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Abstract: We report on a small bowel transplant patient, donor/recipient seropositive (D+/R+) for cytomegalovirus (CMV), with a clinical course complicated by CMV disease. Anti-CMV prophylaxis was given for 100 days. Immunosuppression consisted of alemtuzumab, tacrolimus, mycophenolate mofetil and prednisolone. Five months posttransplant, CMV tissue invasive disease of the upper gastrointestinal tract was evident without the presence of viremia, tested by quantitative polymerase chain reaction (PCR). Complete viral load suppression was achieved with intravenous ganciclovir, followed by valganciclovir for secondary prophylaxis. Mycophenolate mofetil and prednisolone were discontinued. Shortly thereafter the patient presented with recurrent CMV and candida esophagitis. While on ganciclovir and caspofungin, the patient developed CMV tissue invasive disease of the ileal graft, with persistent absence of viremia. Foscarnet and CMV immunoglobulin were added. Viral load declined to undetectable levels; however, clinical improvement did not occur due to occurrence of graft rejection. Despite infliximab and high dose prednisolone, graft rejection was progressive, requiring surgical explantation of the graft. This case highlights the importance of additional diagnostic tools such as endoscopy including PCR analysis of tissue samples. Extension of primary antiviral prophylaxis interval up to 6 months and prolonged retreatment for recurrent CMV disease may be useful to avoid severe CMV-related complications. [Copyright &y& Elsevier]

Current strategies for immunosuppression in liver transplant ( LT) recipients include the design of protocols targeting a more individualized approach to reduce risk factors such as renal failure, cardiovascular complications and malignancies. Renal injury in LT recipients may be often multifactorial and is associated with increased risk of post-transplant morbidity and mortality. The quest for low toxicity immunosuppressive regimens has been challenging and resulted in CNI minimization protocols or CNI withdrawal and conversion to mycophenolate mofetil ( MMF) and/or mammalian target of rapamycin inhibitor-based immunosuppressive regimens. Use of antibody induction to delay CNI administration may be an option in particular in immunocompromized, critically ill patients with high MELD scores. Protocols including MMF introduction and concomitant CNI minimization have the potential to recover renal function even in the medium and long term after LT. We review on hot topics in the prevention and management of acute and chronic renal injury in LT patients . For this purpose, we present and critically discuss results from immunosuppressive studies published in the current literature or presented at recent LT meetings. [ABSTRACT FROM AUTHOR]

Background Biliary strictures after liver transplantation ( LT) are a major cause of morbidity and reduced graft survival. Aims The purpose of this study was to investigate genetic, immunological and clinical risk factors for the occurrence of post- LT ischaemic type biliary lesions ( ITBLs) and biliary anastomotic strictures ( AS). Methods Clinical and laboratory data, chemokine receptor ( CCR) genotypes, chemotactic cytokines and anti-major-histocompatibility complex antibodies in serum were investigated in 162 LT patients. Results In the univariate analysis, older donor and recipient age, partial LT, high peak aspartate aminotransaminase ( AST) levels and CC chemokine receptor 5 delta32 loss-of-function mutation ( CCR5Δ32) were associated with ITBL, whereas LT for acute liver failure ( ALF), ABO-compatible non-identical LT, presence of donor-specific anti-human leucocyte antigen ( HLA) class II antibodies and fractalkine receptor ( CX3 CR1)-249 II allele were associated with AS. In the multivariate analysis, CCR5Δ32 was an independent risk factor for ITBL, whereas LT for ALF, ABO-compatible non-identical LT, and CX3 CR1-249 II allele remained predictive for AS. Serum levels of interferon-gamma and interleukin ( IL)-6 as well as IL-10 were significantly increased in patients with biliary strictures. Conclusion Specific chemokine receptor polymorphisms of the recipient are associated with development of post- LT biliary strictures. Altered cytokine profile may contribute to enhanced fibrotic tissue remodelling and biliary stricture formation. Screening of anti- HLA antibodies might be useful for early identification of at-risk patients who could benefit from closer surveillance and tailored immunosuppressive regimen. Our findings may have relevance for prediction and management of post- LT biliary strictures. [ABSTRACT FROM AUTHOR]

Acute kidney injury (AKI) has a major impact on short- and long-term survival in liver transplant (LT) patients. There is no currently accepted uniform definition of AKI, which would facilitate standardization of the care of patients with AKI and to improve and enhance collaborative research efforts. New promising biomarkers such as neutrophil gelatinase-associated lipocalin or kidney injury molecule-1 have been developed for the prevention of delayed AKI treatment. Early dialysis has been shown to be beneficial in patients with AKI stage III according to the classification of the Acute Kidney Injury Network, whereas treatment with loop diuretics or dopamine is associated with worse outcome. The mainstay for the prevention of AKI seems to be avoidance of volume depletion and maintenance of a mean arterial pressure >65 mmHg. Although the aetiology of chronic kidney disease in transplant recipients may be multifactorial, calcineurin-inhibitor (CNI)-induced nephrotoxicity significantly contributes to the development of renal dysfunction over time after LT. The delayed introduction of CNI at minimal doses has shown to be safe and effective for the preservation of kidney function. Other strategies to overcome CNI nephrotoxicity include CNI minimization protocols or CNI withdrawal and conversion to mycophenolate mofetil or the mammalian target of rapamycin inhibitor-based immunosuppressive regimens. However, CNI avoidance may bear a higher rejection risk. Thus, more results from randomized-controlled studies are urgently warranted to determine which drug combinations are the most beneficial approaches for the potential introduction of CNI-free immunosuppressive regimens. [ABSTRACT FROM AUTHOR]

Background: Micro-ribonucleic acid (miRNA)-199a-5p has been reported to be decreased in hepatocellular carcinoma (HCC) compared to normal tissue. Discoidin domain receptor-1 (DDR1) tyrosine kinase, involved in cell invasion-related signaling pathway, was predicted to be a potential target of miR-199a-5p by the use of miRNA target prediction algorithms. The aim of this study was to investigate the role of miR-199a-5p and DDR1 in HCC invasion. Methods: Mature miR-199a-5p and DDR1 expression were evaluated in tumor and adjacent non-tumor liver tissues from 23 patients with HCC undergoing liver resection and five hepatoma cell lines by the use of real-time quantitative RT-PCR (qRT-PCR) analysis. The effect of aberrant miR-199a-5p expression on cell invasion was assessed in vitro using HepG2 and SNU-182 hepatoma cell lines. Luciferase reporter assay was employed to validate DDR1 as a putative miR-199a-5p target gene. Regulation of DDR1 expression by miR-199a-5p was assessed by the use qRTPCR and western blotting analysis. Results: A significant down-regulation of miR-199a-5p was observed in 65.2% of HCC tissues and in four of five cell lines. In contrast, DDR1 expression was significantly increased in 52.2% of HCC samples and in two of five cell lines. Increased DDR1 expression in HCC was associated with advanced tumor stage. DDR1 was shown to be a direct target of miR-199a-5p by luciferase reporter assay. Transfection of miR-199a-5p inhibited invasion of HepG2 but not SNU-182 hepatoma cells. Conclusions: Decreased expression of miR-199a-5p contributes to increased cell invasion by functional deregulation of DDR1 activity in HCC. However, the effect of miR-199a-5p on DDR1 varies among individuals and hepatoma cell lines. These findings may have significant translational relevance for development of new targeted therapies as well as prognostic prediction for patients with HCC. [ABSTRACT FROM AUTHOR]

Mass spectrometric analysis identified the peptide recognized by a cytotoxic T lymphocyte (CTL) specific for the chemically induced BALB/c Meth A sarcoma as derived from a 17β-hydroxysteroid dehydrogenase type 12 (Hsd17b12) pseudogene present in the BALB/c genome, but only expressed in Meth A sarcoma. The sequence of the peptide is TYDKIKTGL and corresponds to Hsd17b12114–122 with threonine instead of isoleucine at codon 114 and is designated Hsd17b12114T. Immunization of mice with an Hsd17b12114T peptide-pulsed dendritic cell-based vaccine or a non-viral plasmid construct expressing the Hsd17b12114T peptide protected the mice from lethal Meth A tumor challenge in tumor rejection assays. A Hsd17b12114–122 peptide-pulsed vaccine was ineffective in inducing resistance in mice to Meth A sarcoma. These results confirm the immunogenicity of the identified tumor peptide, as well as demonstrate the efficacies of these vaccine vehicles. These findings suggest that the role of the human homolog of Hsd17b12, HSD17B12, as a potential human tumor antigen be explored. [ABSTRACT FROM AUTHOR]

Dendritic cells (DCs) induce and regulate T-cell-mediated immune responses. Circulating precursor (p)DC1 and pDC2 from patients with chronic hepatitis B virus (HBV) infection were quantified by flow cytometry. To assess their function, DC1 were cultured from patients and compared to those of healthy volunteers. HBV patients exhibited a significant decrease in the proportion of freshly isolated pDC1 to pDC2. DC1 propagated from patients showed lower expression of costimulatory molecules and impaired allostimulatory capacity in comparison to controls. After exposure to proinflammatory cytokines, expression of costimulatory molecules, secretion of interleukin-12 (IL-12) and allostimulatory properties increased, but capacity for T-cell stimulation and IL-12 production remained inferior to that of control DCs. HBV-DNA was amplified by polymerase chain reaction in DC1 cultured from all patients. Viral particles were visible in DC1 by electron microscopy. These results suggest that intracellular presence of HBV impairs DC1 functional maturation and subsequent deficits in T-lymphocyte activation may contribute to viral persistence. [Copyright &y& Elsevier]

Summary Dendritic cells (DC) are the most potent antigen-presenting cells and play a central role in the induction of antiviral immune responses. Recently, we have shown that monocyte-derived DC (MoDC) from patients with chronic hepatitis B virus (HBV) infection are functionally impaired. In our present study MoDC from healthy subjects were propagated in vitro and inoculated with HBV particles to investigate the precise mechanisms that underly MoDC dysfunction. T-cell proliferation assays revealed an impaired allostimulatory capacity of HBV-inoculated MoDC (HBV-MoDC) as well as a lower potential of stimulating autologous T cells against a recall antigen in comparison to control-MoDC. Interleukin-2, tumour necrosis factor-α and interferon-γ production by T cells in proliferation assays with HBV-MoDC was significantly lower than with control-MoDC and correlated with lower IL-12 production in HBV-MoDC cultures. The presence of the nucleoside analogue lamivudine (3TC), an inhibitor of HBV replication, restored impaired allostimulatory function of HBV-MoDC and up-regulated major histocompatibility complex class II expression. These results show that HBV infection compromises the antigen-presenting function of MoDC with concomitant impairment of T helper cell type 1 responses. This may play an important role for viral immune escape leading to chronic HBV infection. However, 3TC treatment can overcome HBV-MoDC-related T-cell hyporeactivity and this underscores its important role in enhanced immune responses to HBV. [ABSTRACT FROM AUTHOR]

Background/Aims: The mammalian target of rapamycin (mTOR) inhibitors play a key role in regulating signal transduction by blocking the mTOR pathway and combining anticancer and immunosuppressive properties. This study was undertaken to determine the prevalence and clinicopathological relevance of phospho-p70S6 (p-p70S6) kinase in hepatocellular carcinoma (HCC) and to investigate the effects of rapamycin on HCC in vitro. Methods: A total of 196 patients with HCCs were treated either with surgical resection ( n=106) or liver transplantation ( n=90). Tumour tissue was investigated for p-p70S6, phospho-AKT, Ki-67, Cyclin-D1 and apoptosis, and staining results were correlated with clinicopathologically relevant parameters. Results: Overall, p-p70S6 was detected in 24.5% (48/196) of HCCs. In the resection group, 26.4% (28/106) of HCC were positive and 22.2% (20/90) in the transplant group. p-p70S6 was significantly associated with elevated Cyclin-D1 immunoexpression and was correlated with decreased overall survival ( P=0.011) in patients resected with a clear margin. In multivariate COX regression analysis, p-p70S6 was identified as an independent prognostic parameter in patients resected with a clear margin. Rapamycin induced apoptosis and growth inhibition by G0/G1 cell cycle arrest in vitro. However, in HCC patients p-p70S6 kinase was not associated with proliferation or apoptosis. Conclusions: Activation of p70S6 kinase indicates aggressive tumour behaviour in patients with clear margin-resected HCC. Identification of p-p70S6 kinase in HCC selects high-risk patients who may benefit from drugs targeting the mTOR pathway. [ABSTRACT FROM AUTHOR]

This report describes how donor- and recipient-derived immunity was influenced by immunosuppressive treatment of ABO incompatibility (rituximab and immunoadsorption/plasmaphereses) in the long-term. We present an 8-year course of Hepatitis B virus (HBV) immunity, isohemagglutinins and B cell numbers. Whereas cellular HBV immunity was transferred from the HBV vaccinated donor (blood group A1) to the HBV naïve recipient (blood group 0), humoral HBV specific immune transfer was lacking. Starting at month 17 after transplantation, the recipient was vaccinated six times against HBV. Anti-HBs did not appear until the sixth vaccination at month 44. Immunoadsorption prior to transplantation reduced anti-A1 IgG titers from 256 to 2. Titers after transplantation remained low (⩽64). B cell numbers were below standard values up to month 26, then normalized and exceeded normal values from year 7 to 8 post transplantation. In conclusion, donor-derived B cell immunity was lost but recipient-derived immunity persisted after ABO incompatible transplantation. [ABSTRACT FROM AUTHOR]

Background & Aims: Oncogene polycomb group protein enhancer of zeste homolog 2 (EZH2) has been proposed to be a target gene of putative tumor suppressor microRNA-101 (miR-101). The aim of our study was to investigate the functional role of both miR-101 and EZH2 in human hepatocellular carcinoma (HCC). Methods: MiR-101 and EZH2 expressions were evaluated in tumor tissues of 99 HCC patients and 7 liver cancer cell lines by real-time PCR. Luciferase reporter assay was employed to validate whether EZH2 represents a target gene of miR-101. The effect of miR-101 on HCC growth as well as programmed cell death was studied in vitro and in vivo. Results: MiR-101 expression was significantly downregulated in most of HCC tissues and all cell lines, whereas EZH2 was significantly overexpressed in most of HCC tissues and all cell lines. There was a negative correlation between expression levels of miR-101 and EZH2. Luciferase assay results confirmed EZH2 as a direct target gene of miR-101, which negatively regulates EZH2 expression in HCC. Ectopic overexpression of miR-101 dramatically repressed proliferation, invasion, colony formation as well as cell cycle progression in vitro and suppressed tumorigenicity in vivo. Furthermore, miR-101 inhibited autophagy and synergized with either doxorubicin or fluorouracil to induce apoptosis in tumor cells. Conclusion: Tumor suppressor miR-101 represses HCC progression through directly targeting EZH2 oncogene and sensitizes liver cancer cells to chemotherapeutic treatment. Our findings provide significant insights into molecular mechanisms of hepatocarcinogenesis and may have clinical relevance for the development of novel targeted therapies for HCC. [Copyright &y& Elsevier]

The Bismuth-Corlette (BC) classification is the current preoperative standard to assess hilar cholangiocarcinomas (HC). The aim of this study is to evaluate the accuracy, sensitivity, and prognostic value of the BC classification. Data of patients undergoing resection for HC were analyzed. Endoscopic retrograde cholangiography and standard computed tomography were undertaken in all cases. Additional 3D-CT-reconstructions, magnetic resonance imaging, and percutaneous transhepatic cholangiography were obtained in selected patients. A systematic review and meta-analysis of the literature was performed. Ninety patients underwent resection of the hilar bile duct confluence, with right or left hemihepatectomy in 68 instances. The overall accuracy of the BC classification was 48 per cent. Rates of BC under- and over-estimation were 29 per cent and 23 per cent, respectively. The addition of MRI, 3D-CT-reconstructions, or percutaneous transhepatic cholangiography improved the accuracy to 49 per cent (P = 1.0), 53 per cent (P = 0.074), and 64 per cent (P< 0.001), respectively. Lowest sensitivity rates were for BC Type IIIA/IIIB tumors. Meta-analysis of published BC data corresponding to 540 patients did not reach significance. The BC classification has low accuracy and no prognostic value in cases of HC undergoing resection. [ABSTRACT FROM AUTHOR]

Background: The lack of sufficient donors to satisfy the waiting list requirements has prompted many to expand the acceptance criteria. The purpose of this study was to evaluate our liver transplantation (LT) experience with donors beyond the average lifespan. Patients and methods: From January 2008 to December 2009, we received 75 liver offers involving donors Z75 years of age. Donor and recipient data were analysed by both uni- and multivariate Cox proportional hazard model analyses. Results: We performed 32 adult liver transplants (43%). Half of the patients received organs through rescue allocations. Seven recipients (22%) developed initial poor function. Two had primary graft non-function (PNF). Four recipients were re transplanted (two PNF and two ischaemic-type bile lesions). One- and 3-year cumulative survival was 62 and 51% respectively. PNF, lab model for endstaged liver disease (MELD), post-LT haemodialysis, post-LT re-operations and post-LT sepsis were significant predictors by univariate analysis.Only PNF reached multivariate significance (P = 0.0307). Rescue offer allocation reached significance as a predictor of PNF by general linear model forward analysis. One- and 3-year ‘MELD based allocation’ (n = 16) vs ‘rescue allocation’ (n = 16) survival rates were 44 and 29% vs 82 and 76% respectively (P = 0.0197). Conclusions: Although grafts from donors Z75 years allow for an expansion of the donor pool, long-term recipient survival is inferior to that encountered with younger donors. Acceptable results could be obtained by identifying ‘preferred’ recipients for rescue allocations. [ABSTRACT FROM AUTHOR]

The aim of this study was to determine the efficacy, safety, and immunosuppressant adherence in 125 stable liver transplant (LT) patients converted from twice-daily tacrolimus (TAC BID) to once-daily TAC (TAC OD). Tacrolimus trough levels, laboratory parameters, metabolic disorders, selected patient reported outcomes, and adverse events were assessed. Mean TAC trough level concentration was 6.1 ± 2.3 ng/ml at study entry, decreased to 5.5 ± 2.1 ng/ml ( P = 0.016) and 5.5 ± 2.2 ng/ml ( P = 0.019) after 1 and 2 weeks, respectively, and tended to equal the baseline value during further follow-up. At week 1, TAC concentrations were lower in 62.4% of patients and higher in 36.0% when compared with baseline. Renal and cardiovascular risk factors remained stable and no rejection episodes occurred over 12 months. Adverse events were consistent with the safety profile known from previous studies with TAC BID. Nonadherence measured by the 'Basel Assessment of Adherence Scale to Immunosuppressives' was evident in 66.4% at study entry and decreased to 30.9% postconversion ( P < 0.0001). Prevalence of nonadherence at baseline was significantly higher in patients converted >2 years after LT and in those ≤60 years of age. Conversion to TAC OD is safe, enhances immunosuppressant adherence and should be accompanied by a close TAC level monitoring during the initial period. [ABSTRACT FROM AUTHOR]

Abstract: This study examined the performance of a tuberculosis (TB)-specific enzyme-linked immunospot assay in 48 patients awaiting liver transplantation. They were tested with T-SPOT.TB, tuberculin skin test (TST), and lymphocyte transformation test (LTT) using tuberculin as a stimulus. A questionnaire was used to gain information on TB exposure. Four patients were defined as positive by T-SPOT.TB, 6 by TST, and 28 by LTT. The patients displaying positive results to T-SPOT.TB were also positive in the TST and LTT. We considered them to have latent TB because they were repeatedly (two or three times) positive to the T-SPOT.TB and reported TB exposure. Active TB was excluded by negative multislice computed tomography, negative culture, and absence of symptoms. In 1 patient, T-cell reactivity toward TB peptides was lost 1 and 2 months posttransplantation. Another patient, however, tested 8 and 13 months posttransplantation, displayed measurable cellular TB immune responses. This finding suggests that the measurement of cellular TB immune responses shortly after transplantation may fail. If possible, patients with end-stage liver disease should be screened for TB prior to transplantation. Our data are the first evidence that T-SPOT.TB may be useful to diagnose latent TB in patients awaiting liver transplantation. [Copyright &y& Elsevier]

The aim of this study was to evaluate the accuracy of pretransplant imaging in patients with hepatocellular carcinoma (HCC) considering small pulmonary nodules, and to determine whether preoperatively diagnosed small pulmonary nodules should be considered ‘nodules at risk’. We evaluated 10 consecutive liver transplant patients with a diagnosis of HCC and pulmonary nodules detected by preoperative computerized tomography (CT) scanning. Pretransplant CT evaluation of pulmonary nodules showed a 90% accuracy rate. There was only one incorrect reading in the case of a patient, where a metastasis was misdiagnosed as a pulmonary fibroma. Two patients died from multifocal tumor recurrence with pulmonary metastases 17 and 19 months post-transplant. One more patient died 29 months post-transplantation on account of diffuse metastatic prostate carcinoma. Seven patients are currently alive with no evidence of tumor after a median follow-up period of 48 months post-transplantation. Small pulmonary nodules in high-risk HCC patients (low tumor grading, exceeding Milan criteria) may be characterized as nodules at risk, and evaluated very closely prior to listing and during the pre- and post-transplant periods. [ABSTRACT FROM AUTHOR]

A letter to the editor is presented in response to an article on live donor liver transplantation for hepatocellular carcinoma.

Background: Reference genes, which are often referred to as housekeeping genes are frequently used to normalize mRNA levels between different samples in quantitative reverse transcription polymerase chain reaction (qRT-PCR). The selection of reference genes is critical for gene expression studies because the expression of these genes may vary among tissues or cells and may change under certain circumstances. Here, a systematic evaluation of six putative reference genes for gene expression studies in human hepatocellular carcinoma (HCC) is presented.Methods: Six genes, beta-2-microglobulin (B2M), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), hydroxymethyl-bilane synthase (HMBS), hypoxanthine phosphoribosyl-transferase 1 (HPRT1), succinate dehydrogenase complex, subunit A (SDHA) and ubiquitin C (UBC), with distinct functional characteristics and expression patterns were evaluated by qRT-PCR. Inhibitory substances in RNA samples were quantitatively assessed and controlled using an external RNA control. The stability of selected reference genes was analyzed using both geNorm and NormFinder software.Results: HMBS and GAPDH were identified as the optimal reference genes for normalizing gene expression data between paired tumoral and adjacent non-tumoral tissues derived from patients with HCC. HMBS, GAPDH and UBC were identified to be suitable for the normalization of gene expression data among tumor tissues; whereas the combination of HMBS, B2M, SDHA and GAPDH was suitable for normalizing gene expression data among five liver cancer cell lines, namely Hep3B, HepG2, HuH7, SK-HEP-1 and SNU-182. The determined gene stability was increased after exclusion of RNA samples containing relatively higher inhibitory substances.Conclusion: Of six genes studied, HMBS was found to be the single best reference gene for gene expression studies in HCC. The appropriate choice of combination of more than one reference gene to improve qRT-PCR accuracy depends on the kind of liver tissues or cells under investigation. Quantitative assessment and control of qRT-PCR inhibitors using an external RNA control can reduce the variation of qRT-PCR assay and facilitate the evaluation of gene stability. Our results may facilitate the choice of reference genes for expression studies in HCC. [ABSTRACT FROM AUTHOR]