Background. Natural killer (NK) cells play important roles in killing tumor and virus-infected cells. Immunosuppression used after organ transplantation is thought to increase the risk of tumor recurrence and viral infections. However, the effect of immunosuppressive drugs on NK cells has not yet been clearly established. Therefore, we examined the effect of immunosuppression on NK cells.Methods. NK cells were cultured for 7 days in the presence of interleukin-2 (100 U/mL) with or without the following immunosuppressive drugs: tacrolimus, cyclosporine A, corticosteroid (methylprednisolone [MP]), mycophenolate mofetil, and rapamycin. The effect of the drugs on NK cell activation was tested on the basis of the following: NK cell phenotype, NK cell proliferation, cytotoxicity against K562 cells, cytokine production by NK cells, and anti-hepatitis C virus (HCV) activity with HCV genomic replicon cells.Results. NK cells showed relatively robust functions in the presence of tacrolimus and cyclosporine A. Mycophenolate mofetil and rapamycin significantly prevented only NK cell proliferation (P < .05). In contrast, MP significantly inhibited the proliferation, cytotoxicity, and anti-HCV effect (10.9%, 18.5%, and 1.9%, respectively) of NK cells. Furthermore, MP specifically inhibited the expression of NK cell activation markers and the production of interferon-gamma (P < .05).Conclusions. Corticosteroids have distinct effects on NK cells, which may have important implications for NK cell function in cytotoxicity and HCV effect after transplantation.

The management of severe hepatic artery vasospasm soon after liver transplantation (LT) is challenging because it can lead to hepatic artery thrombosis and subsequent graft failure. A 61-year-old man with hepatitis C cirrhosis and portal vein thrombosis received a deceased donor LT. On postoperative day 1, Doppler ultrasonography revealed a high-resistance waveform in the hepatic artery. Angiography showed severe vasospasm of the donor hepatic artery on postoperative day 3. Strong hepatic arterial buffer response (HABR) was considered for this etiology due to high portal vein velocity. Therefore, vasodilators, including nitroglycerin and prostaglandin E1, were initiated. The waveform of the hepatic artery vasospasm gradually improved as portal vein velocity decreased by Doppler ultrasonography within 7 days after LT. In conclusion, hepatic arterial buffer response can induce hepatic artery vasospasm immediately after LT. This vasospasm type may be managed conservatively with a positive outcome.

To determine uterine and ovarian viability with microsurgical anastomoses of utero-ovarian vessels alone in a baboon model for translation to future human uterine transplantation efforts. Prospective observational study of uterine and ovarian viability in Papio hamadryas baboons with surgically-altered uterine perfusion. Three baboons underwent laparotomy to alter uterine perfusion. Bilateral uterine arteries and veins were surgically ligated. A circumferential colpotomy was made and repaired. One utero-ovarian artery and vein were identified on each side, divided, and re-anastomosed end-to-end using a microsurgical technique. Intra-operative perfusion of the uterus was documented with near-infrared perfusion angiography. Resumption of menstrual blood flow with appearance of uterus on transabdominal ultrasound and cervical biopsies performed 6-10 weeks post-laparotomy showed viability of the uterus, while cyclical changes of the sex skin demonstrated continued ovarian function. All surgeries occurred without incident, and near-infrared perfusion angiography confirmed intra-operative uterine perfusion after completion of microsurgical anastomoses. One baboon acquired cellulitis of the skin incision which resolved with antibiotics. Trans-abdominal ultrasound confirmed presence of uterus in all animals 6-10 weeks after surgery, and simultaneous cervical biopsies verified normal cervical tissue. Within the first 60 days post-laparotomy, all animals demonstrated at least one menstrual bleed, and cyclical pattern of sex-skin changes in accordance with normal baboon physiology. The baboon uterus can be adequately perfused by bilateral microsurgical anastomosis of the utero-ovarian vessels in the absence of the uterine artery and veins, and cervico-vaginal vessel branches. This technique did not disrupt ovarian function and shows promise for future human transplantation trials to occur without meticulous uterine artery and vein dissection, and instead to rely solely on utero-ovarian vasculature.

BACKGROUND AND PURPOSE:Small infants with biliary atresia and hypoplastic portal veins (PV) are at risk for portal vein thrombosis (PVT) after liver transplantation (LT), which can lead to graft loss and mortality. Extra-anatomical PV reconstruction techniques have been established for adult cases of PVT; however, they have not been widely accepted for infants.METHODS:Here, we report the successful use of an extra-anatomical meso-portal venous jump graft to treat early PVT after LT in a 6-month-old infant with biliary atresia and PV hypoplasia. At the time of LT, despite a reduced-sized left lateral graft, we had to create a temporary abdominal closure with silastic mesh.FINDINGS:On postoperative day 1, PVT was detected by Doppler ultrasound of the liver. Surgical thrombectomy was attempted. We removed the blood clots and reconstructed the PV using an interposition venous graft. As the PV flow was still not sufficient, we performed an extra-anatomical meso-portal venous jump graft procedure from the recipient superior mesenteric vein to the donor PV. This resulted in a significant improvement in PV flow.CONCLUSION:For small infants at high risk for PVT, a detailed pretransplantation surgical plan and treatment options for possible early PVT are mandatory. An extra-anatomical meso-portal venous jump graft is a viable surgical technique for early PVT in infants.

To contribute to a novel surgical approach for uterine transplantation by assessing uterine viability after interruption of the bilateral uterine arteries and veins in a baboon model. Prospective observational study of uterine viability in Papio hamadryas baboons undergoing surgical interruption of the bilateral uterine arteries and veins. Three baboons underwent laparotomy during which uterine arteries and veins were isolated, ligated, and transected bilaterally. A circumferential colpotomy was made and the cervix was reattached to the vagina. Intra-operative perfusion of the uterus and the cervico-vaginal junction was documented with near-infrared perfusion angiography following intravenous administration of indocyanine green dye. Postoperatively the animals were monitored for resumption of menses and changes in sex skin turgescence. All baboons underwent transabdominal ultrasonography, vaginoscopy, and cervical biopsy 7 weeks post-laparotomy. Surgeries were performed without complication and intraoperative near-infrared angiography in all cases confirmed prompt blood flow throughout the entire uterus and along the cervico-vaginal anastomosis. Recovery of all animals was also uncomplicated. A normal appearing uterus with a thin and homogenous endometrial stripe was visualized on ultrasound performed 7 weeks after surgery in all subjects. Post-operative vaginoscopy permitted visualization of well approximated cervico-vaginal anastomoses and cervical biopsies revealed normal cervical tissue without evidence of necrosis. For each of the baboons, the first post-operative menstrual bleed occurred within 30 days of surgery and lasted for 2-3 days, consistent with normal baboon menses. Sex skin turgescence occurred in predictable cyclic patterns postoperatively, in accordance with each baboon’s respective menstrual cycle. Histopathology results, ultrasound imaging, and resumption of menstrual cycles demonstrated postoperative uterine viability in all 3 baboons, establishing that bilateral uterine artery and vein ligation does not affect uterine function. The surgical technique of interrupting uterine vasculature, combined with disconnection and reanastamosis of the cervix and vagina, is designed to simulate implantation of a donor uterus that is connected to the recipient exclusively by utero-ovarian vessels. Eliminating dissection of the uterine artery and vein from donor hysterectomy shows great potential for future human uterine transplant trials, as it offers a more efficient, less risky, and less technically challenging technique to live donor uterus procurement.

We present the case of a child who underwent a combined liver, pancreas and double kidney transplant following complications of Wolcott-Rallison syndrome (WRS) a rare genetic disorder that causes infantile insulin-dependent diabetes mellitus (IDDM) and often death in childhood from fulminant liver and concomitant kidney failure. WRS is characterized clinically through infantile IDDM, propensity for liver failure following viral infections, bone dysplasia and growth failure and developmental delay. Fewer than 60 cases with WRS are reported in the literature, mostly from consanguineous parents. Future episodes of liver failure, the main contributor to the increased mortality in WRS, may be prevented through timely liver transplantation. To the best of our knowledge, transplantation has not been utilized to manage complications of WRS prior to this report.

An exciting time to become a transplant surgeon

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.

Uterine transplantation in the sheep model has been described as a partial or whole orthotopic graft from a living donor with vascular anastomoses. As an alternative to surrogate pregnancy or adoption uterus transplantation might be indicated for cases of infertility of uterine origin. The main complications might be rejection and thrombosis. The objective of this work was to develop a model of whole uterus transplantation that was applicable to the human setting, using grafts obtained from brain-dead donors, and suitable for immunologic and viability follow-up with a reduced risk of thrombosis. Two donors and 1 recipient were operated. The first graft was used for an anatomic study; the second was used for transplantation. The donor operation consisted of an en bloc harvest of the uterus, adnexa, and proximal vagina with the distal aorta and cava. After harvest the donor sheep was humanely killed. In the recipient ewe, heterotopic implantation was performed in the lower abdomen. An End-to-side anastomoses of aorta and cava were performed below the recipient's renal vessels. A cutaneous vaginal stoma was performed in the right lower quadrant. The recipient ewe was humanely killed for an autopsy study. The anatomy of uterine veins of the ewe differs from the human. The uterine and ovarian veins join, forming the utero-ovarian vein, which drains at the confluence of the common iliac to the cava. En bloc harvesting allows for rapid graft preparation, with vascular cuffs easily anastomosed with a low risk of thrombosis. The vaginal stoma seems appropriate to facilitate follow-up and graft biopsy. This approach can be a suitable experimental model applicable to humans using grafts from brain-dead donors.

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.

Background: Liver transplantation (LT) has been accepted as the treatment of choice for early, unresectable hepatocellular carcinoma (HCC) in cirrhotic patients. Still, HCC recurrence after transplant remains a serious problem. Currently, there are no evidence-based guidelines for post-transplant surveillance for HCC recurrence. In this study we review our experience with LT for HCC and determine the characteristics and patterns of recurrence. Our purpose is to generate a surveillance strategy for HCC recurrence after LT. Methods: We retrospectively reviewed our experience with LT for HCC, focusing on those patients that experienced HCC recurrence. The setting was a single, high volume center in the US operating under the MELD allocation system. The risk factors that predicted recurrence and the incidence of recurrence were determined. Data regarding the timing and first site of recurrence, alpha-fetoprotein (AFP) level, treatment, and survival after the diagnosis of recurrent HCC were collected and analyzed. With this information we propose surveillance guidelines. Results: Between March 2002 and December 2010, 275 adults underwent primary LT for cirrhosis and HCC including 53 with incidental tumors. The 5 year recurrence-free survival for the cohort was 83.4%. At a median follow-up of 44.0 months, 42 patients developed recurrent HCC for an actual recurrence rate of 15.2%. The factors most predictive of HCC recurrence included an elevated peak AFP prior to transplant (>50 ng/ml) and unfavorable explant pathology (poorly differentiated tumors and/or presence of lymphovascular invasion-LVI). Nine (21%) patients that recurred had none of these factors while 5 (11.9%) of patients that recurred originally had incidental tumors. The median time to recurrence was 20.5 months (n=42), with the presence of LVI predicting earlier recurrence (median 7.5 months, n=24). Nineteen patients (46%) experienced recurrence more than 2 years after LT. The site of first recurrence was the lung (38%) followed by the liver (33%). Serum AFP was elevated (>50 ng/ml) at the time of recurrence diagnosis in 26 patients (61.9%). Median survival after recurrence was 10.0 months; 11 patients are currently alive with recurrent HCC. Patients with recurrent tumors able to undergo resection or loco-regional therapy experienced improved survival. Conclusions: These data suggest that surveillance for HCC recurrence after LT is warranted, including patients thought to be at low risk for recurrence. Surveillance imaging should include the chest and abdomen. Surveillance should be more rigorous for those patients that had HCC with LVI, especially during the first year after LT. The duration of surveillance for HCC recurrence should extend at least 4 years. AFP is a useful adjunct for surveillance. Early detection of HCC recurrence may allow for aggressive treatment and improved survival.

Introduction: Long term survival of intestinal transplant patients is hampered by rejection episodes. Currently protocol endoscopy of the graft and biopsies are the only way to predict a rejection episode. Symptoms and clinical findings frequently appear when injury to the graft is irreversible. In this report we present our initial data of putative candidate biomarkers of graft rejection in peripheral blood of intestinal transplant patients. Materials and methods: Gene expression analysis was performed in peripheral blood of intestinal transplant patients. The results were matched with concurrent graft biopsies using bioinformatics. Peripheral blood samples of intestinal transplant patients were collected in Tempus Blood RNA tubes. Concurrent graft endoscopies were performed and graft biopsies were obtained at the time of the blood samples, to match the results of the gene expression analysis with the state of the graft. Whole genome microarray analysis using the Illumina-HT12 Expression beadchip microarray was performed. The gene expression levels in patient samples were compared to those in a pool of healthy volunteers. Bioinformatics analysis was performed using the MetaCore 6.3 software from GeneGo Inc. and the Pathway Studio 7.1 software from Ariadne Genomics. Results: Peripheral blood samples (n=11), of 3 adult patients [transplant day (n=1), no rejection (n=1), minimal rejection (n=2), mild rejection (n=5) and severe rejection (n=2)] were collected. Bioinformatics: Enrichment Analysis: The three most affected pathways differentially expressed in rejection versus a pool of healthy volunteers were related to protein translation: translation initiation, translation elongation termination, and translation in mitochondria, with p-values for all rejection stages in all patients in the 10-4 to 10-18 range. No significant enrichment was observed for these categories in the day of transplant sample. In addition to translation, significant enrichment of several immune response categories was observed in rejection samples. Subsequent gene set enrichment analysis verified these results. The level of enrichment was very high (p-values of 10-5 - 10-60) and increased with the level of rejection in all patients. Genes significantly down-regulated in translation related gene sets included ribosomal proteins RPL13A, RP L22, RPS23, RPL13 and RPL10A, that could be used as potential biomarkers for future experiments. These results were further verified with the use of additional samples of 2 more small bowel transplant patients. Conclusion: In this pilot study we found a list of genes (involved in translation) significantly down-regulated in the peripheral blood of intestinal transplant patients during rejection. Our hypothesis, derived from this project, is that down-regulation of translation in peripheral blood mononuclear cells occurs early during the course of rejection and persists and progresses with the severity of the rejection episode as determined by concurrent graft biopsies.

Introduction: The present study provides the long term follow up of our adult liver transplant recipients induced with alemtuzumab (Campath 1H, C1H). Methods: This is a retrospective analysis of patients who underwent liver transplantation using C1H induction at our Center from 2001 until 2010. C1H was not administered to patients with Hepatitis C (HCV). Maintenance immunosuppression was with half the usual dose of tacrolimus. Patients receiving steroids for their underlying (autoimmune) disease were maintained on the same dose after transplantation. No other steroids were given except for acute rejection episodes. Patients transplanted before April 2004 received four doses of C1H starting pre-operatively, whereas those transplanted afterwards received two or a single dose of C1H starting immediately after the transplant. Results: Three hundred and four patients (192 male, 112 female), received a liver allograft with C1H induction immunosuppression, Seventy three patients received 4 doses of C1H, the first one pre-operatively, 231 patients received two (n=77) or one (n=154) dose, starting immediately post-operatively. One, three, five and seven year patient survival was 91.4%, 87.35%, 84.62% and 82.83%. One, three, five and seven year graft survival was 89.47%, 84.69%, 80.84% and 78.36%. One, three, five and seven year freedom from rejection was 57.79%, 52.32%, 47.43%, 46.02%. There was no significant difference in incidence of rejection between patients that received the first dose of Campath-1H before or after the transplant. Average tacrolimus trough levels were between 5-7 ng/ml for the first 2 years post transplantation and less than 5 ng/ml thereafter. Twelve patients have required long term renal replacement therapy to date, five requiring kidney transplantation and seven additional patients dialysis. One, three, five and seven year freedom from renal replacement was 99.64%, 98.89%, 96.03%, 92.77%. Seven patients were weaned off immunosuppression 3-7 years post-transplantation. Conclusion: C1H induction with low dose tacrolimus maintenance immunosuppression leads to good outcomes in non-HCV liver transplant recipients. Postoperative administration of C1H is as effective as preoperative exposure.