Identification of miR-31-5p, miR-141-3p, miR-200c-3p, and GLT1 as human liver aging markers sensitive to donor-recipient age-mismatch in transplants.

To understand why livers from aged donors are successfully used for transplants, we looked for markers of liver aging in 71 biopsies from donors aged 12-92 years before transplants and in 11 biopsies after transplants with high donor-recipient age-mismatch. We also assessed liver function in 36 age-mismatched recipients. The major findings were the following: (i) miR-31-5p, miR-141-3p, and miR-200c-3p increased with age, as assessed by microRNAs (miRs) and mRNA transcript profiling in 12 biopsies and results were validated by RT-qPCR in a total of 58 biopsies; (ii) telomere length measured by qPCR in 45 samples showed a significant age-dependent shortage; (iii) a bioinformatic approach combining transcriptome and miRs data identified putative miRs targets, the most informative being GLT1, a glutamate transporter expressed in hepatocytes. GLT1 was demonstrated by luciferase assay to be a target of miR-31-5p and miR-200c-3p, and both its mRNA (RT-qPCR) and protein (immunohistochemistry) significantly decreased with age in liver biopsies and in hepatic centrilobular zone, respectively; (iv) miR-31-5p, miR-141-3p and miR-200c-3p expression was significantly affected by recipient age (older environment) as assessed in eleven cases of donor-recipient extreme age-mismatch; (v) the analysis of recipients plasma by N-glycans profiling, capable of assessing liver functions and biological age, showed that liver function recovered after transplants, independently of age-mismatch, and recipients apparently 'rejuvenated' according to their glycomic age. In conclusion, we identified new markers of aging in human liver, their relevance in donor-recipient age-mismatches in transplantation, and offered positive evidence for the use of organs from old donors.

Lifelong maintenance of composition, function and cellular/subcellular distribution of proteasomes in human liver.

Owing to organ shortage, livers from old donors are increasingly used for transplantation. The function and duration of such transplanted livers are apparently comparable to those from young donors, suggesting that, despite some morphological and structural age-related changes, no major functional changes do occur in liver with age. We tested this hypothesis by performing a comprehensive study on proteasomes, major cell organelles responsible for proteostasis, in liver biopsies from heart-beating donors. Oxidized and poly-ubiquitin conjugated proteins did not accumulate with age and the three major proteasome proteolytic activities were similar in livers from young and old donors. Analysis of proteasomes composition showed an age-related increased of ß5i/α4 ratio, suggesting a shift toward proteasomes containing inducible subunits and a decreased content of PA28α subunit, mainly in the cytosol of hepatocytes. Thus our data suggest that, proteasomes activity is well preserved in livers from aged donors, concomitantly with subtle changes in proteasome subunit composition which might reflect the occurrence of a functional remodelling to maintain an efficient proteostasis. Gender differences are emerging and they deserve further investigations owing to the different aging trajectories between men and women. Finally, our data support the safe use of livers from old donors for transplantation.

Long-term antiviral treatment for recurrent hepatitis C after liver transplantation.

BACKGROUND AND AIMS: The management of patients treated for hepatitis C recurrence after liver transplantation and not achieving virological response following treatment with interferon plus ribavirin is controversial. METHODS: A retrospective analysis of the outcomes of 70 patients non-responders to antiviral treatment after liver transplantation was performed. Twenty-one patients (30.0%; Group A) were treated for ≤ 12 months and 49 (70.0%; Group B) for more than 12 months. RESULTS: The 2 groups were comparable for main demographic, clinical and pathological variables. Median duration of antiviral treatment was 8.2 months in Group A and 33.4 months in Group B. No patient achieved a complete virological response. The 5-year patient hepatitis C-related survival rate was 49.2% in Group A and 88.3% in Group B (P=0.002), while the 5-year graft survival rate was 49.2% in Group A and 85.9% in Group B (P=0.007). The median yearly fibrosis progression rate was 1.21 per year in Group A and 0.40 per year in Group B (P=0.001). CONCLUSIONS: Prolonged antiviral treatment showed an overall beneficial effect in transplanted patients with a recurrent hepatitis C infection and not responding to conventional therapy. The treatment should be continued as long as it is permitted, in order to improve clinical and histological outcomes.

Prognostic factors for tumor recurrence after a 12-year, single-center experience of liver transplantations in patients with hepatocellular carcinoma.

Background. Factors affecting outcomes after orthotopic liver transplantation (OLT) for hepatocellular carcinoma (HCC) have been extensively studied, but some of them have only recently been discovered or reassessed. Methods. We analyzed classical and more recently emerging variables with a hypothetical impact on recurrence-free survival (RFS) in a single-center series of 283 patients transplanted for HCC between 1997 and 2009. Results. Five-year patient survival and RFS were 75% and 86%, respectively. Thirty-four (12%) patients had HCC recurrence. Elevated preoperative alpha-fetoprotein (AFP) levels, preoperative treatments of HCC, unfulfilled Milan and up-to-seven criteria at final histology, poor tumor differentiation, and tumor microvascular invasion negatively affected RFS by univariate analysis. Milan and up-to-seven criteria applied preoperatively, and the use of m-TOR inhibitors did not reach statistical significance. Cox's proportional hazard model showed that only elevated AFP levels (Odds Ratio = 2.88; 95% C.I. = 1.43-5.80; P = .003), preoperative tumor treatments (Odds Ratio = 4.84; 95% C.I. = 1.42-16.42; P = .01), and microvascular invasion (Odds Ratio = 4.82; 95% C.I. = 1.87-12.41; P = .001) were predictors of lower RFS. Conclusions. Biological aggressiveness and preoperative tumor treatment, rather than traditional and expanded dimensional criteria, conditioned the outcomes in patients transplanted for HCC.

Safety and prognostic role of regional lymphadenectomy for primary and metastatic liver tumors.

Routine regional lymphadenectomy for colorectal liver metastases and primary liver tumors is still a subject for debate. During 2001-2005, we performed a prospective study of cases in which regional lymphadenectomy around the hepato-duodenal ligament and common hepatic artery was applied (group R+ LN) or not (group R- LN). Pre-operative clinical features of patients were comparable among groups as well as the operative data. There were 108 (67%) males; the median age was 66 years; 124 cases had a single lesion (77%), and the median diameter was 4 cm. The type of lesion was: 77 (48%) colorectal liver metastases (M-CR), 75 (46%) hepatocellular carcinomas (HCC) and 10 (6%) cholangiocellular carcinomas. In the R+ LN group, the mean number of lymph nodes removed was 6.7 ± 4.8 (range 4-26), and seven cases (8.6%) presented lymph node metastasis. The median follow-up was 3.5 years. M-CR patients showed comparable hospital mortality (R+ LN 0% vs. R- LN 2.6%) and morbidity (R+ LN 17.9% vs. R- LN 21.1%), but R+ LN had higher 5-year disease-free survival (31 vs. 16%, p < 0.05). HCC cases in the R+ LN group presented higher hospital mortality (13.5 vs. 0%, p < 0.05) without any improvement in disease-free survival (it was at 5-year disease-free survival 34 vs. 33%, respectively, p = n.s.). Routine regional lymphadenectomy should be performed for colorectal liver metastases, and avoided in patients with hepatocellular carcinoma.

Effect of different immunosuppressive schedules on recurrence-free survival after liver transplantation for hepatocellular carcinoma.

BACKGROUND: Tumor recurrence represents the main limitation of liver transplantation in patients with hepatocellular carcinoma (HCC) and can be favored by exposure to calcineurin inhibitors. METHODS: We investigated the effect of an immunosuppressant schedule that minimizes the exposure to calcineurin inhibitors on patients transplanted for HCC to ascertain whether this can reduce the tumor recurrence rate. For this purpose, we conducted a matched-cohort study: 31 patients with HCC transplanted between 2004 and 2007 who received sirolimus as part of their immunosuppression (group A) were compared with a control group of 31 patients (group B) transplanted in the same period who had the same prognostic factors but were given standard immunosuppression based on tacrolimus. RESULTS: Three-year recurrence-free survival was 86% in group A and 56% in group B (P=0.04). Although the prevalence of microvascular invasion G3-G4 grading and alpha-fetoprotein more than 200 ng/mL was identical in the two groups, exposure to tacrolimus was significantly higher in patients of group B (median, 8.54; range, 5.5-13.5) in comparison with those of group A (median, 4.6; range, 1.8-9.1) (P=0.0001). CONCLUSIONS: By using sirolimus, exposure to calcineurin inhibitors can be minimized, reducing the risk of HCC recurrence.

Survival benefit after liver transplantation: a single European center experience.

BACKGROUND: The evaluation of the survival achieved with liver transplantation (LT) compared with remaining on the waiting list, the transplant benefit, should be the underlying principle of organ allocation. METHODS: During 2004 to 2007 with an allocation system based on Model for End-Stage Liver Disease (MELD) score with exceptions, we prospectively evaluated the transplant benefit and its relation to the match between recipient and donor characteristics. RESULTS: Among 575 patients listed for chronic liver disease, 218 (37.9%) underwent LT and 115 (20%) were removed from the list (76 deaths, 25 tumor progressions, and 14 sick conditions). The 1- and 3-year survival rates on the list were significantly related to MELD score more than or equal to 20 (57% and 33% vs. 88% and 66%, P<0.001) and to its progression during the waiting time, such as s-Na levels less than or equal to 135 mEq/L (73% and 48% vs. 86% and 69%, P<0.001). These two variables had no impact on survival after LT, except in hepatitis C virus positive recipients. The multivariate Cox model confirmed a positive transplant benefit for all cases with MELD score more than or equal to 20 and without hepatocellular carcinoma (HR 2.9; CI 1.3-6.2) independently of the type of donors. Only hepatocellular carcinoma patients with low MELD scores showed a positive transplant benefit (MELD <15; HR 2; CI 1.1-5.1). CONCLUSIONS: LT should be reserved for cirrhotic patients with MELD score more than or equal to 20 independently of other recipient and donor matches or for cases with lower MELD score but with hepatocellular carcinoma.

Impact of induction therapy on bacterial infections and long-term outcome in adult intestinal and multivisceral transplantation: a comparison of two different induction protocols: daclizumab vs. alemtuzumab.

INTRODUCTION: Induction therapy with daclizumab or alemtuzumab has been recently introduced for intestinal transplantation; however, the impact of such induction therapy on bacterial infections remains to be clarified. The purpose of this study was to evaluate the impact of induction therapy on the incidence of bacterial infections and long-term patient survival. PATIENTS AND METHODS: Over the past seven yr, we performed 39 intestinal (ITx) and multivisceral (MTVx) transplants in 38 adult patients. In the early period, daclizumab was used for induction, and tacrolimus and steroids were administered for maintenance [daclizumab and tacrolimus (DT) group; n = 11]. From 2002, we used alemtuzumab for induction, with low-dose tacrolimus maintenance [alemtuzumab and tacrolimus (AT) group; n = 23]. The incidence of bacterial infections and patient outcome were compared between the two groups. RESULTS: There were no significant differences in recipient and donor demographics, procedure (ITx vs. MTVx), and cold and warm ischemic time between the two groups. Within 30 d after ITx, bacterial infections were observed in seven patients (64%) in the DT and in 14 patients (64%) in the AT group. Between 30 and 180 d after ITx, a total of 17 episodes of bacterial infections were observed in the DT and 26 episodes in the AT group. Three patients in the DT and eight in the AT group died, and all of the deaths were related to infectious complications except one each in DT and AT. CONCLUSION: There was no difference in incidence of bacterial infections and long-term patient survival between the two groups.

Liver transplantations with donors aged 60 years and above: the low liver damage strategy.

According to transplant registries, grafts from elderly donors have lower survival rates. During 1999-2005, we evaluated the outcomes of 89 patients who received a liver from a donor aged > or = 60 years and managed with the low liver-damage strategy (LLDS), based on the preoperative donor liver biopsy and the shortest possible ischemia time (group D > or = 60-LLDS). Group D > or = 60-LLDS was compared with 198 matched recipients, whose grafts were not managed with this strategy (89 donors < 60 years, group D < 60-no-LLDS and 89 donors aged > or =60 years, group D > or = 60-no-LLDS). In the donors proposed from the age group of > or =60 years, the number of donors rejected decreased during the study period and the LLDS was found to be responsible for this in a significant manner (47% vs. 60%, respectively P < 0.01). Among the recipients transplanted, the clinical features (age, gender, viral infection, child and model for end-stage liver disease score) were comparable among groups, but group D > or = 60-LLDS had a lower mean ischemia time: 415 +/- 106 min vs. 465 +/- 111 (D < 60-no-LLDS), P < 0.05 and vs. 476 +/- 94 (D > or = 60-no-LLDS), P < 0.05. After a median follow-up of 3 years, the 1- and 3-year graft survival rates of group D > or = 60-LLDS (84% and 76%) were comparable with group D < 60-no-LLDS (89% and 76%) and were significantly higher than group D > or = 60-no-LLDS (71% and 54%), P < 0.005. In conclusion, the LLDS optimized the use of livers from elderly donors.

Incidence and management of abdominal closure-related complications in adult intestinal transplantation.

BACKGROUND: We sought to determine the best strategy to overcome difficult abdominal wall closures in intestinal transplantation (ITx). METHODS: Among 38 adult recipients of 39 ITxs from deceased donors, the median number of previous laparotomies was 2.0 per patient, with a median donor-to-recipient body weight ratio of 1.1. Eight patients (21%) had full residual intestinal length before transplant. Abdominal wall closure after transplant was considered difficult in 15 (39.5%) patients (group A). To overcome size mismatching, we performed two graft reductions, five skin-only closures, one two-step abdominal wall closure, four prosthetic mesh closures, and three abdominal wall transplants. In the remaining 23 (60.5%) patients, a regular abdominal closure was performed (group B). RESULTS: Twelve patients (32%) experienced complications related to abdominal wall closure, 10 (67%) in group A and 2 (8.7%) in group B (P<0.0001). Abdominal closure-related mortality was 6.7% (1/15) and 4.3% (1/23), respectively (P=1.0). In group A, there were six incisional hernias (one of them after abdominal wall transplant), although all four patients with mesh experienced mesh infection. Two of them developed intestinal fistulae, leading to patient death in one case. In group B, one patient with unfavorable donor/recipient size matching had fatal vascular thrombosis of a multivisceral graft caused by compression after abdominal closure. CONCLUSIONS: A careful evaluation of abdominal cavity is necessary in candidates for ITx. In our experience, closure with mesh should be avoided because of the high rate of complications. Abdominal wall transplantation is a feasible option when a difficult abdominal wall closure is expected.

Preliminary results of a "prope" tolerogenic regimen with thymoglobulin pretreatment and hepatitis C virus recurrence in liver transplantation.

BACKGROUND: Recent reports demonstrate the efficacy of induction immunosuppression with Thymoglobulin, a potent antithymocyte polyclonal antibody, in allowing acquired tolerance by means of a tolerogenic regimen of recipient pretreatment and low-dose postoperative immunosuppression. The effect of this novel approach on recurrence of hepatitis C viral disease after liver transplantation has never been investigated. We report the preliminary results of a retrospective analysis aimed at discovering any relationship between Thymoglobulin immunosuppression and the pattern of recurrence of hepatitis C. METHODS: Thymoglobulin induction plus tacrolimus monotherapy was used in a group of 22 hepatitis C virus (HCV)+ patients receiving liver transplantation; 30 HCV+ patients receiving transplants within the same year received conventional tacrolimus plus steroid immunosuppression and represented the comparison group. RESULTS: Patient survival and acute rejection rate did not differ between the two groups. Significantly lower dosages and levels of tacrolimus were possible with Thymoglobulin, and a progressive weaning of tacrolimus monotherapy was accomplished in most patients, without major rejection complications. The HCV recurrence rate was similar in both groups, although significantly lower HCV RNA loads were obtained with Thymoglobulin pretreatment. The mean time to histologic recurrence was shorter in Thymoglobulin-treated patients; however, no significant difference was observed in mean Ishak's histologic grading and staging of HCV recurrence. CONCLUSIONS: In our preliminary experience, a "prope" tolerogenic regimen with Thymoglobulin pretreatment and low-dose immunosuppression in liver-transplant recipients gave good protection against rejection and permitted lower HCV viral loads, whose significance in the long-term outcome of HCV patients deserves further investigation.

Minimization of immunosuppression with thymoglobuline pre-treatment and HCV recurrence in liver transplantation.

Induction with thymoglobuline, a potent anti-thymocyte polyclonal antibody, has been recently reported to allow minimization of postoperative immunosuppression in organ transplantation. The relationship with recurrence of hepatitis C virus (HCV) after liver transplantation (LTx) has never been investigated. We report here on the outcome in 22 HCV+ patients receiving thymoglobuline pre-treatment and minimal immunosuppression after LTx. Patient survival and acute rejection rates were good, and remarkably low dosages and levels of immunosuppression were achieved with thymoglobuline, without exposing patients to an elevated risk of rejection. A progressive weaning of the primary immunosuppressor was also possible in the majority of patients without complications. The HCV recurrence rate was similar to what is reported in the literature, although lower HCV-RNA viral loads were obtained with thymoglobuline, with a mild histologic course. Although our results need to be validated in large cohort studies, our experience shows that minimization of immunosuppression with thymoglobuline is effective in protecting against rejection and demonstrated a positive impact on HCV recurrence that deserves further investigation.

Use of a branch patch with the cystic artery in living-related liver transplantation.

Technical aspects in living-related liver transplantation are still under debate: the main pitfall is the arterial reconstruction due to the small diameter and the discrepancy between stumps, with a subsequent increased risk of arterial thrombosis. The gold standard is the microsurgical technique, that reports the lowest risk of thrombosis, but it is a time consuming procedure requiring a long training. Our method of choice reconstructing hepatic artery in right lobe is the use of the cystic artery as a branch patch with the recipient hepatic artery by loop magnification, saving time and with a low incidence of hepatic artery thrombosis.