associated with significantly longer hospital and intensive care unit stays (p<0.001), and death occurred in more cases earlier after transplantation (90-day mortality 21.6% vs. 13.0%; p=0.029). Portal vein thrombosis at transplantation was significantly associated with 90-day mortality after transplantation in patients with MELD scores ≥30 (p=0.041), but this was not the case for patients with MELD scores <30, although portal vein thrombosis was equally frequent in individuals of both groups (3.0% vs. 3.4%, p=0.824)., Conclusions: Results of this study suggest that liver transplant recipients with portal vein thrombosis at transplantation should be transplanted before reaching a MELD score ≥30.

Background. Surgical resection remains the best treatment option for intrahepatic cholangiocarcinoma (ICC). Two-stage liver resection combining in situ liver transection with portal vein ligation (ALPPS) has been described as a promising method to increase the resectability of liver tumors also in the case of ICC. Presentation of Case. A 46-year-old male patient presented with an ICC-typical lesion in the right liver. The indication for primary liver resection was set and planed as a right hepatectomy. In contrast to the preoperative CT-scan, the known lesion showed further progression in a macroscopically steatotic liver. Therefore, the decision was made to perform an ALPPS-procedure to avoid an insufficient future liver remnant (FLR). The patient showed an uneventful postoperative course after the first and second step of the ALPPS-procedure, with sufficient increase of the FLR. Unfortunately, already 2.5 months after resection the patient had developed new tumor lesions found by the follow-up CT-scan. Discussion. The presented case demonstrates that an intraoperative conversion to an ALPPS-procedure is safely applicable when the FLR surprisingly seems to be insufficient. Conclusion. ALPPS should also be considered a treatment option in well-selected patients with ICC. However, the experience concerning the outcome of ALPPS in case of ICC remains fairly small.

Renal transplantation is the treatment of choice for patients suffering end-stage renal disease, but as the long-term renal allograft survival is limited, most transplant recipients will face graft loss and will be considered for a retransplantation. The goal of this study was to evaluate the patient and graft survival of the 61 renal transplant recipients after second or subsequent renal transplantation, transplanted in our institution between 1990 and 2010, and to identify risk factors related to inferior outcomes. Actuarial patient survival was 98.3%, 94.8%, and 88.2% after one, three, and five years, respectively. Actuarial graft survival was 86.8%, 80%, and 78.1% after one, three, and five years, respectively. Risk-adjusted analysis revealed that only age at the time of last transplantation had a significant influence on patient survival, whereas graft survival was influenced by multiple immunological and surgical factors, such as the number of HLA mismatches, the type of immunosuppression, the number of surgical complications, need of reoperation, primary graft nonfunction, and acute rejection episodes. In conclusion, third and subsequent renal transplantation constitute a valid therapeutic option, but inferior outcomes should be expected among elderly patients, hyperimmunized recipients, and recipients with multiple operations at the site of last renal transplantation.

Acute rejection (AR) remains a major cause for long-term kidney allograft failure. Reliable immunological parameters suitable to define the pre-transplant immune state and hence the individual risk of graft rejection are highly desired to preferably adapt the immunosuppressive regimen in advance. Donor and third party alloreactivities were determined by mixed lymphocyte cultures. Soluble forms of CD25, CD30, and CD44 were detected in patients' serum by ELISA. Various lymphocyte subpopulations were measured using flow cytometry. All patients received triple immunosuppression (tacrolimus/mycophenolate mofetil/steroids) and were grouped according to biopsy results within the first year: rejection-free (RF, n = 13), borderline (BL, n = 5), or acute rejection (AR, n = 7). Patients with AR showed the highest pre-transplant alloreactivities and serum levels (sCD25/sCD30/sCD44) according to the pattern RF < BL < AR. Relying on serum analysis only, multivariate logistic regression (logit link function) yielded a prognostic score for prediction of rejection with 75.0% sensitivity and 69.2% specificity. Patients with rejection showed markedly higher pre-transplant frequencies of CD4(+) /CD8(+) T cells lacking CD28, but lower numbers of CD8(+) CD161(bright) T cells and NK cells than RF individuals. Pre-transplant immune state defined by alloreactivity, serum markers, and particular lymphocyte subsets seems to correlate with occurrence of graft rejection after kidney transplantation. A prognostic score based on pre-transplant serum levels has shown great potential for prediction of rejection episodes and should be further evaluated., (© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Background: Patients with liver failure could potentially be bridged with porcine xenogeneic liver cell transplantation. We examined species-specific differences between primary human and porcine hepatocytes in the regulation of coagulation protein expression and function., Methods: Isolated primary human and porcine hepatocytes were stimulated with either porcine or human interleukin (IL)-6 (10 ng/ml), IL-1β (10 ng/ml), and tumor necrosis factor-alpha (TNF-α, 30 ng/ml). mRNA expression of coagulation factors were measured by RT-PCR and real-time PCR. Cell culture supernatants were used for the measurement of fibrinogen by ELISA and determination of fibrin clot generation., Results: Fibrinogen expression in human hepatocytes increased after IL-6 treatment (P = 0.010) and decreased after TNF-α treatment (P = 0.005). Porcine hepatocytes displayed a lower increase in fibrinogen expression after IL-6 treatment as compared to hepatocytes of human origin (P = 0.021). Porcine hepatocytes responded contrarily following TNF-α treatment with an increased expression of fibrinogen resulting in a significant species-specific difference between human and porcine hepatocytes (P = 0.029). Fibrin polymer generation by human hepatocytes was stable and widely branched after IL-6 treatment, while stimulation with TNF-α displayed no fibrin generation at all. In contrast, treatment of porcine hepatocytes with TNF-α resulted in generation of a stable and widely branched fibrin polymer, and stimulation with IL-6 only leads to generation of partial fibrin aggregates., Conclusion: We identified species-specific differences in the regulation of fibrinogen mRNA expression and fibrin generation under inflammatory stimuli. In hepatic xenotransplantation of porcine origin, these interspecies differences might lead to a loss of physiological coagulation function and a loss of transplanted cells., (© 2014 John Wiley & Sons A/S Published by John Wiley & Sons Ltd.)

Objectives: Filoviruses such as Ebola virus and Marburg virus cause a severe haemorrhagic fever syndrome in humans for which there is no specific treatment. Since filoviruses use a complex route of cell entry that depends on numerous cellular factors, we hypothesized that there may be drugs already approved for human use for other indications that interfere with signal transduction or other cellular processes required for their entry and hence have anti-filoviral properties., Methods: We used authentic filoviruses and lentiviral particles pseudotyped with filoviral glycoproteins to identify and characterize such compounds., Results: We discovered that amiodarone, a multi-ion channel inhibitor and adrenoceptor antagonist, is a potent inhibitor of filovirus cell entry at concentrations that are routinely reached in human serum during anti-arrhythmic therapy. A similar effect was observed with the amiodarone-related agent dronedarone and the L-type calcium channel blocker verapamil. Inhibition by amiodarone was concentration dependent and similarly affected pseudoviruses as well as authentic filoviruses. Inhibition of filovirus entry was observed with most but not all cell types tested and was accentuated by the pre-treatment of cells, indicating a host cell-directed mechanism of action. The New World arenavirus Guanarito was also inhibited by amiodarone while the Old World arenavirus Lassa and members of the Rhabdoviridae (vesicular stomatitis virus) and Bunyaviridae (Hantaan) families were largely resistant., Conclusions: The ion channel blockers amiodarone, dronedarone and verapamil inhibit filoviral cell entry., (© The Author 2014. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Background: Abdominal complications after thoracic transplantation (Tx) are potentially associated with an increased risk of mortality. We recently reported about the severe outcome after bowel perforation in patients following lung transplantation (LuTx). The aim of the present study was to likewise identify the risk factors with an impact on patient survival following heart transplantation (HTx)., Methods: A retrospective analysis for the frequency and outcome of abdominal interventions following HTx was performed in 342 patients, and these data thereafter compared to a re-evaluated pool of 1,074 patients following LuTx. All patients were transplanted at Hanover Medical School, Germany, between January 2000 and October 2011., Results: The incidence for abdominal surgery was comparable between patients following HTx (n = 33; 9.6 %) and LuTx (n = 90; 8.4 %). Elective operations were more frequently performed in patients after HTx (8.5 vs. 5.1 %). In contrast, the incidence of emergency interventions was higher after LuTx (5.3 %) than that following HTx (2.3 %). Herewith associated was the mortality observed in these transplant recipients (15.3 and 9.9 % for LuTx and HTx, respectively). Leading diagnosis for emergency surgery was bowel perforation (n = 18, regarding all cases). In 11 of these patients, perforation occurred within the first 6 months after Tx and eight of them died in the course of this complication (one patient after HTx and seven patients after LuTx)., Conclusions: Abdominal complications after HTx are less frequently than after LuTx but equally correlate with a high mortality rate. In finding or even reasonable suspicion of an acute abdomen after thoracic Tx, a broad practice for extended diagnostics and a low barrier for an early explorative laparotomy thus are recommended.

Being an integral part of basic, translational and clinical research, the demand for primary human hepatocytes (PHH) is continuously growing while the availability of tissue resection material for the isolation of metabolically competent PHH remains limited. To overcome current shortcomings, this study evaluated the use of explanted diseased organs from liver transplantation patients as a potential source of PHH. Therefore, PHH were isolated from resected surgical specimens (Rx-group; n = 60) and explanted diseased livers obtained from graft recipients with low labMELD-score (Ex-group; n = 5). Using established protocols PHH were subsequently cultured for a period of 7 days. The viability and metabolic competence of cultured PHH was assessed by the following parameters: morphology and cell count (CyQuant assay), albumin synthesis, urea production, AST-leakage, and phase I and II metabolism. Both groups were compared in terms of cell yield and metabolic function, and results were correlated with clinical parameters of tissue donors. Notably, cellular yields and viabilities were comparable between the Rx- and Ex-group and were 5.3±0.5 and 2.9±0.7×106 cells/g liver tissue with 84.3±1.3 and 76.0±8.6% viability, respectively. Moreover, PHH isolated from the Rx- or Ex-group did not differ in regards to loss of cell number in culture, albumin synthesis, urea production, AST-leakage, and phase I and II metabolism (measured by the 7-ethoxycoumarin-O-deethylase and uracil-5'-diphosphate-glucuronyltransferase activity). Likewise, basal transcript expressions of the CYP monooxygenases 1A1, 2C8 and 3A4 were comparable as was their induction when treated with a cocktail that consisted of 3-methylcholantren, rifampicin and phenobarbital, with increased expression of CYP 1A1 and 3A4 mRNA while transcript expression of CYP 2C8 was only marginally changed. In conclusion, the use of explanted diseased livers obtained from recipients with low labMELD-score might represent a valuable source of metabolically competent PHH which are comparable in viability and function to cells obtained from specimens following partial liver resection.

Helicobacter hepaticus can lead to chronic hepatitis and hepatocellular carcinoma in certain strains of mice. Until now the pathogenic role of Helicobacter species on human liver tissue is still not clarified though Helicobacter species identification in human liver cancer was successful in case controlled studies. Therefore we established an in vitro model to investigate the interaction of primary human hepatocytes (PHH) with Helicobacter hepaticus. Successful co-culturing of PHH with Helicobacter hepaticus was confirmed by visualization of motile bacteria by two-photon-microscopy. Isolated human monocytes were stimulated with PHH conditioned media. Changes in mRNA expression of acute phase cytokines and proteins in PHH and stimulated monocytes were determined by Real-time PCR. Furthermore, cytokines and proteins were analyzed in PHH culture supernatants by ELISA. Co-cultivation with Helicobacter hepaticus induced mRNA expression of Interleukin-1 beta (IL-1β), Tumor necrosis factor-alpha, Interleukin-8 (IL-8) and Monocyte chemotactic protein-1 (MCP-1) in PHH (p<0.05) resulting in a corresponding increase of IL-8 and MCP-1 concentrations in PHH supernatants (p<0.05). IL-8 and IL-1β mRNA expression was induced in monocytes stimulated with Helicobacter hepaticus infected PHH conditioned media (p<0.05). An increase of Cyclooxygenase-2 mRNA expression was observed, with a concomitant increase of prostaglandin E2 concentration in PHH supernatants at 24 and 48 h (p<0.05). In contrast, at day 7 of co-culture, no persistent elevation of cytokine mRNA could be detected. High expression of intercellular adhesion molecule-1 on PHH cell membranes after co-culture was shown by two-photon-microscopy and confirmed by flow-cytometry. Finally, expression of Cytochrome P450 3A4 and albumin mRNA were downregulated, indicating an impairment of hepatocyte synthesis function by Helicobacter hepaticus presence. This is the first in vitro model demonstrating a pathogenic effect of a Helicobacter spp. on human liver cells, resulting in an inflammatory response with increased synthesis of inflammatory mediators and consecutive monocyte activation.

Salinomycin raised hope to be effective in anti-cancer therapies due to its capability to overcome apoptosis-resistance in several types of cancer cells. Recently, its effectiveness against human hepatocellular carcinoma (HCC) cells both in vitro and in vivo was demonstrated. However, the mechanism of action remained unclear. Latest studies implicated interference with the degradation pathway of autophagy. This study aimed to determine the impact of Salinomycin on HCC-autophagy and whether primary human hepatocytes (PHH) likewise are affected. Following exposure of HCC cell lines HepG2 and Huh7 to varying concentrations of Salinomycin (0-10 µM), comprehensive analysis of autophagic activity using western-blotting and flow-cytometry was performed. Drug effects were analyzed in the settings of autophagy stimulation by starvation or PP242-treatment and correlated with cell viability, proliferation, apoptosis induction, mitochondrial mass accumulation and reactive oxygen species (ROS) formation. Impact on apoptosis induction and cell function of PHH was analyzed. Constitutive and stimulated autophagic activities both were effectively suppressed in HCC by Salinomycin. This inhibition was associated with dysfunctional mitochondria accumulation, increased apoptosis and decreased proliferation and cell viability. Effects of Salinomycin were dose and time dependent and could readily be replicated by pharmacological and genetic inhibition of HCC-autophagy alone. Salinomycin exposure to PHH resulted in transient impairment of synthesis function and cell viability without apoptosis induction. In conclusion, our data suggest that Salinomycin suppresses late stages of HCC-autophagy, leading to impaired recycling and accumulation of dysfunctional mitochondria with increased ROS-production all of which are associated with induction of apoptosis.

Reference genes (RG) as sample internal controls for gene transcript level analyses by quantitative RT-PCR (RT-qPCR) must be stably expressed within the experimental range. A variety of in vitro cell culture settings with primary human hepatocytes, and Huh-7 and HepG2 cell lines, were used to determine candidate RG expression stability in RT-qPCR analyses. Employing GeNorm, BestKeeper and Normfinder algorithms, this study identifies PSMB6, MDH1 and some more RG as sufficiently unregulated, thus expressed at stable levels, in hepatocyte-like cells in vitro. Inclusion of multiple RG, quenching occasional regulations of single RG, greatly stabilises gene expression level calculations from RT-qPCR data. To further enhance validity and reproducibility of relative RT-qPCR quantifications, the ΔCT calculation can be extended (e-ΔCT) by replacing the CT of a single RG in ΔCT with an averaged CT-value from multiple RG. The use of two or three RG--here identified suited for human hepatocyte-like cells--for normalisation with the straightforward e-ΔCT calculation, should improve reproducibility and robustness of comparative RT-qPCR-based gene expression analyses.

Background: T cells are known to participate in the response to tumor cells and react with cytotoxicity and cytokine release. At the same time tumors established versatile mechanisms for silencing the immune responses. The interplay is far from being completely understood. In this study we show contacts between tumor cells and lymphocytes revealing novel characteristics in the interaction of T cells and cancer cells in a way not previously described., Methods/ Findings: Experiments are based on the usage of a hydrophilic fluorescent dye that occurs free in the cytosol and thus transfer of fluorescent cytosol from one cell to the other can be observed using flow cytometry. Tumor cells from cell lines of different origin or primary hepatocellular carcinoma (HCC) cells were incubated with lymphocytes from human and mice. This exposure provoked a contact dependent uptake of tumor derived cytosol by lymphocytes--even in CD4⁺ T cells and murine B cells--which could not be detected after incubation of lymphocytes with healthy cells. The interaction was a direct one, not requiring the presence of accessory cells, but independent of cytotoxicity and TCR engagement. Electron microscopy disclosed 100-200 nm large gaps in the cell membranes of connected cells which separated viable and revealed astonishing outcome. While the lymphocytes were induced to proliferate in a long term fashion, the tumor cells underwent a temporary break in cell division. The in vitro results were confirmed in vivo using a murine acute lymphoblastic leukemia (ALL) model. The arrest of tumor proliferation resulted in a significant prolonged survival of challenged mice., Conclusions: The reported cell-cell contacts reveal new characteristics i.e. the enabling of cytosol flow between the cells including biological active proteins that influence the cell cycle and biological behaviour of the recipient cells. This adds a completely new aspect in tumor induced immunology.

Survival of critically ill patients is significantly affected by prolonged ventilation. The goal of this study was the development of a respiratory risk score (RRS) for the prediction of 3-month mortality and prolonged ventilation after liver transplantation (LT). Two hundred fifty-four consecutive LT patients from a single center were retrospectively randomized into a training group for model design and a validation group. A receiver operating characteristic (ROC) curve analysis was used to test sensitivity and specificity. The accuracy of the predictions was assessed with the Brier score, and the model calibration was assessed with the Hosmer-Lemeshow test. Cutoff values were determined with the best Youden index. The RRS was calculated in the first 24 hours as follows: (laboratory Model for End-Stage Liver Disease score > 30 = 2.36 points) + (fresh frozen plasma > 13.5 U = 2.70 points) + (partial pressure of arterial oxygen/fraction of inspired oxygen ratio < 200 mm Hg = 2.23 points) + (packed red blood cells > 10.5 U = 3.50 points) + (preoperative mechanical ventilation = 3.87 points) +  (preoperative dialysis = 2.83 points) + (donor steatosis hepatis > 40% = 2.95 points). The RSS demonstrated high predictive accuracy, good model calibration, and c statistics > 0.7 in the training and validation groups. The RSS was able to predict 3-month mortality [cutoff = 6.64, area under the (ROC) curve (AUROC) = 0.794] and prolonged ventilation (cutoff = 3.69, AUROC = 0.798) with sensitivities of 69% and 81%, specificities of 83% and 73%, and overall model correctness of 76% and 77%, respectively. In conclusion, this study provides the first prognostic model for the prediction of 3-month mortality and prolonged ventilation after LT with high sensitivity and specificity and good model accuracy. The application of the RRS to an external cohort would be desirable for its further validation and introduction as a clinical tool for intensive care resource planning and prognostic decision making., (© 2013 American Association for the Study of Liver Diseases.)

Objectives: Prolonged nucleoside reverse transcriptase inhibitors (NRTI) exposure can lead to microvesicular steatosis. We hypothesized that thymidine analogues might interfere with autophagy in hepatocytes, a lysosomal degradation pathway implicated in cell survival and regulation of hepatocyte lipid metabolism., Design: Using HepG2 and HUH7 cell lines and primary human hepatocytes, we performed a comprehensive analysis of NRTI-mediated effects on autophagy., Methods: The impact of zidovudine (ZDV), stavudine (d4T) and lamivudine (3TC) on constitutive and induced autophagy was analyzed by fluorescent and electron microscopy, western blotting and flow cytometry. Effects on hepatocyte autophagy were correlated to cellular viability, mitochondrial dysfunction and intracellular lipid accumulation., Results: ZDV and d4T, but not 3TC, significantly inhibited both constitutive as well as stimulated autophagic activity in a dose-dependent and time-dependent manner. Inhibition of autophagy at therapeutic drug concentrations led to accumulation of dysfunctional mitochondria, increased ROS production, increased apoptosis, decreased proliferation and increased intracellular lipid accumulation. These NRTI effects could be readily resembled by pharmacological and genetic inhibition of hepatocyte autophagy., Conclusion: Our data suggest that thymidine analogues inhibit autophagy in hepatocytes, which in turn leads to increased ROS production, lipid accumulation and hepatic dysfunction. This novel mechanism could contribute to nonalcoholic fatty liver disease in HIV-infected patients.

Clinical trials have pointed out the promising role of co-stimulation blocker Belatacept for improvement of graft function and avoidance of undesired side-effects associated with calcineurin-inhibitors (CNI). However, due to the worldwide limited availability of appropriate patients, almost no data exist to assess the effects of sustained application of this immunomodulator on the recipient's immune system. The aim of this study was to reveal specific alterations in the composition of immunologic subpopulations potentially involved in development of tolerance or chronic graft rejection following long-term Belatacept therapy. For this, peripheral lymphocyte subsets of kidney recipients treated with Belatacept (n=5; average 7.8years) were determined by flow-cytometry and compared with cells from matched patients on CNI (n=9) and healthy controls (n=10). T cells capable of producing IL-17 and serum levels of soluble CD30 were quantified. Patients on CNI showed a higher frequency of CD4(+) CD161(+) Th(17) -precursors and IL-17-producing CD4(+) T cells than Belatacept patients and controls. Significantly higher serum levels of soluble CD30 were observed in CNI patients, indicating a possible involvement of the CD30/CD30L-system in Th(17) -differentiation. No differences were found concerning CD4(+) CD25(+) CD127(low) FoxP3(+) regulatory T cells. In conclusion, patients on therapy with Belatacept did not show a comparable Th(17) -profile to that seen in individuals with chronic intake of CNI. The distinct effects of Belatacept on Th(17) -immunity might prove beneficial for the long-term outcome following kidney transplantation., (© 2012 The Authors. Transplant International © 2012 European Society for Organ Transplantation.)

Background: Pancreatic endocrine tumours are often diagnosed at an advanced stage with hepatic metastasis. This study investigated whether extended resections for advanced malignant pancreatic endocrine tumours influenced disease-free and disease-specific survival., Methods: Patients who had curative resection of pancreatic endocrine tumours were analysed retrospectively for disease-free and disease-specific survival, with a focus on the role of extended surgical resection., Results: Forty-one patients were included in the analysis, 13 of whom underwent extended surgical resection in addition to pancreatic resection. This included partial liver resection in nine patients, portal vein resection in three, partial gastric resection in five and liver transplantation in three patients. There were no deaths in hospital or within 30 days. Median follow-up was 40 (range 2-239) months. Thirty-five, 24 and 13 patients survived more than 1, 3 and 5 years respectively. Patients who underwent extended resection had similar disease-specific survival to those who had pancreatic resection alone (hazard ratio (HR) 1·50, 95 per cent confidence interval (c.i.) 0·35 to 6·35; P = 0·581) but with a higher frequency of complications (odds ratio (OR) 4·28, 95 per cent c.i. 1·04 to 17·62; P = 0·044). Among patients with liver metastases, the mortality rate was higher in those in whom liver resection was not possible than in patients who had liver resection (HR 9·24, 1·00 to 85·18; P = 0·049). Patients who had liver resection had similar disease-specific survival to those without liver metastases (HR 0·84, 0·09 to 7·57; P = 0·877)., Conclusion: Extended surgical resection for locally advanced and metastatic pancreatic endocrine tumours is feasible with encouraging disease-specific survival., (Copyright © 2011 British Journal of Surgery Society Ltd. Published by John Wiley & Sons, Ltd.)

Purpose: Abdominal complications after lung transplantation (LuTx) are associated with a high mortality risk. Aim of the present study was to analyse frequency and outcome of abdominal interventions following LuTx., Methods: Retrospective analysis of the requirement of abdominal surgery including data of 754 patients undergoing LuTx at the Hannover Medical School, Germany, between January 2000 and December 2008., Results: In the course of lung transplantation, 55 patients (7%) were in need of surgical interventions due to abdominal complications. Following LuTx, 35 individuals were operated in 43 cases of emergency indication. The leading diagnosis was bowel perforation (n = 10) with surgery performed 10.4 months after LuTx, although 7 of 10 patients were operated within the first 4 weeks post-transplantation. Emergency interventions were associated with a mortality rate of 28%, 42% thereof after bowel perforation. Elective surgical treatments (n = 31) were diverse and had no influence on mortality., Conclusions: Early abdominal complications after LuTx correlate with a high mortality rate. Perforation of the bowel was the leading diagnosis with a severe impact on the outcome. Thus, in cases of an acute abdomen after LuTx, we recommend the broad use of further diagnostic measures as well as an early decision for explorative laparotomy.

Acute rejection (AR) is an important factor for the development of chronic allograft dysfunction following kidney Tx. Identification of patients who would benefit from closer clinical surveillance to allow individual tailoring of immunosuppression and hence reducing the rate of AR is highly desired. Aim of this study was to investigate the association of pre-transplant alloreactivity and frequency of regulatory T cells (T(regs) ) with AR following living-donor kidney Tx. Peripheral blood mononuclear cells were isolated from 40 patients prior to Tx. T-cell alloreactivity against donor and third-party antigen was assessed by proliferative responses in mixed lymphocyte culture and enzyme linked immunospot technique. Pre-transplant frequency of CD4(+) CD25(+) CD127(low) FoxP3(+) T(regs) was determined by flow cytometry. Experimental data were correlated with occurrence of AR. We found that patients with rejection-free (RF) post-Tx courses showed significantly lower pre-transplant alloreactivity to donor antigen compared to individuals with borderline findings (BL) or AR. For RF patients, the proliferative T-cell responses to third-party antigen were significantly higher than for stimulation with donor cells whereas lymphocytes of the AR group showed the inverse pattern. A significantly higher expression of FoxP3 within the CD4(+) CD25(+) CD127(low) subset for RF and BL compared to the AR group was observed. In conclusion, pre-transplant anti-donor alloreactivity and FoxP3 expression within the CD4(+) CD25(+) CD127(low) subpopulation might prove useful to further define the patient's risk for AR., (© 2010 John Wiley & Sons A/S.)

Monoclonal anti-CD25-antibodies are successfully applied in organ transplantation to reduce the incidence of acute graft rejection. However, targeting the CD25 molecule might not only affect activated T-cells but also regulatory T-cells (T(regs)) constitutively expressing the CD4(+)CD25(+)CD127(low)FoxP3(+) phenotype. In this study, we investigated the influence of the anti-CD25-antibody Basiliximab on the frequency of T(regs) early after kidney transplantation comparing individuals receiving/not receiving induction therapy (n = 14 and n = 7). Following Basiliximab administration, a distinct loss of CD4(+)CD25(high) T-cells was observed lasting for at least 6 weeks. This was not accompanied by a disappearance of the entire CD4(+)CD25(+)FoxP3(+) T(regs) but rather a decreased expression density of CD25 on the latter. In addition, a transient rise in CD4(+)CD25(-)FoxP3(+) T-cells was found which expressed the CD127(low) phenotype. Thus, a phenotypic shift of T(regs) from the CD25(+) to the CD25(-) compartment was suggested. This was supported by in vitro findings showing that the disappearance of CD4(+)CD25(high) cells in the presence of Basiliximab was due to down-regulation of CD25 expression meanwhile the suppressive function of these cells was maintained. In conclusion, Basiliximab therapy directly affects CD4(+)CD25(+)CD127(low)FoxP3(+) T(regs) but does not seem to be associated with functional consequences. Thus, it is unlikely that Basiliximab treatment negatively influences strategies involving T(regs) to promote tolerance after organ transplantation.

Transplantation of primary human hepatocytes is a promising approach in certain liver diseases. For the visualization of the hepatocytes during and following cell application and the ability of a timely response to potential complications, a non-invasive modality for imaging the transplanted cells has to be established. The aim of this study was to label primary human hepatocytes with micron-sized iron oxide particles (MPIOs), enabling the detection of cells by clinical magnetic resonance imaging (MRI). Primary human hepatocytes isolated from 13 different donors were used for the labelling experiments. Following the dose-finding studies, hepatocytes were incubated with 30 particles/cell for 4 hrs in an adhesion culture. Particle incorporation was investigated via light, fluorescence and electron microscopy, and labelled cells were fixed and analysed in an agarose suspension by a 3.0 Tesla MR scanner. The hepatocytes were enzymatically resuspended and analysed during a 5-day reculture period for viability, total protein, enzyme leakage (aspartate aminotransferase [AST], lactate dehydrogenase [LDH]) and metabolic activity (urea, albumin). A mean uptake of 18 particles/cell could be observed, and the primary human hepatocytes were clearly detectable by MR instrumentation. The particle load was not affected by resuspension and showed no alternations during the culture period. Compared to control groups, labelling and resuspension had no adverse effects on the viability, enzyme leakage and metabolic activity of the human hepatocytes. The feasibility of preparing MPIO-labelled primary human hepatocytes detectable by clinical MR equipment was shown in vitro. MPIO-labelled cells could serve for basic research and quality control in the clinical setting of human hepatocyte transplantation.

Demands for primary human hepatocytes are continuously increasing, while supply is insufficient due to limited cell sources. To improve cell availability, the present study investigates the influence of donor liver characteristics on the outcome of hepatocyte isolation from surgically removed liver tissue (n = 50). Hepatocytes were isolated from liver specimens using a standardized two-step collagenase perfusion technique. The patient's sex, previous chemotherapy, or histopathology have shown no influence. Donor age significantly affected the isolation outcome, but was not found suitable for predicting cell yields. Preoperative blood parameters did not correlate with cell yield, although cell function was affected: total protein, albumin synthesis, and cell viability were significantly decreased for serum gamma-glutamyl-transferase (GGT) levels >60 U/L. Specimens from patients with benign diseases gave significantly higher cell yields than tissue removed due to secondary and primary tumors, respectively. The indication for surgery is a valuable basis for identifying the most yielding specimens. Hepatocytes from donors with high GGT levels appear to show reduced functional properties.

The transplantation of primary human hepatocytes is a promising approach in the treatment of specific liver diseases. However, little is known about the fate of the cells following application. Magnetic resonance imaging (MRI) could enable real-time tracking and long-term detection of transplanted hepatocytes. The use of superparamagnetic iron oxide particles as cellular contrast agents should allow for the non-invasive detection of labelled cells on high-resolution magnetic resonance images. Experiments were performed on primary human hepatocytes to transfer the method of detecting labelled cells via clinical MRI into human hepatocyte transplantation. For labelling, Tat-peptide modified nano-sized superparamagnetic MagForce particles were used. Cells were investigated via a clinical MR scanner at 3.0 Tesla and the particle uptake within single hepatocytes was estimated using microscopic examinations. The labelled primary human hepatocytes were clearly detectable by MRI, proving the feasibility of this new concept. Therefore, this method is a useful tool to investigate the effects of human hepatocyte transplantation and to improve safety aspects of this method.

Certain cell types, especially primary human cells, favor a well-defined culture environment offering continuous supply of nutrients and oxygen and waste product removal. Several bioreactors based on special matrices or hollow fibers have been developed that provide such conditions. However, characterization of matrix re-organization or growth of tissue within these systems is possible only after culture termination. Evaluation of the influence of certain medium additives or culture conditions (e.g., temperature, oxygenation) on cell viability, expansion, and differentiation within these systems remains a challenging task. The SlideReactor, a miniaturized hollow fiber-based bioreactor, was developed to enable the observation of cells during culture. An operation concept offering predefined conditions for various cell types has been designed. For proof of concept, primary human cells (hepatocytes, fibroblasts, keratinocytes) and cell lines (HepG2, HuH7, C3A, WiDr, SkHep1) were cultured and observed. A series of experiments (n=40) showed the feasibility of the set-up; determination of process parameters and continuous observation is possible. The SlideReactor may serve as a simple and cost-efficient tool for cell characterization and optimization of cell-culture conditions.