Impact of Steroids on Natural Killer Cells Against Cytotoxicity and Hepatitis C Virus Replication.

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-γ (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.

A Case Report of Severe Hepatic Artery Vasospasm Induced by Hepatic Arterial Buffer Response After Liver 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.

Extra-anatomical Meso-portal Venous Jump Graft Repair for Early Portal Vein Thrombosis After Liver Transplant in an Infant With a Hypoplastic Portal Vein: A Case Report.

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.

Liver, pancreas and kidney transplantation for the treatment of Wolcott-Rallison syndrome.

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.

Uterus transplantation model in sheep with heterotopic whole graft and aorta and cava anastomoses.

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.

Use of the recipient celiac trunk for hepatic artery reconstruction in orthotopic liver transplantation.

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.

Factor V Leiden mutation is a risk factor for hepatic artery thrombosis in liver transplantation.

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.

Comparative analysis of T-cell depletion method for clinical immunotherapy-anti-hepatitis c effects of natural killer cells via interferon-γ production.

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.

Citrulline level is a potent indicator of acute rejection in the long term following pediatric intestinal/multivisceral transplantation.

Citrulline has been advocated as a marker for acute cellular rejection (ACR) in intestinal transplantation; however, its significance as a forewarning in the long-term follow-up remains unknown. This study aimed to investigate the association between citrulline levels and the grading of ACR to establish a cutoff point that accurately predicts ACR beyond 3 months posttransplant in the pediatric patient population. During a 16-year period (1995-2011), a total of 13 499 citrulline samples were prospectively collected from 111 consecutive pediatric intestinal/multivisceral transplant recipients: 2155 were obtained concurrently with intestinal biopsies. There were 185 ACR episodes observed among 74/111 (67%) patients (median follow-up: 4.4 years). Citrulline levels were inversely proportional to the severity of ACR. Negative predictive values for any type of ACR (cutoff, 20 µmol/L) and moderate/severe ACR (cutoff, 10 µmol/L) were 95% and 99%, respectively. When patients were divided according to graft size, diagnostic accuracy using the same cutoff was identical. Similarly, subgroup analysis by the timing of citrulline measurement prior to biopsy varying from 1 to 7 days demonstrated comparable results. Citrulline is a potent indicator as a danger signal for ACR, being an exclusionary, noninvasive biomarker with excellent negative predictive values in the long term after pediatric intestinal/multivisceral transplant.

MicroRNA signature of intestinal acute cellular rejection in formalin-fixed paraffin-embedded mucosal biopsies.

Despite continuous improvement of immunosuppression, small bowel transplantation (SBT) is plagued by a high incidence of acute cellular rejection (ACR) that is frequently intractable. Therefore, there is a need to uncover novel insights that will lead to strategies to achieve better control of ACR. We hypothesized that particular miRNAs provide critical regulation of the intragraft immune response. The aim of our study was to identify miRNAs involved in intestinal ACR. We examined 26 small intestinal mucosal biopsies (AR/NR group; 15/11) obtained from recipients after SBT or multivisceral transplantation. We investigated the expression of 384 mature human miRNAs and 280 mRNAs associated with immune, inflammation and apoptosis processes. We identified differentially expressed 28 miRNAs and 58 mRNAs that characterized intestinal ACR. We found a strong positive correlation between the intragraft expression levels of three miRNAs (miR-142-3p, miR-886-3p and miR-132) and 17 mRNAs including CTLA4 and GZMB. We visualized these miRNAs within cells expressing CD3 and CD14 proteins in explanted intestinal allografts with severe ACR. Our data suggested that miRNAs have a critical role in the activation of infiltrating cells during intestinal ACR. These differences in miRNA expression patterns can be used to identify novel biomarkers and therapeutic targets for immunosuppressive agents.

Association between panel reactive antibodies and acute small bowel rejection: analysis of a series of 324 intestinal transplants.

INTRODUCTION: Panel reactive antibodies (PRA) to class I and II HLA molecules have been associated with acute kidney graft rejection, but their role in small bowel transplantation has not been characterized. METHODS: Since 1994, 324 SBT, alone or as multivisceral transplantation (MVT), have been performed in 286 patients. Routine and surveillance biopsies were performed to rule out or confirm acute rejection (AR), and PRA quantification was performed at varying intervals. We obtained data from 110 patients and 651 PRA measurements. While AR grade (mild to severe, grades 1-3) was determined by histopathological analysis, the status of no AR was determined also by clinical data. When biopsy samples or PRA measurements were frequent around an AR episode within periods of 7 days, the highest value was used. RESULTS: A comparison could be made between 259 instances in which there was a PRA measurement and simultaneous rejection evaluation. Positive PRA showed association with AR (P < 0.001). The positive and negative predictive values were 44% and 79%, respectively. No correlation was found in the severity of rejection. CONCLUSION: The presence of increased levels of PRA is a risk factor of rejection in small bowel transplantation. Alloantibody-mediated injury to the graft contributes frequently to acute rejection of small bowel, and it is associated with cell-mediated immunity in variable proportion.

Citrulline evaluation in bowel transplantation.

INTRODUCTION: The purpose of this study was to evaluate the correlation of plasma citrulline and rejection episodes in intestinal transplantation. METHODS: From January 2007 until present, we performed citrulline assays on our small bowel patients. We investigated the correlation of these assays with the rejection status of the patients. The rejection status of the graft was defined based on graft biopsies. RESULTS: Of 5195 citrulline samples, average serum citrulline levels decreased significantly when the patients presented a rejection episode. We found the following: no rejection, 17.38 microm/L; mild rejection, 13.05 microm/L; moderate rejection, 7.98 microm/L; and severe rejection, 6.05 microm/L. Our current emphasis is to determine the predictive power of citrulline with other biomarkers versus as a separate and isolated measurement. CONCLUSIONS: In our study, citrulline levels correlated significantly with the rejection status of the graft. Serial follow-up of the patients using this assay may alert us to the possibility of increased alloreactivity and rejection episodes.

International grading scheme for acute cellular rejection in small-bowel transplantation: single-center experience.

A standardized grading scheme for the assessment of acute cellular rejection (ACR) in small-intestine transplantation was proposed in 2003 at the Eighth International Small Intestinal Transplantation Symposium. We have implemented the current grading scheme for ACR in small-bowel transplantation since October 2003. The pathologic diagnoses of those small-intestine biopsy samples, including ACR grade and other supplementary findings were evaluated. A total of 3484 small intestine biopsy samples from 155 patients were available for evaluation in this study. Frequency of grades 0, indeterminate, 1, 2, and 3 acute cellular rejection was 33.9%, 49.1%, 12.6%, 3.7%, and 0.8%, respectively. Duration of ACR episode strongly correlated with grade of ACR episode (P < .001). Other supplementary findings included acute vascular rejection component, 2.2%; increase in lymphoplasmacytic infiltrate in lamina propria, 15.7%; mucosal fibrosis, 0.4%; and regenerative changes, 0.3%. Our data substantiate that this grading system is reliable and useful for clinical decision making in bowel transplantation. We suggest that an assessment and quantification of supplementary findings be considered a component of the International Pathology Grading Scheme.

Immediate antibody-mediated (hyperacute) rejection in small-bowel transplantation and relationship to cross-match status and donor-specific C4d-binding antibodies: case report.

BACKGROUND: The role of preformed donor-specific antibodies (DSAs) as a barrier to isolated intestinal transplantation (ITx) remains ambiguous; thus, a positive cross-match has not been a contraindication to ITx. OBJECTIVE: To report the case of a patient with Crohn's disease who underwent ITx and developed immediate antibody-mediated rejection on reperfusion of the allograft. METHODS: Percent reactive antibody testing was performed using pretransplantation serum samples and at transplantation using bead-based assays (Luminex, Luminex Corp, Austin, Tex) and flow cytometry solid-phase assays (FlowPRA single-antigen beads (One Lambda, Inc, Canoga Park, Calif). Serologic tests, flow cytometry cross-matching, and flow cytometry assays of C4d-binding serum antibodies were also performed. Histologic and immunofluorescent analysis of biopsy specimens was performed. RESULTS: HLA typing revealed no sharing of class I or II antigens between donor and recipient. Pretransplantation donor-specific antibodies (DSA) were present at transplantation. Cross-matching (performed during surgery) was positive for class I and II by serologic testing and flow cytometry. After reperfusion, the graft immediately developed severe ischemic injury and arteritis on mucosal biopsy specimens, with immunoglobulin deposition. The DSA C4d binding antibodies were also present. After intense immunosuppression and plasmapheresis, the graft and the biopsy histologic findings showed marked improvement (day 2). By day 7 posttransplantation, patient and graft status were stable. The patient has remained clinically stable for more than a year after transplantation. CONCLUSIONS: Pretransplant DSA in ITx can be a risk factor for immediate (hyperacute) but potentially reversible antibody-mediated rejection. Thus, pretransplantation DSA and cross-match results are critical components to be considered in patients awaiting or undergoing ITx.

Abdominal wall transplantation: surgical and immunologic aspects.

Abdominal wall transplantation is a type of composite tissue allograft that can be utilized to reconstitute the abdominal domain of patients undergoing intestinal transplantation. We have presented herein combined experience and long-term follow-up results of a series of abdominal wall transplants performed at 2 institutions. A total of 15 abdominal wall transplants from cadaveric donors were performed in 14 patients at the end of intestinal transplant surgery or, in 2 cases, a few days after the primary intestinal transplant. The vascular supply was through the inferior epigastric vessels, from the iliac vessels in 12 cases and via a microsurgical technique in 3 cases. Immunosuppression consisted of induction with alemtuzumab and maintenance treatment with tacrolimus monotherapy. Two grafts lost to vascular thrombosis were removed. Five patients are still alive, although all deaths were unrelated to the abdominal wall transplant. There were 3 episodes of abdominal wall graft rejection, treated with steroids; the abdominal wall graft and the intestinal grafts experienced rejection independent from each other. In summary, abdominal wall transplantation is a feasible technique for recipients of intestinal or multivisceral transplants, when the closure of the abdominal cavity by primary intention is technically impossible.

Effect of pretransplant hepatitis C virus RNA status on posttransplant outcome.

Undetectable hepatitis C virus (HCV) RNA [RNA(-)] before liver transplantation (OLT) has been shown to decrease the rates of disease recurrence. We sought to determine whether RNA(-) subjects differ in post-OLT recurrence (virological/VR, histological/HR), graft failure (GF), or patient survival from RNA(+) patients using a retrospective review. From 1995 to 2004, a total of 49 patients were RNA(-) at OLT as a result of interferon-based therapy: 22 SVR and 27 with end-of-treatment response (ETR) transplanted when RNA(-) within 6 months of ET. Forty-eight RNA(+) patients were analyzed as controls. Virological recurrence (VR) was seen in 55% of RNA(-) subjects with no difference in HR between RNA(-) vs (+) groups, namely 36.7% versus 56.3% (P = .068), respectively. The RNA(+) subjects showed a lower time to HR (5.6 vs 11 months; P = .027). The SVR subjects displayed lower VR (36.4%) and histological recurrence (HR) (13.6%) compared to ETR (VR 70.4%, P = .023; HR 55.6%, P = .003) or RNA(+) (HR 56%, P = .0008). The SVR subjects, who were identified with a sensitive assay (SVR(S), lower limit <600 IU/mL) showed no VR, HR, or GF. The 1- and 5-year survivals were 87.8%/75.6% and 89.6%/77.8% for RNA(-) and (+) groups, respectively (P = .77). In conclusion, RNA(-)-transplanted patients displayed lower VR and longer time to HR. The SVR patients showed lower VR and HR compared to ETR and RNA(+) patients.

Hepatic gas gangrene following orthotopic liver transplantation: three cases treated with re-transplantation and a review of the literature.

Gas gangrene is a rare and devastating infectious process that can occur after liver transplantation, most often following hepatic artery thrombosis. We here report 3 cases of gas gangrene following orthotopic liver transplantation. Blood cultures were positive for Clostridium clostridiiforme in one case. In 2 other cases liver tissue from explanted specimens was positive for Enterobacter cloacae. Ultrasound demonstrated hepatic artery thrombosis and computed tomography imaging revealed diffuse liver necrosis with gas formation in each case. All 3 patients were successfully treated with a combination of antibiotics and emergent re-transplantation. We review previously published cases of gas gangrene after liver transplant and emphasize the importance of hepatic artery thrombosis in the development of this syndrome as well as the frequent involvement of non-clostridial organisms. Early diagnosis and aggressive combined medical and surgical treatment including re-transplantation are essential for successful treatment of these rare and catastrophic infections.

Analysis of acute cellular rejection episodes in recipients of primary intestinal transplantation: a single center, 11-year experience.

Intestinal transplantation has evolved over the years with major improvements in patient and graft survival. Acute cellular rejection of the intestine, however, still remains one of the most challenging aspects of postoperative management. We analyzed retrospectively collected data from 209 recipients of primary intestinal grafts at our institution over the past 11 years. A total of 290 episodes of biopsy-proven rejection requiring clinical treatment were analyzed. Rejection episodes doubled in length, on average, with each increasing grade (mild, moderate, severe). We observed increased incidence of overall rejection and particularly severe rejection in recipients of isolated intestinal and liver-intestine grafts in comparison with multivisceral grafts. Two rejection history variables had a significant negative impact on graft survival: the occurrence of a severe rejection episode and a rejection episode lasting >or=21 days. The lower incidence rate of severe rejection in recipients of multivisceral grafts might be due to a combination of increased donor lymphatic tissue and larger load of donor-derived immune competent cells present in the graft. The development of more effective monitoring and treatment protocols to prevent the occurrence of severe and/or lengthy rejection episodes is of critical importance for intestinal graft survival.

Ten-year experience in porto-caval hemitransposition for liver transplantation in the presence of portal vein thrombosis.

Porto-caval hemitransposition (PCH) in liver transplantation allows revascularization of the liver when the porto-mesenteric axis is thrombosed. We, here, review our experience over an 11-year period. A total of 23 patients underwent liver transplantation using PCH. Immunosuppression was based on tacrolimus, with sirolimus used in case of renal insufficiency. Most common diagnoses were hepatitis C, Laennec's, Budd-Chiari and cryptogenic cirrhosis. Six patients needed splenectomy prior to transplant, 5 during transplant, 1 post-transplant, 11 had no splenectomy. Overall survival was 60% at 1 year and 38% at 3 years, with 10 of 23 patients currently alive and the longest survivor at 9.3 years. Most common cause of death was sepsis/multisystem organ failure, followed by pulmonary embolism. A total of 7/23 patients experienced post-operative gastrointestinal bleeding episodes, 6/23 patients developed thrombosis of the vena cava (median 162 days post-op). Post-operative ascites was noted in almost all patients. Renal dysfunction was commonly seen even after the first month post-transplant. PCH offers a feasible option for liver transplantation in those patients with complex thrombosis of the mesenteric and portal circulation.