Errata.

Bioluminescence Imaging Visualizes Activation of Nuclear Factor-kappaB in Mouse Cardiac Transplantation.

BACKGROUND.: The purpose of the current study was to evaluate the role of bioluminescence imaging (BLI) in the determination of nuclear factor (NF)-kappaB activation in cardiac allograft rejection and ischemia-reperfusion injury. METHODS.: To visualize NF-kappaB activation, luciferase transgenic mice under the control of a mouse NF-kappaB promoter (NF-kappaB-Luc) were used as donors or recipients of cardiac grafts. Alternatively, NF-kappaB-Luc spleen cells were adoptively transferred into Rag2 mice with or without cardiac allografts. BLI was performed posttransplantation to detect luciferase activity that represents NF-kappaB activation. RESULTS.: The results show that luciferase activity was significantly increased in the cardiac allografts when NF-kappaB-Luc mice were used as recipients as well as donors. Luciferase activity was also elevated in the wild-type cardiac allografts in Rag2 mice that were transferred with NF-kappaB-Luc spleen cells. CD154 monoclonal antibody (mAb) therapy inhibited luciferase activity and induced long-term survival of cardiac allografts. toll-like receptor-9 ligand, CpG DNA, enhanced luciferase activity and abrogated tolerance induction by CD154 mAb. Luciferase activity was also increased in ischemia-reperfusion injury of the cardiac grafts. CONCLUSION.: BLI using Luc-NF-kappaB mice is a noninvasive approach to visualize the activation of NF-kappaB signaling in mouse cardiac allograft rejection and ischemia-reperfusion injury. CD154 mAb can inhibit NF-kappaB activation, which is reversed by toll-like receptor engagement.

Ma L, Xiang Z, Sherrill TP, Wang L, Blackwell TS, Williams P, Chong A, Chari R, Yin DP.

1 Department of Surgery, Division of Hepatobiliary Surgery and Liver Transplantation, Vanderbilt University Medical Center, Nashville, TN. 2 Department of Medicine, Division of Allergy/Pulmonary and Critical Care Medicine, Vanderbilt University Medical Center, Nashville, TN. 3 Department of Surgery, Section of Transplantation, University of Chicago, Chicago, IL.

SV40 Infection Associated With Rituximab Treatment After Kidney Transplantation in Nonhuman Primates.

BACKGROUND.: A regimen consisting of polyclonal anti-T-cell antibody, sirolimus (SRL), and donor bone marrow (DBM) infusion induces robust transplantation tolerance to skin allografts in mice. We investigated the effect of a similar regimen in a nonhuman primate (NHP) model. METHODS.: Cynomolgus macaques (Macaca fascicularis) were transplanted with mismatched kidney allografts. Recipients were treated with 7 doses of antithymocyte globulin (Thymoglobulin, day 1 to 9), sirolimus, and DBM infusion (day 14). Anti-CD20 antibody, rituximab, was given on days 0 and 5. RESULTS.: A regimen of Thymoglobulin, 30 days of SRL, and DBM infusion induced significantly greater prolongation of graft survival with a mean survival time of 88 days compared with the control regimen (no DBM) with an mean survival time of 53 days (P=0.022). Unlike the murine skin allograft model, all grafts were rejected within 111 days. A combination of Thymoglobulin, continuous SRL, and rituximab caused graft and systemic SV40 infection and failed to achieve further extension of graft survival. C4d deposition was observed in 50% of recipients as early as 18 days, suggesting antidonor antibody production. A transient, low-to-moderate degrees of multilineage chimerism was observed after DBM infusion. Treatment with Thymoglobulin resulted in profound depletion of CD4 and CD8 T cells, whereas addition of rituximab achieved prolonged (up to 3 months) depletion of CD20 B cells. CONCLUSION.: The Thymoglobulin, SRL, and DBM protocol is simple and produces long-term kidney allograft survival in NHP although additional treatment modalities may be necessary for induction of long-term tolerance.

Maki T, Carville A, Stillman IE, Sato K, Kodaka T, Minamimura K, Ogawa N, Kanamoto A, Gottschalk R, Monaco AP, Marr-Belvin A, Westmoreland SV, Sehgal P.

1The Transplant Center, Department of Surgery, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA. 2 New England Primate Research Center, Harvard Medical School, Framingham, MA. 3 Department of Pathology, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA.

Novel Immunosuppression: R348, a JAK3- and Syk-Inhibitor Attenuates Acute Cardiac Allograft Rejection.

BACKGROUND.: Janus kinase (JAK)3 is crucial for signal transduction downstream of various cytokine receptors in immune cells. This is the first report on the novel JAK3 inhibitor R348. METHODS.: (1) Detailed pharmacokinetic data were obtained in rats; (2) multiple in vitro enzyme inhibition assays were performed to characterize the drug; (3) prevention of acute rejection was investigated in animals treated with different doses of R348 or rapamycin for 5 days; and (4) cardiac allograft survival after a 10-day treatment period was studied for various regimens of R348, tacrolimus, or rapamycin; combination indices were calculated to evaluate drug interactions. RESULTS.: (1) Plasma levels of R348\’s active metabolite R333 sustained high for 8 hr or more, depending on the dose. (2) In vitro enzyme assays showed potent inhibition of JAK3- and Syk-dependent pathways. (3) R348 40 mg/kg preserved graft function, significantly reduced graft infiltration, and decreased histologic ISHLT rejection scores on postoperative day 5. Results were similar to those of rapamycin 3 mg/kg. Likewise, both drugs significantly reduced the cellular Th1 and Th2 immune responses, as determined by enzyme-linked immunosorbent assays. Intragraft inflammatory cytokine upregulation was similarly suppressed by R348 and rapamycin. R348 10 mg/kg was subtherapeutic. (4) Allograft survival was similar for R348 20 and 40 mg/kg, which was comparable with therapeutically dosed tacrolimus or rapamycin. In combination regimens, R348 demonstrated highly beneficial synergistic interactions with tacrolimus. CONCLUSIONS.: R348 is a promising novel JAK3/Syk-inhibitor with favorable pharmacokinetics and biological activity. It effectively diminishes acute cardiac allograft rejection and is suitable for combination regimens with tacrolimus.

Deuse T, Velotta JB, Hoyt G, Govaert JA, Taylor V, Masuda E, Herlaar E, Park G, Carroll D, Pelletier MP, Robbins RC, Schrepfer S.

1 Department of Cardiothoracic Surgery, Stanford University School of Medicine, Stanford, CA. 2 Rigel Pharmaceuticals, 1180 Veterans Boulevard, South San Francisco, CA.

Alleviating Intestinal Ischemia-Reperfusion Injury in an In Vivo Large Animal Model: Developing an Organ-Specific Preservation Solution.

INTRODUCTION.: This study investigated the role of a novel nutrient-rich preservation solution in alleviating intestinal ischemia-reperfusion (IR) injury in a large animal model. MATERIALS AND METHODS.: Porcine intestines were treated in vivo with the following intraluminal flush solutions: group 1, none; group 2, University of Wisconsin solution; group 3, an amino acid-based solution, previously shown to be effective in reducing IR injury in rodent models. Intestinal ischemia was induced in vivo for 60 min, followed by 180 min reperfusion. Key metabolic aspects were assessed in relation to two fundamental kinase mechanisms that govern cell fate, AMP kinase, and Jun kinase. RESULTS.: After 180 min reperfusion, groups 1 and 2 exhibited clefting, denudation, and mucosal hemorrhage, whereas injury was markedly reduced in group 3 (median grades 4.5 and 5 vs. 0; P<0.05). In contrast to groups 1 and 2, group 3 tissues exhibited a full recovery of adenylates (ATP, total adenylates) and an effective control of oxidative stress throughout reperfusion. Neutrophil-mediated inflammation was abrogated in group 3. An up-regulation of two key enzymes (glutaminase and alanine aminotransferase) provided a mechanism for the superior recovery of energetics and the preservation of mucosal integrity in group 3. A strong activation of AMP-activated protein kinase resulting in the up-regulation of a primary proapoptotic kinase mechanism, Jun kinase, was evident in groups 1 and 2. DISCUSSION.: A strategy of intraluminal administration of a nutrient-rich solution represents a potential therapy for alleviating intestinal IR injury; these findings suggest a more effective method for the ischemic storage of intestine.

Salehi P, Bigam DL, Ewaschuk JB, Madsen KL, Sigurdson GT, Jewell LD, Churchill TA.

1 Department of Surgery, Division of Transplantation, University of Illinois, Chicago, IL. 2 Department of Surgery, University of Alberta Hospital, Edmonton, Alberta, Canada. 3 Division of Gastroenterology, Department of Medicine, University of Alberta, Edmonton, Alberta, Canada. 4 Department of Laboratory Medicine and Pathology, Division of Anatomical Pathology, University of Alberta Hospital, Edmonton, Alberta, Canada. 5 Surgical-Medical Research Institute, University of Alberta, Edmonton, Alberta, Canada.

Prevention and Inhibition But Not Reversion of Chronic Allograft Vasculopathy by FK778.

BACKGROUND.: This study aimed at investigating the efficacy of the novel immunosuppressant FK778 to prevent the development and progression of chronic allograft vasculopathy (CAV). METHODS.: Orthotopic aortic transplantations were performed in the PVG-to-ACI rat model and followed over the course of 120 days. Immunosuppression with FK778 (20 mg/kg) or sirolimus (2 mg/kg) was either started early or delayed when CAV was already present. Trough levels were monitored. Aortic luminal obliteration was quantified using computer morphometry and intragraft cytokine profiles were analyzed with Western Blotting. Donor-reactive antibodies were quantified by flow cytometry. RESULTS.: Untreated animals developed CAV with luminal obliteration of 25.2+/-13.6% and 41.4+/-23.3% after 80 and 120 days, respectively. Continuous immunosuppression with FK778 or sirolimus effectively prevented the development of vasculopathy. When the start of the immunosuppressive regimen was delayed until postoperative day 80, FK778 and sirolimus inhibited a progression of established CAV but did not reverse the luminal obliteration. Intragraft tumor growth factor-beta activity increased over the course of time in untreated recipients but was significantly suppressed after continuous immunosuppression with either agent. Expression of platelet-derived growth factor, intercellular adhesion molecule-1, and vascular adhesion molecule-1 also was moderately suppressed. A stable elevation of donor-reactive IgG-antibody levels was found over 120 days in the absence of treatment. With FK778 or sirolimus, antibody levels were effectively decreased. FK778 was very well tolerated and only sirolimus showed side effects with elevation of BUN, cholesterol, triglycerides, and ALT after 120 days. CONCLUSIONS.: FK778 prevents the development of CAV and inhibits a progression of established disease. It shows a similar efficacy but a safer drug profile when compared to sirolimus.

Deuse T, Hoyt G, Koyanagi T, Robbins RC, Schrepfer S.

1 Department of Cardiothoracic Surgery, Stanford University School of Medicine, Stanford, CA. 2 Department of Chemical and Systems Biology, Stanford University School of Medicine, Stanford, CA.

Intraoperative blood salvage during liver transplantation in patients with hepatocellular carcinoma: efficiency of leukocyte depletion filters in the removal of tumor cells.

BACKGROUND.: Intraoperative blood salvage (IBS) reduces homologous transfusion in orthotopic liver transplantation (OLT), but may carry with it the risk of reinfusing tumor cells in patients with hepatocellular carcinoma (HCC). The use of leukocyte depletion filters (LDFs) for the removal of tumor cells is rarely reported in clinical OLT. The aims of this study were to evaluate the frequency of tumor cell contamination in surgical field during OLT for HCC recipients and to investigate the efficiency of additional LDFs for eliminating tumor cells from IBS. METHODS.: Thirty-two HCC patients with preoperatively elevated serum alpha-fetoprotein (AFP) underwent OLT. The blood from the surgical field was collected and processed by an autotransfusion device (Cell Saver 5), followed by 2 consecutive LDF filtrations. The HCC cells in IBS samples and filtered samples were determined using a nested RT-PCR technique to detect the AFP mRNA. RESULTS.: The shed blood samples from 20 (62.5%) of the 32 HCC patients were contaminated with HCC cells and 15 of them remained positive after Cell Saver processing. Patients within the Milan or UCSF criteria were less likely to have HCC cell contamination and the contaminated HCC cells were more likely to be removed by the Cell Saver in these patients as compared to other patients (P<0.01). After filtration through an additional LDF, most cases (13/15) became negative except for those with ruptured tumors (P<0.05). CONCLUSIONS.: Our results suggest that blood filtration with the LDF can efficiently remove tumor cells and the use of an additional LDF after use of the Cell Saver could markedly reduce the risk of tumor cell reintroduction during the OLT in HCC recipients with nonruptured tumors.

Liang TB, Li DL, Liang L, Li JJ, Bai XL, Yu W, Wang WL, Shen Y, Zhang M, Zheng SS.

Department of General Surgery, Key Laboratory of Multi-organ Transplantation of Ministry of Public Health, the First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, P.R. China.

Limited Benefit of Biochemical Response to Combination Therapy for Patients With Recurrent Hepatitis C After Living-Donor Liver Transplantation.

BACKGROUND.: Effective management of patients with recurrent hepatitis C after liver transplantation has not been established. In this study, we tested the significance of biochemical response (BR) as well as sustained virological response (SVR) to combination therapy with interferon alpha-2b and ribavirin for recurrent hepatitis C after living-donor liver transplantation. METHODS.: Forty patients received therapy and formed 3 groups by response: SVR (n=14, 35%) and BR (n=14, 35%), defined when serum alanine aminotransferase normalized during therapy in non-SVR patients, and no response (NR; n=12, 30%). Histological changes of these groups after the combination therapy were investigated. RESULTS.: Activity grade of liver histology improved significantly in patients with an SVR at 1 and at 2-4 years after the initiation of treatment, showing difference from the BR and NR groups in which the activity was not improved by interferon therapy. Fibrosis deteriorated significantly in both the BR and the NR groups at two to four years. The rates of patients whose fibrosis stage deteriorated to stage F2 or more were significantly greater in the BR and NR groups than in the SVR group. Fibrosis decreased or remained stable in five of eight patients (63%) with an SVR at two to four years, whereas it increased in 12 of 15 patients (80%) with a BR or NR. CONCLUSIONS.: Patients with an SVR showed histological improvement, but the benefit of a BR for non-SVR patients to improve the activity grade or to inhibit the progression of fibrosis was limited.

Ueda Y, Takada Y, Haga H, Nabeshima M, Marusawa H, Ito T, Egawa H, Tanaka K, Uemoto S, Chiba T.

1 Department of Gastroenterology and Hepatology, Graduate School of Medicine, Kyoto University, Kyoto, Japan. 2 Department of Surgery, Graduate School of Medicine, Kyoto University, Kyoto, Japan. 3 Department of Pathology, Graduate School of Medicine, Kyoto University, Kyoto, Japan.

Successful Treatment of BK Viremia Using Reduction in Immunosuppression Without Antiviral Therapy.

BACKGROUND.: Treatment of BK virus (BKV) infection in renal transplant recipients remains controversial. This retrospective analysis evaluated efficacy and safety of reducing immunosuppression without antiviral therapy. METHODS.: This single center analysis included 24 patients diagnosed with BK viremia between September 2001 and December 2003. Sixteen patients (66%) presented with BKV nephritis and eight patients (34%) presented with viremia without evidence of nephritis on renal biopsy. RESULTS.: At time of diagnosis, mean plasma BKV DNA (copies/mL) was 460,409 (range 10,205-1,920,691). Mean doses reduction of mycophenolate mofetil and tacrolimus were 44% and 41%, respectively, from time of diagnosis of BKV infection to complete resolution of viremia. A decline in BK viral load was noticed within 15 to 30 days, with successful elimination of viremia over a mean period of 5.8 months (range, 1-9.5). Mean serum creatinine at time of diagnosis of BK viremia was 1.8 mg/dL (range, 1.2-2.8). Mean follow-up period is 30.9 months postdiagnosis. At the most recent visit, serum creatinine was 2.0 mg/dL (range, 1.0-3.6) (P=0.14). With reduction in immunosuppressive therapy, three patients (13%) developed acute cellular rejection and were treated successfully with intravenous bolus steroids. During follow-up, one patient had a relapse of BKV nephritis during pregnancy and lost her graft. After mean follow-up period of 43.5 months posttransplantation, all 24 patients are alive and 23 have a functioning graft. Seventeen patients (71%) have stable or improved graft function. CONCLUSION.: Our analysis shows that reduction in immunosuppression therapy alone results in clearance of the BK viremia with good long-term outcome.

Saad ER, Bresnahan BA, Cohen EP, Lu N, Orentas RJ, Vasudev B, Hariharan S.

1 Department of Medicine, Division of Nephrology, Medical College of Wisconsin, Milwaukee, WI. 2 Division of Biostatistics, Medical College of Wisconsin, Milwaukee, WI. 3 Department of Pediatrics, Division of Hematology/Oncology, Medical College of Wisconsin, Milwaukee, WI.

ABO Blood Group-Related Waiting List Disparities in Liver Transplant Candidates: Effect of the MELD Adoption.

BACKGROUND.: Blood group O candidates remain on the waiting list for a liver transplant for a longer time than candidates of other blood groups. Herein, we analyzed potential factors affecting waiting times in the period that preceded the introduction of the model for end-stage liver disease (MELD) and in MELD era, remarking possible corrections introduced by the adoption of the MELD. METHODS.: Our analysis was entirely based on data obtained from the \”Organ Procurement and Transplantation Network\”, referring to the periods before and after the adoption of the MELD. RESULTS.: In the MELD era, taking into consideration all candidates, the cumulative probability of remaining on the waiting list significantly diminished whereas that of undergoing transplantation significantly increased when compared with the pre-MELD era. However, group O candidates maintained the lowest cumulative probability of undergoing liver transplant, in all MELD classes, and the highest percentage of list removal for death/too sick. What caused the highest disadvantage for group O, in both eras, was the use of group O organs for ABO-compatible transplants, even in the absence of urgency. In candidates receiving ABO-compatible organs a significantly lower graft survival rate was observed compared with candidates receiving ABO-identical organs, even when the analysis was adjusted for the MELD score. CONCLUSIONS.: The introduction of the MELD significantly reduced the waiting time for all candidates as also the shift of group O organs. Limiting ABO-compatible organs exclusively to urgent cases would have a positive effect not only in terms of individual justice, but also terms of in general utility, considering the effect of ABO-matching on graft survival.

Barone M, Avolio AW, Di Leo A, Burra P, Francavilla A.

1 Section of Gastroenterology, D.E.T.O., University of Bari, Piazza G. Cesare, Bari, Italy. 2 Transplantation Service, Department of Surgery, Catholic University “A. Gemelli”, L.go Gemelli, Roma, Italy. 3 Section of Gastroenterology, Department of Surgical Science and Gastroenterology, University of Padova, Via Giustiniani, Padova, Italy.