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Colorectal cancer (CRC) is one of the most common malignancies worldwide. Patients with CRC may need chemotherapy (CTx) in a neoadjuvant, adjuvant or palliative setting through the course of the disease. Unfortunately, its effect is limited by chemoresistance and chemotoxicity. Novel more effective and non‑toxic CTx regimens are needed to further improve CRC treatment outcomes. Thus, the present study was designed to test the hypothesis that non‑toxic sulforaphane (SF) is effective against CRC and has additive effects in combination with conventional 5‑fluorouracil, oxaliplatin and folinic acid (FOLFOX) CTx in vitro . Highly metastatic human colon cancer cells, CX‑1, and fibroblasts were treated with FOLFOX ± SF. Cell viability was assessed using an MTT assay. The level of apoptosis and the expression of apoptotic proteins were measured by TUNEL assay and quantitative PCR analysis. Aldehyde dehydrogenase isoform 1 (ALDH1) and multidrug resistance protein 2 (MRP2) levels were evaluated. The ability of cells to form spheroids was measured in three‑dimensional cell culture. SF alone and in combination with FOLFOX effectively decreased the viability of the CX‑1 cells, promoted apoptosis within the CX‑1 cells, prevented cellular spheroid formation and decreased ALDH1 activity. However, SF promoted MRP2 expression and protein levels. In conclusion, SF together with conventional FOLFOX has additive anticancer effects against highly metastatic human CRC in vitro .

Aim of the Study: Hepatocyte transplantation has been discussed as an alternative to liver transplantation in selected cases of acute and chronic liver failure and metabolic diseases. Immediately after infusion of hepatocytes, hypoxia-related cell injury is inevitable. N-acetylcysteine (NAC) has been suggested to attenuate hypoxic damage. This study's objective was to evaluate NAC's protective effect in a model of hypoxia-related hepatocyte injury., Material and Methods: HepG2 cells were used as a model for hepatocytes and were cultured under standardized hypoxia or normoxia for 24 hours with or without NAC. Growth kinetics were monitored using trypan blue staining. The activation of apoptotic pathways was measured using quantitative real-time PCR for Bcl-2/Bax and p53. The proportions of vital, apoptotic and necrotic cells were verified by fluorescence activated cell sorting using annexin V-labelling. The expression of hypoxia inducible factor 1 (HIF-1) was measured indirectly using its downstream target vascular endothelial growth factor A (VEGF-A)., Results: After NAC, cell proliferation increased under both hypoxia and normoxia by 528% and 320% ( p < 0.05), while VEGF-A expression decreased under normoxia by 67% and 37% ( p < 0.05). Compared to cells treated without NAC under hypoxia, the Bcl-2/Bax ratio increased significantly in cells treated with NAC. This finding was confirmed by an increased number of vital cells in FACS analysis., Conclusions: NAC protects hepatocytes from hypoxic injury and ultimately activates anti-apoptotic pathways.

Background: After gastrectomy or esophagectomy, esophagogastrostomy and esophagojejunostomy are commonly used for reconstruction. Water-soluble contrast swallow is often used as a routine screening to exclude anastomotic leakage during the first postoperative week. In this retrospective study, the sensitivity and specificity of oral water-soluble contrast swallow for the detection of anastomotic leakage and its clinical symptoms were analysed., Material/methods: Records of 104 consecutive total gastrectomies and distal esophagectomies were analysed. In all cases, upper gastrointestinal contrast swallow with the use of a water-soluble contrast agent was performed on the 5 th postoperative day. Extravasation of the contrast agent was defined as anastomotic leakage. When anastomotic insufficiency was suspected but no extravasation was present, a computed tomography (CT) scan and upper endoscopy were performed., Results: Oral contrast swallow detected 7 anastomotic leaks. Based on CT-scans and upper endoscopy, the true number of anastomotic leakage was 15. The findings of the oral contrast swallow were falsely positive in 4 and falsely negative in 12 patients, respectively. The sensitivity and specificity of the oral contrast swallow was 20% and 96%, respectively., Conclusions: Routine radiological contrast swallow following total gastrectomy or distal esophagectomy cannot be recommended. When symptoms of anastomotic leakage are present, a CT-scan and endoscopy are currently the methods of choice., Competing Interests: Statement There is no conflict of interest. There was no funding of this original research. An ethical approval was not required.

BACKGROUND The model for end-stage liver disease (MELD) is a laboratory-based scoring system for assessing the severity of liver disease. Since 16 December 2006, MELD-based organ allocation for liver transplantation (LT) has been established in Germany to prioritize the sickest patients. The present study was designed to evaluate the effect of using different laboratories on the calculated MELD score, despite the use of mandatory round-robin testing for comparable of inter-laboratory results. MATERIAL AND METHODS Blood samples were taken from 10 patients with liver disease. Bilirubin, creatinine, and INR were measured in 6 different laboratories. The patients' MELD scores were calculated and the inter-laboratory variability was compared. RESULTS The highest discrepancy between the laboratories was 5 MELD score points and the highest inter-laboratory difference for bilirubin, INR, and creatinine was 4.6, 1.2, and 0.99 mg/dl, respectively. Pairwise comparison of the laboratory values showed a significant inter-laboratory difference (p<0.05). CONCLUSIONS Results clearly demonstrate, for the first time, that liver allocation for LT in Germany is based on nationwide noncomparable laboratory readouts for the MELD score.

Neo-angiogenesis is important for tumor growth. Glycine is a non-toxic amino acid with suspected anti-angiogenic effects. This study was designed to evaluate anti-angiogenic effects of glycine in colorectal cancer. Glycine was added to cultures of human and rat colorectal cancer cells (CRC), and endothelial cells (HUVEC). Glycine's direct impact was monitored using MTT assays. Angiogenesis in HUVEC was monitored using 3D sprouting and migration assays. VEGF and CRC-conditioned media were used to stimulate angiogenesis. The glycine receptor (GlyR) was detected using Western blotting and inhibited using strychnine. The WAG-Rij/CC-531 model of metastatic CRC was used to evaluate glycine's impact in vivo. Tumor growth and vessel density were monitored in rats fed with or without 5 % glycine for 14 days. VEGF and conditioned media significantly increased proliferation, migration, and capillary formation to up to 267 %. Glycine completely neutralized this effect and strychnine completely blunted glycine's effect. GlyR was detected in HUVEC. Tumor volume, weight, and vessel density decreased by 35 % (p = 0.02), 34 % (p = 0.03), and 55 % (p = 0.04) in glycine-fed animals. Glycine inhibits angiogenic signaling of endothelial cells and tumor growth. Glycine would be a promising additive to standard and targeted cancer therapies.

Background . After pancreaticoduodenectomy (PD), pancreatic fistulas (PF) are a frequent complication. Infusions may compromise anastomotic integrity. This retrospective analysis evaluated associations between intraoperative fluid excess and PF. Methods . Data on perioperative parameters including age, sex, laboratory findings, histology, infusions, surgery time, and occurrence of grade B/C PF was collected from all PD with pancreaticojejunostomy (PJ) performed in our department from 12/2011 till 02/2015. The glomerular filtration rate (GFR), infusion rate, and the ratio of both and its association with PF were calculated. ROC analysis was employed to identify a threshold. Results . Complete datasets were available for 83 of 86 consecutive cases. Median age was 66 years (34-84; 60% male), GFR was 93 mL/min (IQR 78-113), and surgery time was 259 min (IQR 217-307). Intraoperatively, 13.6 mL/min (7-31) was infused. In total, n = 18 (21%) PF occurred. When the infusion : GFR ratio exceeded 0.15, PF increased from 11% to 34% ( p = 0.0157). No significant association was detected for any of the other parameters. Conclusions . This analysis demonstrates for the first time an association between intraoperative fluid excess and PF after PD with PJ even in patients with normal renal function. A carefully patient-adopted fluid management with due regard to renal function may help to prevent postoperative PF., Competing Interests: The authors declare that they have no competing interests.

BACKGROUND Data on low-exposure calcineurin inhibitor therapy with mycophenolate mofetil (MMF) in de novo liver transplant patients are limited and restricted to tacrolimus. MATERIAL AND METHODS Twenty-eight patients receiving cyclosporine and MMF at a single center were identified retrospectively and categorized as low-exposure or standard-exposure CsA (median concentration <80 ng/mL [n=16] or ≥80 ng/mL [n=12] during days 1-7) and analyzed to 12 weeks post-transplant. RESULTS Biopsy-proven acute rejection (Banff ≥4) occurred in 3 low-CsA patients and no standard-CsA patients (p=0.238); graft failure occurred in 4 and zero patients, respectively (p=0.113); no graft loss was attributable to rejection. Mean (SD) estimated GFR at baseline and week 12 was 79.5 (45.3) and 79.3 (24.5) mL/min/1.73 m2 in the low-CsA group (p=0.508), and 106.0 (66.9) and 86.7 (23.2) mL/min/1.73 m2 in the standard-CsA group (p=0.093). Estimated GFR decreased significantly in patients with good baseline renal function (≥80 mL/min/1.73 m2) in the standard-CsA (p=0.028) and increased markedly in patients with poor function (≤60 mL/min/1.73 m2) given low-CsA (p=0.043). There was no significant between-group difference regarding incidence of infections. CONCLUSIONS These preliminary findings suggest that immunosuppressive efficacy is maintained with low-exposure CsA and MMF in de novo liver transplant patients and good baseline renal function may be better preserved, but no benefit for infections was observed.

Background: In patients with end-stage liver disease, liver transplantation is the only available curative treatment. Although the outcome and quality of life in the patients have improved over the past decades, primary dys- or nonfunction (PDF/PNF) can occur. Early detection of PDF and PNF is crucial and could lead to individual therapies. This study was designed to identify early markers of reperfusion injury and PDF in liver biopsies taken during the first hour after reperfusion., Methods: Biopsies from donor livers were prospectively taken as a routine during the first hour after reperfusion. Recipient data, transaminases and outcome were routinely monitored. In total, 10 biopsy specimens taken from patients with 90-day mortality and PDF, and patients with long-term survival but without PDF were used for DNA microarrays. Markers that were significantly up- or down-regulated in the microarray were verified using quantitative real-time PCR., Results: Age, indications and labMELD score were similar in both groups. Peak-transaminases during the first week after transplantation were significantly different in the two groups. In total, 20 differentially regulated markers that correlated to PDF were identified using microarray analysis and verified with quantitative real-time PCR., Conclusions: The markers identified in this study could predict PDF at a very early time point and might point to interventions that ameliorate reperfusion injury and thus prevent PDF. Identification of patients and organs at risk might lead to individualized therapies and could ultimately improve outcome.

Background: Although animal studies models are frequently used for the purpose of attenuating ischemia reperfusion injury (IRI) in liver transplantation (LT), many of pharmacological agents have not become part of clinical routine., Methods: A search was performed using the PubMed database to identify agents, from which 58 articles containing 2700 rat LT procedures were selected. The identified pharmacological agents were categorized as follows: I - adenosine agonists, nitric oxide agonists, endothelin antagonists, and prostaglandins, II - Kupffer cell inactivator, III - complement inhibiter, IV - antioxidant, V - neutrophil inactivator, VI -anti-apoptosis agent, VII - heat shock protein and nuclear factor kappa B inducer, VIII - metabolic agent, IX - traditional Chinese medicine, and X - others. Meta-analysis using 7-day-survival rate was also performed with Mantel-Haenszel's Random effects model., Results: The categorization revealed that the rate of donor-treated experiments in each group was highest for agents from Group II (70%) and VII (71%), whereas it was higher for agents from Group V (83%) in the recipient-treated experiments. Furthermore, 90% of the experiments with agents in Group II provided 7-day-survival benefits. The Risk Ratio (RR) of the meta-analysis was 2.43 [95% CI: 1.88-3.14] with moderate heterogeneity. However, the RR of each of the studies was too model-dependent to be used in the search for the most promising pharmacological agent., Conclusion: With regard to hepatic IRI pathology, the categorization of agents of interest would be a first step in designing suitable multifactorial and pleiotropic approaches to develop pharmacological strategies.

Background: New strategies for immunosuppression (IS) after liver transplantation (LTx) are in part responsible for the increased patient and graft survival seen over time. With a few basic exceptions-notably the continued use of steroids and calcineurin inhibitors (CNIs)-IS drugs and regimens being used today are different from those used 30 years ago. While graft loss due to acute or chronic rejection has become rare, the side effect burden of IS drugs exerts a significant toll on patients., Concepts/trends: CNIs continue to form the backbone of IS regimens, although their use is hampered by nephrotoxicity and other adverse effects. Consequently, a variety of CNI reduction or withdrawal strategies have formed the basis of clinical trials or entered into clinical practice. These trials have included the use of everolimus, an mTOR inhibitor, and anti-interleukin-2 receptor antibodies. Basiliximab, as well as other lymphocyte nondepleting and depleting agents, have shown benefit in induction regimens., Summary: Along with steroid reduction or elimination, current strategies for IS after LTx continue to explore novel combinations of agents, with an aim toward striking a balance between diminution of rejection and the need for avoiding adverse effects of the IS drugs. Long-term maintenance strategies are also discussed in this review, as is development of tolerance and antibody-mediated rejection.

Background and Aims: Liver transplantation is the only curative treatment for end-stage liver disease. While waiting list mortality can be predicted by the MELD-score, reliable scoring systems for the postoperative period do not exist. This study's objective was to identify risk factors that contribute to postoperative mortality., Methods: Between December 2006 and March 2011, 429 patients underwent liver transplantation in our department. Risk factors for postoperative mortality in 266 consecutive liver transplantations were identified using univariate and multivariate analyses. Patients who were <18 years, HU-listings, and split-, living related, combined or re-transplantations were excluded from the analysis. The correlation between number of risk factors and mortality was analyzed., Results: A labMELD ≥20, female sex, coronary heart disease, donor risk index >1.5 and donor Na+>145 mmol/L were identified to be independent predictive factors for postoperative mortality. With increasing number of these risk-factors, postoperative 90-day and 1-year mortality increased (0-1: 0 and 0%; 2: 2.9 and 17.4%; 3: 5.6 and 16.8%; 4: 22.2 and 33.3%; 5-6: 60.9 and 66.2%)., Conclusions: In this analysis, a simple score was derived that adequately identified patients at risk after liver transplantation. Opening a discussion on the inclusion of these parameters in the process of organ allocation may be a worthwhile venture.

Hepatocellular carcinoma (HCC) is a highly vascularized tumor with limited susceptibility to chemotherapy. Modern targeted therapies are aimed at specific properties of this neoplasm. Glycine is a simple non-essential amino acid with potential antiangiogenic effects. In this study, the amino acid's effect on angiogenic signaling in an in vitro model of HCC was evaluated. HepG2 and Huh7 cells were treated with glycine-free DMEM supplemented with 0, 0.01, 0.1, 1.0, 2.0, 5.0 and 10 mM glycine. The direct effects of glycine on the viability of HCC cells were monitored using MTT assay. To detect angiogenic signaling, mRNA and protein levels of vascular endothelial growth factor (VEGF-A) were measured using RT-PCR and Western Blot assays. To determine whether or not glycine receptors (GlyR) played a significant role, the specific antagonist, strychnine, was used as a direct inhibitor. Western Blotting was performed to show the presence of GlyR. While there was no direct pro- or antiproliferative effect of either glycine or strychnine in both cell lines, glycine was shown to significantly decrease VEGF-A expression on mRNA and protein level up to 63 % in both cell lines. This effect was blunted by the presence of strychnine. GlyR was also identified in both cell lines. Glycine decreases GlyR-dependent, VEGF-A-mediated, angiogenic signaling in human HCC and thus might be a promising additive to chemotherapy treatment strategies for highly vascularized tumors.

Background: Machine perfusion (MP) in solid organ transplantation has been a topic of variable importance for decades. At the dawn of organ transplantation, MP was one of the standard techniques for preservation; today's gold standard for organ preservation for transplantation is cold storage (CS). The outcome after transplantation of solid organs has tremendously improved over the last five decades. MP has been continuously under investigation and may be an option for organ preservation in selected cases; however, there is only little evidence from clinical trials that can be used to advocate for MP as a routine organ preservation method., Methods: This article reviews the current knowledge on MP in the field of solid organ transplantation with special focus on findings from clinical trials., Conclusion: Especially in heart and lung transplantation, MP seems to be a promising tool to improve postoperative outcome, but a general evidence-based recommendation for or against an application of MP cannot be given due to the lack of the highest level of clinical evidence. Gold standards such as CS should not be left behind without good reason. Randomized clinical trials are desperately needed to further improve outcome and for better understanding of the underlying pathophysiological mechanisms.

Unlabelled: Hepatocellular carcinoma (HCC) is the fifth most common malignancy worldwide with a poor prognosis and limited therapeutic options. To aid the development of novel immunological interventions, we studied the breadth, frequency, and tumor-infiltration of naturally occurring CD8(+) T-cell responses targeting several tumor-associated antigens (TAA). We used overlapping peptides spanning the entire alpha-fetoprotein (AFP), glypican-3 (GPC-3), melanoma-associated gene-A1 (MAGE-A1) and New York-esophageal squamous cell carcinoma-1 (NY-ESO-1) proteins and major-histocompatibility-complex-class-I-tetramers specific for epitopes of MAGE-A1 and NY-ESO-1 to analyze TAA-specific CD8(+) T-cell responses in a large cohort of HCC patients. After nonspecific expansion in vitro, we detected interferon-γ (IFN-γ)-producing CD8(+) T cells specific for all four TAA in the periphery as well as in liver and tumor tissue. These CD8(+) T-cell responses displayed clear immunodominance patterns within each TAA, but no consistent hierarchy was observed between different TAA. Importantly, the response breadth was highest in early-stage HCC and associated with patient survival. After antigen-specific expansion, TAA-specific CD8(+) T cells were detectable by tetramer staining but impaired in their ability to produce IFN-γ. Furthermore, regulatory T cells (Treg) were increased in HCC lesions. Depletion of Treg from cultures improved TAA-specific CD8(+) T-cell proliferation but did not restore IFN-γ-production., Conclusion: Naturally occurring TAA-specific CD8(+) T-cell responses are present in patients with HCC and therefore constitute part of the normal T-cell repertoire. Moreover, the presence of these responses correlates with patient survival. However, the observation of impaired IFN-γ production suggests that the efficacy of such responses is functionally limited. These findings support the development of strategies that aim to enhance the total TAA-specific CD8(+) T-cell response by therapeutic boosting and/or specificity diversification. However, further research will be required to help unlock the full potential of TAA-specific CD8(+) T-cell responses., (© 2014 The Authors. Hepatology published by Wiley on behalf of the American Association for the Study of Liver Diseases.)

Background: Oxidative stress is a major factor in the development of ischemia reperfusion injury (IRI) after heart transplantation. The isothiocyanate found in broccoli - sulforaphane (SFN) - is an indirect antioxidant that acts by inducing Nrf2-dependent Phase 2 enzymes, such as NAD(P)H-quinone oxidoreductase 1, glutathione reductase, glutathione peroxidase, and the cardioprotective enzymes, haemoxygenase-1 (HO-1) and thioredoxin (Trx-1), participate in adaptive and protective responses to oxidative stress and various inflammatory stimuli. The aim of this study was to ameliorate IRI following heart transplants by recipient preconditioning., Material and Methods: Male Lewis rats were randomly divided into groups of n=10 animals each for heart donation. Heart grafts underwent 18 hours of cold storage in histidine-tryptophanketoglutarate (HTK) solution. Preconditioning of recipients with sulforaphane was performed 24 hours before transplantation., Results: SFN significantly decreased serum levels of TnT, CK, CK-MB, LDH, AST, and ALT, which correlated with better graft function scores and improved survival prognosis. Pretreated recipients showed significantly decreased expression of iNOS, caspase 3, and HIF-1a., Conclusions: Our results indicate that recipient preconditioning with SFN protects the heart from IRI after experimental transplantation.

Background: Sulforaphane is a naturally occuring antioxidative and anti-inflammatory isothiocyanat. In this study, its impact on experimental kidney transplantation was evaluated., Material and Methods: Male Brown Norway rats (n=112) were used as experimental animals. Donor kidneys were harvested and stored for 12 hours in HTK-solution at 4°C. D,L-Sulforaphane (4.4 mg/kg BW; 0.2ml) or normal saline (0.2 ml) was given i.v. to the recipients 24 and 1 hour before, and 6 hours after transplantation. Recipients were nephrectomized bilaterally and subsequently transplantation was performed. After 6 and 48 hours, biopsies were taken and processed for light and electron microscopy. Graft function was monitored using serum values of creatinine and BUN after 6 and 24 hours. Quantitative real-time PCR was used to detect differences in SOD2-gene expression after 6 hours and apoptotic activity was detected after 6 hours using propidium iodide flow cytometry., Results: Recipient preconditioning improved reperfusion damage index from 12.8±1.6 in controls to 8.8±1.8 (p<0.001). Serum levels of creatinine and BUN decreased from 4.29±0.25 mg/dl and 119±23 mg/dl in controls to 3.65±0.7 mg/dl and 81±19 mg/dl (p<0.05). The number of severely injured tubules decreased (p<0.05). Apoptotic activity was increased in SFN-treated rats. Mitochondrial microstructure was better preserved after SFN, while SOD 2 gene expression increased (p<0.05)., Conclusions: SFN ameliorates ischemia/reperfusion injury after KTx, most likely through anti-oxidative effects.

Cell transplantation is a treatment option for diabetes, metabolic liver disease in children, and leukemia. Except for the latter indication, solid organ transplantation is one of the available therapies but can be replaced by cell transplantation. However, due to the limited amount of cells that can be transplanted and due to rejection, results of cell transplants are still inferior to solid organ transplantation; there is a general shortage of donor organs, and cell isolation is limited to organs which cannot be transplanted as a whole for anatomic reasons. Therefore, alternatives to islets and beta cells are needed. There are some cells which can be generated from the recipient and would not be rejected; still, immunosuppression would be required to prevent reoccurrence of type I diabetes unless durable tolerance to beta cells could be induced. Generating beta cells for transplant from the recipient would help to overcome the lack of available organs. Moreover, understanding the underlying mechanisms of differentiation of these cells into beta-like cells would deepen our understanding of both pathophysiology and development of diabetes mellitus type I. This article examines embryonal stem cells, induced pluripotent cells, mesenchymal stromal cells, and hepatocytes as potential alternatives to beta-cell transplantation., (© 2013 John Wiley & Sons A/S.)

Background: Ischemia/reperfusion injury is an obstacle especially in steatotic livers, including those with steatosis induced by acute toxic stress. Recently, a modified histidine-tryptophan-ketoglutarate (HTK) solution, HTK-N, has been developed. This solution contains N-acetylhistidine, amino acids, and iron chelators. This study was designed to test the effects of HTK-N on preservation injury to rat livers after acute toxic injury., Methods: Microvesicular steatosis was induced by a single dose of ethanol (8 g/kg BW). Livers were harvested and stored at 4 °C for 8 h with HTK or HTK-N before transplantation. Tissue and blood samples were taken at 1, 8, and 24 h after reperfusion to compare serum liver enzymes (aspartate aminotransferase, alanine aminotransferase, and lactate dehydrogenase), standard histology, and immunohistochemistry for myeloperoxidase (MPO), caspase-3, and inducible nitric oxide synthase. Survival was compared after 1 week. For statistics, Analysis of Variance and t test were used., Results: HTK-N improved survival from 12.5% in HTK to 87.5% (p < 0.05). Furthermore, liver enzymes were decreased to 2-75% of HTK values (p < 0.05). Necrosis and leukocyte infiltration and MPO, caspase-3, and iNOS expression after transplantation were decreased (p < 0.05)., Conclusions: This study demonstrates that HTK-N protects liver grafts with microvesicular steatosis caused by acute toxic injury from cold ischemic injury better than standard HTK most likely via inhibition of hypoxic injury and oxidative stress and amelioration of the inflammatory reaction occurring upon reperfusion.

Mesenchymal stem cell (MSC) transplantation is suggested for therapy of end-stage liver disease, due to e.g. liver cancer and metastasis. Liver transplantation is the only therapeutic option so far but donor organs are short. Also, the availability of allogeneic human MSCs for liver regeneration is limited. Therefore, we evaluated the suitability of porcine bone marrow MSCs from semi-adult pigs and found that morphology, surface expression pattern and multilineage differentiation are similar to those of human MSCs. Porcine MSCs differentiated to a hepatocyte-like phenotype and expressed porcine mRNA of typical liver proteins. However, hepatocyte-like MSCs failed to express the corresponding proteins and did not produce glycogen and urea as primary porcine hepatocytes do. Porcine MSCs were immunotolerated, since they did not activate resting human PBMCs, and were not attacked by human activated PBMCs. However, porcine MSCs led to enhanced proliferation of human pre-activated PBMCs suggesting that immunotoleration of porcine MSCs in the human system has limitations. Together, the potential of porcine MSCs for xenogenous use in human liver therapy is promising but needs further evaluation prior to clinical use.

Introduction: In 2006, model for end-stage liver disease (MELD)-based allocation was implemented in the Eurotransplant (ET) region. Sick patients, who in general require more resources, are prioritized. In this analysis, the effect of MELD on costs for liver transplantation (LTx) was assessed., Methods: Total costs for LTx before and after implementation of MELD were identified in 256 patients from January 2005-December 2007. Forty-nine patients (Re-LTx, HU listings, and 30-d mortality) were excluded from further analysis. The costs of LTx in 207 patients have been correlated with their corresponding labMELD; 84 and 123 LTx before and after implementation of MELD were compared, and patient survival was monitored., Results: A positive correlation exists between labMELD and costs (r(2) = 0.28; p < 0.05). Only nominal correlation existed between the Child-Pugh classification and costs. The labMELD scores can be stratified into four groups (I: 6-10, II: 11-18, III: 19-24, and IV: >24), with an increase of €15.672 ± 2.233 between each group (p < 0.05). Recipients' labMELD at the time of LTx increased significantly in the MELD-based allocation system. Costs increased by €11.650/patient (p < 0.05), while median survival decreased from 1219 to 869 d (p < 0.05)., Conclusion: LabMELD-based allocation increased total costs of LTx. In accordance with other studies, the sickest patients need the most resources., (© 2011 John Wiley & Sons A/S.)

Experimental data suggest that melatonin decreases inflammatory changes after major liver resection, thus positively influencing the postoperative course. To assess the safety of a preoperative single dose of melatonin in patients undergoing major liver resection, a randomized controlled double-blind pilot clinical trial with two parallel study arms was designed at the Department of General and Transplantation Surgery, Ruprecht-Karls-University, Heidelberg. A total of 307 patients, who were referred for liver surgery, were screened. One hundred and thirteen patients, for whom a major liver resection (≥3 segments) was scheduled, were eligible. Sixty-three eligible patients refused to participate, and therefore, 50 patients were randomized. A preoperative single dose of melatonin (50 mg/kg BW) dissolved in 250 mL of milk was administered through the gastric tube after the intubation for general anesthesia. Controls were given the same amount of microcrystalline cellulose. Primary endpoint was safety. Secondary endpoints were postoperative complications. Melatonin was effectively absorbed with serum concentrations of 1142.8 ± 7.2 ng/mL (mean ± S.E.M.) versus 0.3 ± 7.8 ng/mL in controls (P < 0.0001). Melatonin treatment resulted in lower postoperative transaminases over the study period (P = 0.6). There was no serious adverse event in patients after melatonin treatment. A total of three infectious complications occurred in either group. A total of eight noninfectious complications occurred in five control patients, whereas three noninfectious complications occurred in three patients receiving preoperative melatonin (P = 0.3). There was a trend toward shorter ICU stay and total hospital stay after melatonin treatment. Therefore, a single preoperative enteral dose of melatonin is effectively absorbed and is safe and well tolerated in patients undergoing major liver surgery., (© 2011 John Wiley & Sons A/S.)

Hepatotoxic side effects of neoadjuvant chemotherapy for colorectal liver metastases increase perioperative morbidity and mortality. Glycine protects liver from injury in various animal models. Thus, this study was designed to assess its effect on liver after chemotherapy. Sprague-Dawley rats (200-220 g) were fed a synthetic diet containing 5% glycine for 5 days. Subsequently, chemotherapy (FOLFIRI: irinotecan, folinic acid and fluorouracil, or FOLFOX: oxaliplatin, folinic acid and fluorouracil) was administered at standard doses. Transaminases, histology, immunohistochemistry and in vivo microscopy were used to index liver injury, to monitor intrahepatic microperfusion and activation of Kupffer cells. Glycine significantly decreased transaminases after chemotherapy to 25-50% of control values (p < 0.05). Microvesicular steatosis was significantly reduced from 18.5 ± 3.4 and 57.1 ± 8.6% in controls to 9.5 ± 1.8 and 37.7 ± 4.4% after FOLFIRI and FOLFOX, respectively. Furthermore, phagocytosis of latex beads was reduced by about 50%, while leukocyte adherence in central and midzonal subacinar zones decreased to 60-80% after glycine (p < 0.05). Glycine significantly reduced expression of inducible nitric oxide synthase after chemotherapy, while hepatic microcirculation was increased (p < 0.05). This study shows for the first time that glycine reduces chemotherapy-induced liver injury. The underlying mechanisms most likely include Kupffer cells and an improved intrahepatic microperfusion.

Background: IRI still is a major problem in liver surgery due to warm ischemia and organ manipulation. Steatosis is not only induced by diabetes, hyperalimentation, alcohol and toxins, but also chemotherapy given before resection. Since steatotic livers are prone to Kupffer cell-dependent IRI, protection of steatotic livers is of special interest. This study was designed to compare the effect of taurine and glycine on IRI in steatotic livers., Materials and Methods: Steatosis was induced with ethanol (7 g/kg b.w.; p.o.) in female SD rats. Ten minutes after inactivation of Kupffer cells with taurine or glycine (300 mM; i.v.), left liver lobes underwent 60 minutes of warm ischemia. Controls received the same volume of valine (300 mM; i.v.) or normal saline. After reperfusion, white blood cell-endothelial interactions and latex-bead phagocytosis by Kupffer cells were investigated. Liver enzymes were measured to estimate injury. For statistical analysis, ANOVA and Student's t-test were used., Results: Glycine and taurine significantly decreased leukocyte- and platelet-endothelium interactions and latex-bead phagocytosis (p < 0.05). Liver enzymes were significantly lower after glycine and taurine (p < 0.05)., Conclusions: This study shows that preconditioning with taurine or glycine is equally effective in preventing injury to fatty livers most likely via Kupffer cell-dependent mechanisms., (© 2011 John Wiley & Sons Ltd.)

Aim: To evaluate quality of life (QoL) after curative liver resection and identify variables associated with decreased QoL., Methods: From October 2001 to July 2004, 323 patients underwent liver resection. At 3-36 mo after discharge, 188 patients were disease free. QoL was assessed using the Short Form (SF)-12 Health Survey with mental and physical component scales (SF-12 MCS and PCS), supplemented with generic questions concerning pain and liver-specific items., Results: Sixty-eight percent (128/188) returned the questionnaire, which was completed in 75% (96/128) of cases. Median SF-12 PCS and MCS were 46.7 (interquartile range: 34.2-53.9) and 54.1 (42.8-58.2). Fifty percent were pain free with a median symptom score of 1.75 (1.38-2.13). PCS was higher after major hepatectomy [57% (55/96)] compared to minor resection (P = 0.0049), which represented an improved QoL. QoL was not affected by sex but by age compared to the general German population. MCS was higher after liver surgery for metastatic disease [55.9 (47.5-58.8)] compared to primary carcinoma [49.6 (36.5-55.1)] and benign disease [49.2 (37.7-56.3)] (P = 0.0317). There was no correlation between length of postoperative period and QoL. Pain, deficiencies in everyday life and a high symptom score significantly decreased MCS and PCS., Conclusion: Most patients were only marginally affected even after major liver resection; however, minor complications were associated with decreased SF-12 MCS and PCS and need careful attention.

Reperfusion injury remains one of the major problems in transplantation. Free radicals and disturbance of microcirculation are the supposed main contributors. Recent evidence shows that Danshen, a traditional Chinese drug used in vascular diseases, can scavenge radicals and improve microcirculation. This study investigates its effect on liver transplantation (LTx). Before organ recovery, female Sprague-Dawley rats (210-240 g) received intravenous Danshen or the same volume of Ringer solution as control. LTx was performed after 1 h of cold storage. Microperfusion, leukocyte-endothelium interaction and latex-bead phagocytosis were evaluated with in vivo microscopy. Survival, transaminases and histology were assessed. Immunohistology was used for TNF-alpha levels. anova and Fisher's exact test were employed for statistical analyses as appropriate. Survival increased from 60% in controls to 100% (P < 0.05). AST and LDH decreased from 3969 +/- 1255 U/l and 15444 +/- 5148 U/l in controls to 1236 +/- 410 U/l and 5039 +/- 1594 U/l, respectively (P < 0.05). In vivo microscopy revealed decreased leukocyte-adherence and increased blood flow velocity in sinusoidal zones after administration of Danshen (P < 0.05), while latex-bead phagocytosis was found in 60% of controls (P < 0.05). The TNF-alpha index decreased from 2.08 +/- 0.09 in controls to 1.09 +/- 0.09 (P < 0.05). This study clearly demonstrates hepatoprotective effects after experimental LTx, which can be explained via anti-oxidative effects, improved microcirculation and decreased Kupffer cell activation.

Background: Concomitant treatment in addition to intervention may influence the primary outcome, especially in complex interventions such as surgical trials. Evidence-based standards for perioperative care after distal pancreatectomy, however, have been rarely defined. This study's objective was therefore to identify and analyse the current basis of evidence for perioperative management in distal pancreatectomy., Methods: A standardised questionnaire was sent to 23 European centres recruiting patients for a randomized controlled trial (RCT) on open distal pancreatectomy that would compare suture versus stapler closure of the pancreatic remnant (DISPACT trial, ISRCTN 18452029). Perioperative strategies (e.g., bowel preparation, pain management, administration of antibiotics, abdominal incision, drainages, nasogastric tubes, somatostatin, mobilisation and feeding regimens) were assessed. Moreover, a systematic literature search in the Medline database was performed and retrieved meta-analyses and RCTs were reviewed., Results: All 23 centres returned the questionnaire. Consensus for thoracic epidural catheters (TECs), pain treatment and transverse incisions was found, as well as strong consensus for the placement of intra-abdominal drainages and perioperative single-shot antibiotics. Also, there was consensus that bowel preparation, somatostatin application, postoperative nasogastric tubes and intravenous feeding might not be beneficial. The literature search identified 16 meta-analyses and 19 RCTs demonstrating that bowel preparation, somatostatin therapy and nasogastric tubes can be omitted. Early mobilisation, feeding and TECs seem to be beneficial for patients. The value of drainages remains unclear., Conclusion: Most perioperative standards within the centres participating in the DISPACT trial are in accordance with current available evidence. The need for drainages requires further investigation., Clinical Trial Registration: ISRCTN 18452029.

Free radicals are involved in pathophysiology of ischemia/reperfusion injury (IRI). Melatonin is a potent scavenger of reactive oxygen and nitrogen species. Thus, this study was designed to elucidate its effects in a model of rat kidney transplantation. Twenty Lewis rats were randomly divided into 2 groups (n = 10 animals each). Melatonin (50 mg/kg BW) dissolved in 5 mL milk was given to one group via gavage 2 hr before left donor nephrectomy. Controls were given the same volume of milk only. Kidney grafts were then transplanted into bilaterally nephrectomized syngeneic recipients after 24 hr of cold storage in Histidine-Tryptophan-Ketoglutarate solution. Both graft function and injury were assessed after transplantation through serum levels of blood urea nitrogen (BUN), creatinine, transaminases, and lactate dehydrogenase (LDH). Biopsies were taken to evaluate tubular damage, the enzymatic activity of superoxide dismutase (SOD) and lipid hydroperoxide (LPO), and the expression of NF-kBp65, inducible nitric oxide synthase (iNOS), caspase-3 as indices of oxidative stress, necrosis, and apoptosis, respectively. Melatonin improved survival (P < 0.01) while decreasing BUN, creatinine, transaminases, and LDH values up to 39-71% (P < 0.05). Melatonin significantly reduced the histological index for tubular damage, induced tissue enzymatic activity of SOD while reducing LPO. At the same time, melatonin down-regulated the expression of NF-kBp65, iNOS, and caspase-3. In conclusion, donor preconditioning with melatonin protected kidney donor grafts from IRI-induced renal dysfunction and tubular injury most likely through its anti-oxidative, anti-apoptotic and NF-kB inhibitory capacity.

Recent findings suggest that the presence of cancer stem cells could be linked with patients' survival. We profiled suggested cancer stem cell markers in tissue specimens of hepatocellular carcinoma and colorectal carcinoma liver metastases. About 1% of cells co-expressed cancer stem cell antigens, but there was no correlation between the amount of CD133(+), CD133(+)/CD44(+) or CD133(+)/CD24(-) cells and the patients' clinical-pathological status or with the cancer stem cell marker-positive cells localization. CD133(+) and CD133(-) fractions of Huh7 cells did not differ in migratory properties. Therefore, presence of markers alone should be taken with caution as single prognostic parameters.

Danshen (DS) is used for treatment of various ischemic events in the traditional Chinese medicine. Hence, this study was designed to investigate its effect on ischemia/reperfusion injury (IRI) after experimental kidney transplantation (eKTx). Nephrectomized Sprague-Dawley rats underwent eKTx. Some animals were infused with 1.5 ml DS 10 min before surgery. Kidney grafts were transplanted after cold storage for 20 h in Histidine-Tryptophane-Ketoglutarate solution. After reperfusion blood samples were collected for blood urinary nitrogen (BUN), creatinine, lactate dehydrogenase (LDH), and alanine transaminase. Further, tissue was assessed for morphologic and pathophysiologic changes. Donor preconditioning with DS (DS-d) significantly decreased BUN, creatinine, LDH, and aspartate aminotransferase to 65-97% of controls while preconditioning of the recipient (DS-r) decreased values to 58-82% (P < 0.05). Tubular damage and caspase-3 decreased significantly in both DS-d and DS-r (DS-d: 96% and 67%, DS-r: 83% and 75% of controls) while heat shock protein 72 and superoxide dismutase increased significantly (DS-d: 143% and 173%, DS-r: 166% and 194% of controls). Further, inducible nitric oxide synthase and tumor necrosis factor-alpha decreased (DS-d: 84% and 61%, DS-r: 79% and 67% of controls) after DS. Preconditioning of both donors and recipients with DS significantly reduces IRI and thus improves graft function after eKTx.

Great hope is set in the use of mesenchymal stem cells for gene therapy and regenerative medicine. Since the frequency of this subpopulation of stem cells in bone marrow is low, mesenchymal stem cells are expanded ex vivo and manipulated prior to experimental or clinical use. Different methods for isolation and expansion are available, but the particular effect on the stem cell character is unclear. While the isolation of mesenchymal stem cells by density centrifugation followed by selection of the plastic adherent fraction is frequently used, the composition of expansion media differs. Thus, in the present study we cultured mesenchymal stem cells isolated from five healthy young volunteers in three widely used expansion media and performed a detailed analysis of the effect on morphology, proliferation, clonogenicity, passaging, differentiation and senescence. By this way we clearly show that the type of expansion medium used determines the stem cell character and time of senescence which is critical for future gene therapeutic and regenerative approaches using mesenchymal stem cells.

Objective: The liver is the primary site of hematopoiesis during fetal development; it has been shown that thrombopoietin (TPO) produced by the liver during fetal development is a major regulator of megakaryocytopoiesis. As maximum liver growth and hematopoiesis occur simultaneously, we hypothesized that TPO may act as a growth factor for hepatic progenitors. Therefore, the influence of TPO on the proliferation of fetal hepatic progenitors in vitro compared with that of adult hepatocytes was analyzed. The expression of the TPO receptor, c-mpl, was investigated in fetal and adult liver., Methods: Cell proliferation was measured by bromodeoxyuridine incorporation and total cell counts. TPO and c-mpl gene expression was investigated by reverse transcription polymerase chain reaction. The cell surface expression of c-mpl was analyzed in fetal and adult human liver by immunohistochemistry., Results: Hepatic progenitors of fetal and adult liver but not hepatocytes expressed the TPO receptor, c-mpl, on the cell surface. Fetal hepatic progenitors expressed mRNA for TPO and its receptor. TPO stimulated cell proliferation and increased cell numbers of cultured rat fetal hepatic progenitors but not adult hepatocytes., Conclusion: We conclude that TPO acts in addition to its known role in megakaryocytopoiesis as a growth factor for hepatic progenitors but not hepatocytes in vitro; thus, TPO represents a growth factor for hepatic progenitors during fetal liver development.

The clinical experience using a novel technique of liver resection with vascular staplers for dissection of hepatic parenchyma, was documented most recently in a prospective manner. These data have clearly demonstrated for the first time that stapler hepatectomy is a safe and fast dissection technique in major liver surgery (e.g. hepatectomy) which is feasible in a routine clinical setting.

Today, liver transplantation is still the only curative treatment for liver failure due to end-stages liver diseases. Donor organ shortage, high cost and the need of immunosuppressive medications are still the major limitations in the field of liver transplantation. Thus, alternative innovative cell-based liver directed therapies, e.g. liver tissue engineering, are under investigation with the aim, that in future an artificial liver tissue could be created and be used for the replacement of the liver function in patients. Using cells instead of organs in this setting should permit (i) expansion of cells in an in vitro phase, (ii) genetic or immunological manipulation of cells for transplantation, (iii) tissue typing and cryopreservation in a cell bank, and (iv) the ex vivo genetic modification of patient's own cells prior re-implantation. Function and differentiation of liver cells are influenced by the three-dimensional organ architecture. The use of polymeric matrices permits the three dimensional formation of a neo-tissue and specific stimulation by adequate modification of the matrix-surface which might be essential for appropriate differentiation of transplanted cells. Additionally, culturing hepatocytes on three dimensional matrices permits culture in a flow bioreactor system with increased function and survival of the cultured cells. Based on bioreactor technology, bioartificial liver devices (BAL) are developed for extracorporeal liver support. Although BALs improved clinical and metabolic conditions, increased patient survival rates have not been proven yet. For intra-corporeal liver replacement, a concept which combines Tissue Engineering using three-dimensional, highly porous matrices with cell transplantation could be useful. In such a concept, whole liver mass transplantation, long term engraftment and function as well as correction of a metabolic defect in animal models could be achieved with a principally reversible procedure. Future studies have to investigate, which environmental conditions and transplantation system would be most suitable for the development of artificial functional liver tissue including blood supply for a potential use in a clinical setting.

Aim: To investigate the hepatocytic differentiation of mesenchymal stem cells (MSCs) in co-cultures with fetal liver cells (FLC) and the possibility to expand differentiated hepatocytic cells., Methods: MSCs were marked with green fluorescent protein (GFP) by retroviral gene transduction. Clonal marked MSCs were either cultured under liver stimulating conditions using fibronectin-coated culture dishes and medium supplemented with stem cell factor (SCF), hepatocyte growth factor (HGF), epidermal growth factor (EGF), and fibroblast growth factor 4 (FGF-4) alone, or in presence of freshly isolated FLC. Cells in co-cultures were harvested, and GFP+ or GFP- cells were separated using fluorescence activated cell sorting. Reverse transcription-polymerase chain reaction (RT-PCR) for the liver specific markers cytokeratin-18 (CK-18), albumin, and alpha-fetoprotein (AFP) was performed in different cell populations., Results: Under the specified culture conditions, rat MSCs co-cultured with FLC expressed albumin, CK-18, and AFP-RNA over two weeks. At wk 3, MSCs lost hepatocytic gene expression, probably due to overgrowth of the cocultured FLC. FLC also showed a stable liver specific gene expression in the co-cultures and a very high growth potential., Conclusion: The rat MSCs from bone marrow can differentiate hepatocytic cells in the presence of FLC in vitro and the presence of MSCs in co-cultures also provides a beneficial environment for expansion and differentiation of FLC.

Stem cells are interesting candidates as a new source for cell/organ culture or cell transplantation concepts. So far it is believed that the hepatoblast is the common progenitor cell during fetal liver development. In previous studies two distinct fractions of liver cells were found during development: cells co-expressing Thy1 and CK-18 (cytokeratin-18) and cells expressing CK-18 only. In this study we cultured Thy1-positive and Thy1-negative hepatic progenitors isolated from collagenase digested fetal rat livers after depletion of OX43/OX44-positive hematopoietic cells. The cells were cultured on a collagen-I matrix in a medium containing epidermal growth factor, insulin, and fetal calf serum. Thy1-positive cells isolated from ED16, ED18, or ED20 livers showed significantly enhanced cell growth compared with Thy1-negative cells during the culture period. Both cell types showed expression of the liver-specific genes CK-18, albumin and alpha-feto-protein at the beginning of the culture period, as assessed by reverse-transcription polymerase chain reaction and immunocytochemistry. The growth of Thy1-positive cells was significantly higher when compared with Thy1-negative cells and declined with maturation of the liver. The data suggest a stem cell-like growth potential of Thy1-positive fetal hepatic cells. Thus, these cells might be useful for concepts of cell-based therapies. However, further efforts must be undertaken to define the biological, ethical, and legal aspects of using fetal cells.

Cell transplantation and tissue engineering with liver cells are currently under investigation as experimental therapies for certain liver diseases. In this study we evaluated a fibrin-based gel matrix as carrier for hepatocytes in culture. Furthermore, a novel technique for direct intrahepatic injection of fibrin gel-immobilized hepatocytes was developed and evaluated in a rat model. Hepatocytes were harvested from rats. Fibrin matrix was generated with modified fibrin sealant. Cells, in medium containing epidermal growth factor and insulin, were seeded in a drop of fibrin matrix onto plastic culture dishes. Cell numbers were assessed by DNA content. Hepatocyte differentiation was evaluated by reverse transcription-polymerase chain reaction (RT-PCR) and immunohistology (IH) for cytokeratin (CK)-18 and albumin. PKH26-labeled fibrin gel-immobilized hepatocytes were transplanted into liver by direct injection underneath the capsule. Fluorescence microscopy of explanted liver was performed to identify PKH26+ donor cells. Neotissue was characterized by IH for the markers CK-18, ED1, and desmin. Culture in a fibrin matrix allowed stable cell numbers and three-dimensional neotissue formation. RT-PCR and IH showed preservation of liver-specific markers CK-18 and albumin in vitro. Transplanted cells were identified by fluorescence microscopy after 2 and 7 days. CK-18 and desmin staining showed integration of hepatocytes and hepatic stellate cells into the host liver. Fibrin matrix is an appropriate environment for hepatocytes in culture. Direct intrahepatic injection of fibrin gel-immobilized hepatocytes is technically feasible. We conclude that fibrin gel immobilization is an attractive tool for the development of tissue engineering-based liver support systems.

Aim: The origin of putative liver cells from distinct bone marrow stem cells, e.g. hematopoietic stem cells or multipotent adult progenitor cells was found in recent in vitro studies. Cell culture experiments revealed a key role of growth factors for the induction of liver-specific genes in stem cell cultures. We investigated the potential of rat mesenchymal stem cells (MSC) from bone marrow to differentiate into hepatocytic cells in vitro. Furthermore, we assessed the influence of cocultured liver cells on induction of liver-specific gene expression., Methods: Mesenchymal stem cells were marked with green fluorescent protein (GFP) by retroviral gene transduction. Clonal marked MSC were either cultured under liver stimulating conditions using fibronectin-coated culture dishes and medium supplemented with SCF, HGF, EGF, and FGF-4 alone, or in presence of freshly isolated rat liver cells. Cells in cocultures were harvested and GFP+ or GFP- cells were separated using fluorescence activated cell sorting. RT-PCR analysis for the stem cell marker Thy1 and the hepatocytic markers CK-18, albumin, CK-19, and AFP was performed in the different cell populations., Results: Under the specified culture conditions, rat MSC cocultured with liver cells expressed albumin-, CK-18, CK-19, and AFP-RNA over 3 weeks, whereas MSC cultured alone did not show liver specific gene expression., Conclusion: The results indicate that (1) rat MSC from bone marrow can differentiate towards hepatocytic lineage in vitro, and (2) that the microenvironment plays a decisive role for the induction of hepatic differentiation of rMSC.