Sulforaphane has an additive anticancer effect to FOLFOX in highly metastatic human colon carcinoma cells.

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 non­toxic CTx regimens are needed to further improve CRC treatment outcomes. Thus, the present study was designed to test the hypothesis that non­toxic sulforaphane (SF) is effective against CRC and has additive effects in combination with conventional 5­fluorouracil, oxaliplatin and folinic acid (FOLFOX) CTx in vitro. Highly metastatic human colon cancer cells, CX­1, 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 three­dimensional cell culture. SF alone and in combination with FOLFOX effectively decreased the viability of the CX­1 cells, promoted apoptosis within the CX­1 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.

N-acetylcysteine protects hepatocytes from hypoxia-related cell injury.

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.

Routine Use of Contrast Swallow After Total Gastrectomy and Esophagectomy: Is it Justified?

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 5th 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.

Impact of Inter-Laboratory Variability on Model of End-Stage Liver Disease (MELD) Score Calculation.

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.

Intraoperative Fluid Excess Is a Risk Factor for Pancreatic Fistula after Partial Pancreaticoduodenectomy.

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.

Glycine inhibits angiogenesis in colorectal cancer: role of endothelial cells.

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.

A Retrospective Comparison of Mycophenolate Mofetil with Low-Exposure Cyclosporine Versus Standard Cyclosporine Therapy in De Novo Liver Transplant Patients.

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.

Early markers of reperfusion injury after liver transplantation: association with primary dysfunction.

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.

Prediction of postoperative mortality in liver transplantation in the era of MELD-based liver allocation: a multivariate analysis.

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.

Current strategies for immunosuppression following liver transplantation.

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.

Machine perfusion in solid organ transplantation: where is the benefit?

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.

Glycine inhibits angiogenic signaling in human hepatocellular carcinoma cells.

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.

Immunodominance and functional alterations of tumor-associated antigen-specific CD8+ T-cell responses in hepatocellular carcinoma.

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.

A systematic review of pharmacological treatment options used to reduce ischemia reperfusion injury in rat liver transplantation.

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.

Sulforaphane protects hearts from early injury after experimental transplantation.

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.

Sulforaphane decreases kidney injury after transplantation in rats: role of mitochondrial damage.

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.

Alternatives to islet transplantation: future cell sources of beta-like cells.

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.

HTK-N, a modified HTK solution, decreases preservation injury in a model of microsteatotic rat liver transplantation.

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.

Evaluation of porcine mesenchymal stem cells for therapeutic use in human liver cancer.

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.

LabMELD-based organ allocation increases total costs of liver transplantation: a single-center experience.

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.