Liver transplantation (LT) is a life-saving treatment for liver cirrhosis patients with hepatocellular carcinoma (HCC). However, 10%–20% HCC recurrence rate after LT is due to the immunosuppression inducing tumor growth. We recently reported a novel immunotherapy with donor liver natural killer (NK) cells to prevent HCC and hepatitis C virus (HCV) recurrence after LT. In this cell processing procedure, Muromonab-CD3 (Orthoclone OKT3, an anti-CD3 antibody) was added to the culture medium to deplete CD3+ T cells to prevent graft-versus-host disease. However, the manufacture of OKT3 was discontinued in 2010, when other treatments with similar efficacy and fewer side effects became available. In this study, we examined alternative reagents for T-cell depletion-MACS GMP CD3 pure (GMP CD3), antithymocyte globulin, and alemtuzumab-for NK cell immunotherapy in the allogeneic setting. We observed that GMP CD3 showed exactly the same effects on liver mononuclear cells as OKT3, including activation of NK cells and depletion of T cells. Interestingly, binding of T-cell depletion antibodies to NK cells led to an anti-HCV effect via interferon-γ production. These results with the use of in vitro culture systems suggested that antibodies which produce T-cell depletion affected NK cell function.

Background. Hepatic artery thrombosis (HAT) after orthotopic liver transplantation (OLT) is associated with significant morbidity and mortality. Factor V Leiden (FVL) mutation is the most common genetic defect that predisposes to thrombosis. The reconstruction of hepatic artery with arterial graft is a documented risk factor for HAT. However, the relationship among FVL mutation, arterial graft, and HAT remains to be determined.Methods. We randomly genotyped 485 patients who underwent OLT from April 2002 to January 2011 and studied the incidence of Hepatic artery thrombosis in the presence of FVL mutation.Results. Of 485 patients, 21 patients (4.3%) developed HAT (13 male, 8 female); 10 patients (4 male, 6 female) were heterozygous for the FVL mutation. The incidences of HAT in patients without versus with the FVL mutation were 3.8% and 30% (P = .007). Of patients with HAT, 8 hepatic arteries were reconstructed with infrarenal aortic conduits. All 3 patients (100%) with vs 5 (28%) without FVL who received arterial grafts developed HAT (P = .042).Conclusion. Our study suggested that the FVL mutation may be a risk factor for HAT in liver transplantation; the risk is augmented in the presence of an arterial graft.

Arterial complications contribute to significant morbidity and mortality after liver transplantation (OLT). If hepatic artery inflow to the graft is inadequate, alternative approaches can be considered, such as supraceliac or infrarenal aortic conduits and splenic artery as an arterial inflow. Between January 2005 and January 2012, we performed 928 OLTs. We used the recipient celiac trunk for arterial inflow in 9 patients (1%). evaluated retrospectively, We the indications, results, and outcome of this technique. Doppler ultrasound of the liver was used to evaluate arterial flow. Eight cases are first transplant and 1 case is a second transplant. Five cases are pediatric recipients and four cases are adult recipients. Male to female ratio is 3/6. Average follow-up is 23 months. No complications were encountered as a result of sacrificing the branches of the celiac axis. The conclusion is that the celiac trunk provides an adequate arterial inflow in OLT when the recipient's hepatic artery is not suitable to use.

Background: HCV related end stage liver disease is becoming the leading indication for liver transplant (OLTx), however it's prognosis is still dismal. Even though the relapse of the HCV infection is almost universal, certainly some of the liver recipients with HCV relapse can maintain good graft function as long as the liver recipients without HCV infection. The rate of sustained viral response (SVR) in the treatment of recurrent HCV after OLTx using interferon or pegylated interferon (peg IFN) have been reported as ranging from 5 to 50%, and once SVR was established, the degree of graft inflammation and fibrosis were downgraded. However the long-term outcome after the anti-viral therapy for recurrent HCV have not been addressed yet. The aim of this study is to identify the factor that can affect graft survival after the OLTx for the HCV related patients, and evaluate the impact of the anti-HCV therapy on the long-term graft survivals. Methods: Four handred fifty six patients, who underwent OLTx in University of Miami/Jackson Memorial Hospital since 2002 to 2006, were reviewed retrospectively. Patients with hepatocellular carcinoma were excluded. One handred sixty one recipients had HCV-related liver disease (HepC), and 295 uninfected contolols were identified (control). Graft loss was defined as at time of recipient's death or time of re-transplant. Anti-HCV therapy using peg IFN with ribavirin (pegIFN/Rib) was initiated when the patients showed abnormal allo-graft function and HCV re-infection confirmed by histologically. Median follow-up time was 45 month (range 0-79 months). Statistical analysis was performed using R version 2.7.2. Results: One handred five grafts were lost in total of 456 recipients. Graft survival in HCV was shorter than control, without reaching significance (p=0.56). The cumulative incidence of early graft loss (graft loss within 2 years after OLTx) was higher in HCV than controls (26% in HCV and 16% in controls). High postoperative HCV-RNA titer (more than 500,000 IU/ml), administration of muromonab-CD3 and MELD score greater than 20 appeared to be significant predictive factors for early graft loss from logistic regression analysis among HepC (p=0.04, 0.002 and 0.03, respectively). One handred patients underwent peg IFN/Rib therapy in total of 161 HepC patients, and the SVR in our series was 21%. 5 year graft survival from the patients with or without pegIFN/Rib were 77% and 57%, respectively. The cumulative graft survival of the patients with pegIFN/Rib was significantly longer than the patients without pegIFN/Rib (p< 0.001). Also, lower postoperative HCV-RNA titer appeared to be significant predictors for long-term graft survival from log rank test (p=0.003). Conclusion: High HCV-RNA titer was implicated in early graft loss after OLTx, whereas lower HCV-RNA titer had positive impact for long-term graft survival in patients with HCV. PegIFN with ribavirin therapy after OLTx also had a positive impact for long-term graft survival in patients with HCV.

Abstract Tumor recurrence is the limitation of liver transplant (LT) in patients with hepatocellular carcinoma (HCC). However, there is no prevention or treatment for HCC recurrence after LT. Here, we describe a clinical-scale method for an adoptive immunotherapy that uses natural killer (NK) cells derived from donor liver graft perfusate to prevent HCC recurrence after LT. Liver mononuclear cells (LMNC) that were extracted from the deceased donor liver graft perfusate contained a large percentage of NK cells (45.0% ± 4.0%) compared with peripheral blood mononuclear cells (PBMC) (21.8% ± 5.2%) from the same donor. Furthermore, interleukin (IL)-2-stimulated NK cells showed greater upregulation of activation markers and the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), which is critical for NK cell-mediated anti-tumor cell. Moreover, IL-2 stimulation induced LMNC to exhibit a stronger cytotoxicity against HCC compared with PBMC (p < 0.01). After obtaining approval from the FDA and IRB of our institute, we successfully applied this approach to 10 liver cirrhotic patients (9M / 1F) with HCC (Clinicaltrial.gov #NCT01147380). The average number of administered NK cells was 175 × 106 cells/body. After infusion, cytotoxicity, TRAIL, NKp44, and CD226 expression on NK cells significantly increased in patients’ PBMC (p < 0.05). There are no study related adverse events. In conclusion, the administration of IL-2-stimulated cadaveric donor liver NK cells is well tolerated.

Background: We recently established a novel immunotherapy with activated donor liver NK cells for the liver transplant patients with hepatocellular carcinoma (HCC). In this procedure, we added an anti-CD3 mAb, OKT3, to the culture medium a day before the inoculation in order to inactivate CD3+ alloreactive T cells. Unexpectedly, IL-2/OKT3 treated liver NK/NKT cells could mount anti-hepatitis C virus (HCV) responses in HCV-infected liver transplant recipients (Ohira, et al. JCI 2009; 3226). Since OKT3 was recently out of market, we had been seeking alternative antibodies in this immunotherapy. In this study, we evaluated some alternative reagents to affect the NK cells and T cells in culture systems. Methods: We compared the effect of three commercially available reagents (GMP CD3 pure mAb [GMP CD3, Miltenyi Biotec], Thymoglobulin [ATG], and Alemtuzumab [AL]) in vitro culture. To determine the effect of these agents on NK cell function, freshly isolated peripheral blood mononuclear cells or donor graft liver derived mononuclear cells were cultured for 3 days in the presence of rhIL-2 (1,000 U/ml). One day before the cell analysis, these agents were added in the culture system. The effect of agents was tested on the basis of the following parameters: NK cell phenotype, remaining T cells, cytotoxicity against K562 cells, cytokine production of NK cells, and anti-HCV activity with HCV genomic replicon cells. Results: Addition of OKT3 (1mg/ml) or GMP CD3 (1mg/ml) significantly decreased T cell population. In contrast, ATG only showed a trend toward reduced T cells and AL did not affect T cell population in the culture system (Fig. 1A). IL-2 stimulated LMNCs showed a significantly increase in TRAIL and NKp44 (p< 0.05). OKT3 and GMP CD3 did not affect the NK cell phenotype, but ATG and AL significantly decreased the expression of CD16 on NK cells (p< 0.01) in the dose dependent manner. These CD16+ NK cells were lysed by the complement dependent cytotoxicity. We next analyzed the cytotoxicity and anti-HCV effect of cultured LMNCs and PBMCs. OKT3 and GMP CD3 did not affect NK cell-mediated killing against K562 targets but ATG and AL dampened their cytotoxicity. Interestingly, binding of these T cell depletion antibodies to NK cells lead to anti-HCV effect via interferon-γ production (Fig. 1B).[Figure 1]Conclusions: The effect of GMP CD3 on the ex vivo culture system is the almost same as OKT3. Therefore, a phase I trial has been under way to investigate the safety and anti-tumor effects of donor liver NK cells treated with IL-2/GMP CD3. Our studies also suggest that T cell depletion antibodies impact NK cells in unexpected ways.

Introduction: Portal vein thrombosis (PVT) remains a challenging problem in liver transplantation. Long-term results after transplant in patients with PVT are not well defined. Methods: We reviewed all consecutive adult primary liver transplants performed between 1998 and 2009. Patient demographics, methods of portal vein reconstruction, and the impact of PVT on postoperative morbidities and survival were analyzed in comparison with non-PVT cases. Results: Among 1,381 recipients, 165 (11.9%) had PVT at the time of transplant: 81 (49%) complete (Yerdel classification, Grades 3 and 4) and 85 (51%) partial (Grades 1 and 2). Thrombectomy and end-to-end anastomosis was performed in 115 (69%), venous interposition graft from the confluence of superior mesenteric vein and splenic vein in 16 (10%), venous interposition “jump” graft from the infrapancreatic superior mesenteric vein in 10 (6%), cavoportal hemitransposition in 18 (11%), renoportal anastomosis in 5 (3%), and portal vein arterialization in 1 (1%). Compared with non-PVT patients (N=1,216), PVT group was older (54.2 versus 52.4 years, P=0.028), had longer cold ischemia (451 versus 426 min, P=0.013) and warm ischemia time (41 versus 38 min, P=0.005), increased blood loss (11.0 versus 7.2 L, P=0.002) and extended length of stay (26 versus 19 days, P=0.002). Model for End-Stage Liver Disease (20 versus 21), donor age (40.5 versus 41.8 years) and postoperative mortality rate (5.5% versus 5.2%) were not different between the 2 groups (P>0.05). During a median follow-up of 88 months, PVT group demonstrated lower patient (66% versus 74%, P=0.020) and graft 5-year survival (61% versus 69%, P=0.024: Fig. A) compared with non-PVT. PVT group was more susceptible to prolonged mechanical ventilation (23.6% versus 16.4%, P=0.028) or requiring dialysis (26.1% versus 18.2%, P=0.016) after transplant, but biliary (13.9% versus 13.0%) and arterial complications (7.3% versus 4.7%) and retransplants (10.3% versus 9.5%) were similar (P>0.05). Subset analysis within PVT group revealed that recipients with cavoportal hemitransposition or renoportal anastomosis were at increased risk for re-thrombosis of portomesenteric veins (26% versus 5%, P< 0.001), gastrointestinal bleeding (35% versus 13%, P=0.007), and massive ascites (30% versus 13%, P=0.028) compared with other types of reconstruction; their 5-year patient survival was diminished (48% versus 69%, P=0.020: Fig. B). No significant difference was observed between complete and partial PVTs.[Figure]Conclusions: This is the largest series of liver transplant in the face of PVT. Although satisfactory long-term survival can be achieved, the outcomes are inferior compared to non-PVT in the most severe cases.

Aim: Our aim was to assess the risk of potential transmission of autoimmune diseases from cadaveric donors to recipients of allogeneic solid organ transplantation. Patients and methods: Donors younger than 25 years whose organs were procured between 2001 and 2010 were included in the study. Past history of autoimmune diseases were assessed from the charts. Recipients of any solid organ from these donors were track for the potential occurrence of autoimmune diseases. Results: A total of 449 donors were included, only 5 of them suffered from some type of autoimmune disease: 2 type I diabetes, 2 past history of idiopathic thrombocytopenic purpura, and 1 hypothyroidism. In the 14 recipients beneficed from these donors, organ distribution were as follows: 4 single kidney, 3 liver, 3 heart, 2 both kidneys, 1 both lungs, and 1 multivisceral. Median follow-up time was 4.9 (0.0-9.9) years. Any patient showed any clinical manifestation of related to their respective donor autoimmune disease or any other. Two recipients (1 liver, 1 both lungs) died in the early postoperative period, two died because of rejection in the short term. Conclusions: Past history of acute autoimmune disorders or active chronic autoimmune diseases is not contraindication for solid organ donation.

Background: Children with short bowel syndrome starting in the neonatal period (NB- SBS) are exposed to many live-threatening complications that can affect their outcome before and after transplantation. Our aim was to assess the outcome of this group of children in a single transplant center. Material and methods: Retrospective review of pediatric intestinal transplants performed between 1994 and 2010 at our center. All patients with short bowel syndrome of neonatal onset were included. Those with motility disorders or intestinal epithelium disease but normal bowel length were not included in spite of the congenital character of these diseases. Pre-transplant status, weight, time on the waiting list, type of graft, patient survival, graft survival and immunological complications were compared between patients with NB-SBS and the rest of the children included. T-Student test, Chi-square tests and log-rank were used to compare quantitative, qualitative and survival data respectively. Results: A total of 180 pediatric intestinal transplants were performed in children between 1994 and 2010. Of them, 104 (57%) suffered from short bowel syndrome of neonatal onset, 96 (53%) were male and 85 (46%) females. Patient survival at 1, 5 and 10y were similar between patients with NB-SGS and other children (61, 42, 32% vs 62, 42, 34%; respectively). Graft survival was also similar in the three periods for the two groups (61, 39, 33% vs 51, 35, 32%, respectively). Waiting time was lower but not significantly in the NB-SBS group (77.3 vs 98.8 days) Type of graft distribution was similar in both groups except for the absence of MMV in the group of NB-SBS that contrast to 11% of these grafts in other children. Weight at transplantation was significantly lower in the NB-SGS group (10.2±16.8 vs 17.5±6.8 Kg). Regarding pre-transplant status patients in the NB-SGS were more likely to be in the ICU (24. 2 vs 10.9%) and less at home (44.4 vs 62.2%) compared to patients in the other group. Pre transplant creatinin level was similar the two groups but pre transplant albumin, bilirubin, INR and PT were more seriously impaired in patients in the NB-SGS group compared to other children. Recipient/donor weight rate was similar in both groups (1.2 vs 1.1) in NB-SGS vs others. PTLD was most frequent in children with NB-SBS compared to others (18.1 vs 8.5%). Rejection, GVHD and infectious complications were similar in the two groups. Conclusions: Patients suffering from NB-SGS had similar patient and graft survival compared to the rest of children that received an intestinal transplant. Liver function tests were more impaired in the NB-SGS group, and they were more prone to be in the ICU at the moment of transplant than the rest of children. The rest of pre and post-transplant variables examined did not show any difference.

Introduction: Liver transplantation (LT) is closely associated with blood transfusion practices since the first transplants. Requirement of blood has been reduced by advance and refinements in patient selection, transfusion methods, and surgical technique. LT without any blood product became real clinical practice in patient with stable preoperative condition. The aim of this study was to analyze the pre-operative, intra-operative, post-operative factors associated with LT without intraoperative red blood cell (RBC) transfusion. Methods: Four hundreds forty four adult patients were selected from our database due to the less than 5 units of RBC transfusion. Out of 444, 51 patients were selected as study group due to no RBC transfusion and 389 patients were selected as control group due to the 1-5 units of RBC transfusion during LT surgery. Coagulations were monitored intraoperatively and corrected if clinically indicated. Aprotinin was not used. Auto transfusion of blood salvaged was used routinely following our protocol. Independent factors were analyzed to find a link with no RBC transfusion. Survival curve was generated by Kaplan Meier methods and compared with log rank test. Results: Factors of study group vs. control group were as follows: MELD score was 20 vs. 20. Calculated MELD score was 12 vs. 12. Cold ischemic time was 429 vs. 425 min. Warm ischemic time was 37.0 vs. 37.1 min. Donor age was 41 vs. 39 yrs. Use of piggyback technique (PB) was 94% (n=48) vs. 92% (n=363). Use of Conventional cava reconstruction was 6% (n=3) vs. 8% (n=30). There were no statistically significant differences in demographic factors including donor age, recipient age, primary liver diagnosis, MELD score, CIT, WIT, INR, Cr, bilirubin, and amounts of auto-transfusion, cava reconstruction methods between the groups. Amount of fresh frozen plasma is significantly less in study group (P< 0.05). One, 3, 5 years' patient and graft survival were 92%, 88% 79% and 92%, 88%, 79% in study group and 89%, 82%, 76% and 84%, 78%, 71% in control group respectively. There was no statistical difference in patient and graft survival between the groups. Conclusion: Preoperative factor have limited predictive power for no intra-operative RBC transfusion in patient with stable condition. No transfusion could be achieved by LT. LT without transfusion did not show survival benefit. Surgical technique and auto transfusion of blood salvaged may play roles to decrease the intra-operative RBC requirements.[Figure 1]

Since the adoption of the Model for End-Stage Liver Disease, simultaneous liver/kidney transplants (SLKT) have substantially increased. Recently, unfavorable outcomes have been reported yet contributing factors remain unclear. We retrospectively reviewed 74 consecutive adult SLKT performed at our center from 2000 to 2010 and compared with kidney transplant alone (KTA, N = 544). In SLKT, patient and death-censored kidney graft survival rates were 64 ± 6% and 81 ± 5% at 5 years, respectively (median follow-up, 47 months). Multivariable analyses revealed three independent risk factors affecting patient survival: hepatitis C virus positive (HCV+, hazard ratio [HR] 2.9, 95% confidence interval [CI] 1.1–7.9), panel reactive antibody (PRA) > 20% (HR 2.8, 95% CI 1.1–7.2) and female donor gender (HR 2.9, 95% CI 1.1–7.9). For death-censored kidney graft survival, delayed graft function was the strongest negative predictor (HR 8.3, 95% CI 2.5–27.9), followed by HCV+ and PRA > 20%. The adjusted risk of death-censored kidney graft loss in HCV+ SLKT patients was 5.8 (95% CI 1.6–21.6) compared with HCV+ KTA (p = 0.008). Recurrent HCV within 1 year after SLKT correlated with early kidney graft failure (p = 0.004). Careful donor/recipient selection and innovative approaches for HCV+ SLKT patients are critical to further improve long-term outcomes.

Introduction: Requirement of blood transfusions during liver transplantation (LT) has been reduced by refinements in patient selection, transfusion methods, and surgical technique. LT with limited volume of blood product is the current clinical practice. The aim of this study was to analyze the effect of intra-operative red blood cell (RBC) transfusion volume on liver transplant outcome in adult patients. Methods: Two thousands two hundreds twenty eight adult patients were selected from our database. Out of 2228, 1960 patients were included for this study. Fifty one patients were selected as group 1 (G1) due to no RBC transfusion, 1557 patients as group 2 (G2) due to the 1-20 units of RBC transfusion, and 352 patients as group 3 (G3) due to more than 20 units of RBC transfusion during LT surgery. Aprotinin was not used. Auto transfusion of blood salvaged was used routinely. Independent factors were analyzed with Cox hazard regression analysis to find a link with survival. Survival curve was generated by Kaplan Meier methods and compared with log rank test. Results: There were statistically significant differences in recipient age (G1: 50.5 yrs., G2: 52.9 yrs., G3: 54.3 yrs.), pre-transplant creatinine (G1: 0.9 mg/dl, G2: 1.25 mg/dl, G3: 1.56 mg/dl), cold ischemic time (G1: 429 min, G2: 446 min, G3; 477min), and warm ischemic time (G1: 37.0 min, G2: 37.8 min, G3; 40.5min). There were no statistically significant differences in demographic factors including donor age, primary liver diagnosis, MELD score, INR, bilirubin, volume of auto-transfusion and cava reconstruction methods between the groups. One, 3, 5 years' patient survival were G1: 92% 86% 80% G2: 87% 79%, 73% and G3: 73% 70% 62%. One, 3, 5 years' graft survival were G1: 92% 86% 79% G2: 83% 73% 68% and G3: 70% 63% 59%. There was statistical difference in patient and graft survival between the groups (p< 0.05). Amounts of transfusion between the groups had survival impact (Hazard ratio 1.3, 95% CI 1.12-1.51, P< 0.05). Conclusion: Recipient age and pre-transplant creatinine had impact on the RBC transfusion volume. To the contrary, recipient MELD score, primary liver disease, donor age and method of caval reconstruction had no significant impact. Amount of transfusion had an impact on the patient and graft survival.Figure: [Patient Survival]

Background: Patients with end stage liver disease may have coexisting cardiac disease which requires surgical correction which is associated with high morbidity and mortality unless the liver is replaced. It is unclear whether the cardiac surgery should be performed at the time or following liver transplantation, if at all possible. Methods: Between February 1987 and December 2011, 12 patients (2 female, 10 male) underwent simultaneous cardiac operations and liver transplantation (Group A), 5 patients (2 female, 3 male) underwent cardiac operations after liver transplantation (Group B) at Miami Transplant Institute, University of Miami. The cardiac surgery in Group A includes heart transplantation (6), aortic valve replacement (3), coronary artery bypass grafting (2), and tricuspid and pulmonary valve replacement (1). Patients in Group B patients underwent cardiac surgery 2-138 months after liver transplantation, which includes heart transplantation (2), coronary artery bypass grafting (3). Results: Survival rates at 6 months, 1 year, 2 years, 3 years, 5 years in group A and B are 75%, 58%, 42%, 42%, 42%, vs. 80%, 80%, 80%, 60%, 40% respectively (Kaplan-meier, p=0.02). Age, MELD score, gender, cumulative hospital stay did not differ between two groups.Figure: [survival comparison]Conclusions: Although long term outcome was similar in both groups, short term results were better when the liver transplant preceded the open heart surgery. Consequently, heart surgery should be postponed if at all possible.