No augmentation of indoleamine 2,3-dioxygenase (IDO) activity through belatacept treatment in liver transplant recipients.

Belatacept is a second-generation cytotoxic T lymphocyte antigen (CTLA)-4 immunoglobulin (Ig) fusion protein approved for immunosuppression in renal transplant recipients. It was designed intentionally to interrupt co-stimulation via CD28 by binding to its ligands B7·1 and B7·2. Experimental evidence suggests a potential additional mechanism for CTLA-4 Ig compounds through binding to B7 molecules expressed on antigen-presenting cells (APCs) and up-regulation of indoleamine 2,3-dioxygenase (IDO), an immunomodulating enzyme that catalyzes the degradation of tryptophan to kynurenine and that down-regulates T cell immunity. So far it remains unknown whether belatacept up-regulates IDO in transplant recipients. We therefore investigated whether belatacept therapy enhances IDO activity in liver transplant recipients enrolled in a multi-centre, investigator-initiated substudy of the Phase II trial of belatacept in liver transplantation (IM103-045). Tryptophan and kynurenine serum levels were measured during the first 6 weeks post-transplant in liver transplant patients randomized to receive either belatacept or tacrolimus-based immunosuppression. There was no significant difference in IDO activity, as indicated by the kynurenine/tryptophan ratio, between belatacept and tacrolimus-treated patients in per-protocol and in intent-to-treat analyses. Moreover, no evidence was found that belatacept affects IDO in human dendritic cells (DC) in vitro. These data provide evidence that belatacept is not associated with detectable IDO induction in the clinical transplant setting compared to tacrolimus-treated patients.

Efficacy and Safety Outcomes of Extended Criteria Donor Kidneys by Subtype: Subgroup Analysis of BENEFIT-EXT at 7 Years After Transplant.

The phase III Belatacept Evaluation of Nephroprotection and Efficacy as First-Line Immunosuppression Trial-Extended Criteria Donors Trial (BENEFIT-EXT) study compared more or less intensive belatacept-based immunosuppression with cyclosporine (CsA)-based immunosuppression in recipients of extended criteria donor kidneys. In this post hoc analysis, patient outcomes were assessed according to donor kidney subtype. In total, 68.9% of patients received an expanded criteria donor kidney (United Network for Organ Sharing definition), 10.1% received a donation after cardiac death kidney, and 21.0% received a kidney with an anticipated cold ischemic time ≥24 h. Over 7 years, time to death or graft loss was similar between belatacept- and CsA-based immunosuppression, regardless of donor kidney subtype. In all three donor kidney cohorts, estimated mean GFR increased over months 1-84 for belatacept-based treatment but declined for CsA-based treatment. The estimated differences in GFR significantly favored each belatacept-based regimen versus the CsA-based regimen in the three subgroups (p < 0.0001 for overall treatment effect). No differences in the safety profile of belatacept were observed by donor kidney subtype.

A randomized, controlled study to assess the conversion from calcineurin-inhibitors to everolimus after liver transplantation--PROTECT.

Posttransplant immunosuppression with calcineurin inhibitors (CNIs) is associated with impaired renal function, while mTor inhibitors such as everolimus may provide a renal-sparing alternative. In this randomized 1-year study in patients with liver transplantation (LTx), we sought to assess the effects of everolimus on glomerular filtration rate (GFR) after conversion from CNIs compared to continued CNI treatment. Eligible study patients received basiliximab induction, CNI with/without corticosteroids for 4 weeks post-LTx, and were then randomized (if GFR > 50 mL/min) to continued CNIs (N = 102) or subsequent conversion to EVR (N = 101). Mean calculated GFR 11 months postrandomization (ITT population) revealed no significant difference between treatments using the Cockcroft-Gault formula (-2.9 mL/min in favor of EVR, 95%-CI: [-10.659; 4.814], p = 0.46), whereas use of the MDRD formula showed superiority for EVR (-7.8 mL/min, 95%-CI: [-14.366; -1.191], p = 0.021). Rates of mortality (EVR: 4.2% vs. CNI: 4.1%), biopsy-proven acute rejection (17.7% vs. 15.3%), and efficacy failure (20.8% vs. 20.4%) were similar. Infections, leukocytopenia, hyperlipidemia and treatment discontinuations occurred more frequently in the EVR group. No hepatic artery thrombosis and no excess of wound healing impairment were noted. Conversion from CNI-based to EVR-based immunosuppression proved to be a safe alternative post-LTx that deserves further investigation in terms of nephroprotection.

Treg-therapy allows mixed chimerism and transplantation tolerance without cytoreductive conditioning.

Establishment of mixed chimerism through transplantation of allogeneic donor bone marrow (BM) into sufficiently conditioned recipients is an effective experimental approach for the induction of transplantation tolerance. Clinical translation, however, is impeded by the lack of feasible protocols devoid of cytoreductive conditioning (i.e. irradiation and cytotoxic drugs/mAbs). The therapeutic application of regulatory T cells (Tregs) prolongs allograft survival in experimental models, but appears insufficient to induce robust tolerance on its own. We thus investigated whether mixed chimerism and tolerance could be realized without the need for cytoreductive treatment by combining Treg therapy with BM transplantation (BMT). Polyclonal recipient Tregs were cotransplanted with a moderate dose of fully mismatched allogeneic donor BM into recipients conditioned solely with short-course costimulation blockade and rapamycin. This combination treatment led to long-term multilineage chimerism and donor-specific skin graft tolerance. Chimeras also developed humoral and in vitro tolerance. Both deletional and nondeletional mechanisms contributed to maintenance of tolerance. All tested populations of polyclonal Tregs (FoxP3-transduced Tregs, natural Tregs and TGF-beta induced Tregs) were effective in this setting. Thus, Treg therapy achieves mixed chimerism and tolerance without cytoreductive recipient treatment, thereby eliminating a major toxic element impeding clinical translation of this approach.

Murine mobilized peripheral blood stem cells have a lower capacity than bone marrow to induce mixed chimerism and tolerance.

Allogeneic bone marrow transplantation (BMT) under costimulation blockade allows induction of mixed chimerism and tolerance without global T-cell depletion (TCD). The mildest such protocols without recipient cytoreduction, however, require clinically impracticable bone marrow (BM) doses. The successful use of mobilized peripheral blood stem cells (PBSC) instead of BM in such regimens would provide a substantial advance, allowing transplantation of higher doses of hematopoietic donor cells. We thus transplanted fully allogeneic murine granulocyte colony-stimulating factor (G-CSF) mobilized PBSC under costimulation blockade (anti-CD40L and CTLA4Ig). Unexpectedly, PBSC did not engraft, even when very high cell doses together with nonmyeloablative total body irradiation (TBI) were used. We show that, paradoxically, T cells contained in the donor PBSC triggered rejection of the transplanted donor cells. Rejection of donor BM was also triggered by the cotransplantation of unmanipulated donor T cells isolated from naïve (nonmobilized) donors. Donor-specific transfusion and transient immunosuppression prevented PBSC-triggered rejection and mixed chimerism and tolerance were achieved, but graft-versus-host disease (GVHD) occurred. The combination of in vivo TCD with costimulation blockade prevented rejection and GVHD. Thus, if allogeneic PBSC are transplanted instead of BM, costimulation blockade alone does not induce chimerism and tolerance without unacceptable GVHD-toxicity, and the addition of TCD is required for success.

Alemtuzumab (Campath-1H) and tacrolimus monotherapy after renal transplantation: results of a prospective randomized trial.

The lymphocyte-depleting antibody alemtuzumab was evaluated in a prospective randomized multicenter trial in deceased donor kidney transplantation. The 65 patients in the study group received induction with alemtuzumab followed by delayed tacrolimus monotherapy, while the 66 patients in the control group were started on tacrolimus in combination with mycophenolate mofetil and steroids. Tacrolimus levels of 8-12 ng/mL for the first 6 months and 5-8 ng/mL thereafter were aimed for in both groups. At 12 months the biopsy-proven rejection rate was 20% in the study group and 32% in the control group (p = 0.09). Patient survival at 1 year was 98% for both groups. Graft survival was 96% for the study group versus 90% for the control group (p = 0.18). Graft function was identical in both groups. Adverse events were similar in both groups apart for more CMV infections in the study group. At the end of the first year 82% of the patients in the study group were steroid-free and 71% continued on tacrolimus monotherapy. These results suggest that alemtuzumab induction together with tacrolimus monotherapy is at least as efficient in renal transplantation as is a tacrolimus-based triple-drug regimen with a similar safety profile but more CMV infections.

Cytomegalovirus hyperimmunoglobulin: mechanisms in allo-immune response in vitro.

BACKGROUND: Cytomegalovirus hyperimmunoglobulin (CMVIg) containing drugs are routinely administered in cardiac transplantation for prophylaxis against CMV disease. Yet little is known about their influence on transplant relevant immune functions. The aim of this study was to evaluate the effect of CMVIg on cellular immunity in in vitro experiments and to define their role in tolerance inducing mechanisms. MATERIALS AND METHODS/RESULTS: CMVIg reduces proliferation in mixed lymphocyte reactions and anti-CD3 blastogenesis assays and is related to decreased production of immune modulating cytokines interleukin (IL)-2, interferonr (IFNgamma), IL-10. This antiproliferative effect is associated with a cell-cycle arrest in the G0/G1 phase and induction of apoptosis in CD8+ and natural killer cells. Co-incubation with CMVIg causes down-regulation of cell bound immunoglobulin and FcgammaRIII surface expression on natural killer cells and leads to attenuation of antibody dependent cellular cytotoxicity effector functions. CONCLUSIONS: We conclude that CMVIg induces immunological features on leukocytes in vitro that are known to be related to tolerance induction. Our observations extend the current concept of CMVIg as passive CMV prophylaxis to a therapeutic drug compound capable of reducing allogeneic immune response.

The advantage of allocating kidneys from old cadaveric donors to old recipients: a single-center experience.

BACKGROUND: In January 1999 a new kidney allocation program was launched by the Eurotransplant Foundation, the 'Eurotransplant Senior Program' (ESP). Cadaveric donors above the age of 65 yr are allocated to kidney transplant recipients of the same age group. METHODS: Using a single-center database, 91 patients who underwent first renal transplantation at the age of 65 yr and older in the years 1999-2002 were identified. Fifty-six patients were transplanted through ESP allocation (study group) and 35 patients (control group) via normal Eurotransplant Kidney Allocation System (ETKAS) procedure. RESULTS: Age, sex and comorbid conditions did not differ by group. The rate of acute rejection episodes, primary non-function, delayed graft function, perioperative mortality did not differ by group. Serum creatinine was significantly lower in the ETKAS group (1.3 vs. 1.9 mg/dL; p=0.015) from six months after the transplantation on. Overall graft survival at six yr was 56% in the ETKAS group and 52% in the ESP group. With 73% in the ETKAS group and 71% in the ESP group, cumulative patient survival according to the Kaplan-Meier estimation was not statistically different at five yr. CONCLUSIONS: We did not find a relevant difference in the outcome between young and old kidney transplants in old recipients after this long observation period.

Influence of cumulative number of marginal donor criteria on primary organ dysfunction in liver recipients.

BACKGROUND: The aim of this cohort study was to assess the cumulative effect of marginal donor criteria on initial graft function and patient survival after liver transplantation. METHODS: We included 734 consecutive patients who underwent orthotopic liver transplantation at the Vienna General Hospital between January 1993 and December 2003. We employed the local registry of the Department of Transplant Surgery, where variables of all patients are routinely and prospectively recorded. Primary outcome was initial graft function, secondary outcome was patient survival. RESULTS: Cumulative number of marginal donor criteria was significantly and linearly associated with an increased rate of primary dysfunction (PDF; p = 0.005). In patients with more than three cumulative marginal donor criteria the rate of PDF was 36%. Patient survival was not influenced by the cumulative number of donor criteria (log-rank test, p = 0.81). Independent marginal donor criteria to predict PDF were cold ischemia time >10 h [odds ratio (OR) 0.56; 95% CI 0.32-0.98] and donor peak serum sodium >155 mEq/L (OR 0.44; 95% CI 0.26-0.77), as assessed in a multivariate regression model. CONCLUSIONS: The use of marginal liver donors with more than three marginal donor criteria shows deleterious effects on initial graft function. Noteworthy, patient survival was not associated with marginal donor criteria, which may be explained by early and successful retransplantation of liver recipients with primary non-function.

Pilot study with pegylated liposomal doxorubicin for advanced or unresectable hepatocellular carcinoma.

We performed a pilot-study on pegylated liposomal doxorubicin (PLD) for advanced hepatocellular carcinoma. Seventeen patients received 40 mg/m(2) PLD intravenously every 4 weeks. A clinical benefit response was achieved in 50% (complete remission 7%, minor remission 7%, stable disease 36%). Toxicities were moderate. In view of these encouraging findings, further studies appear warranted.

Post-transplant acute myeloid leukemia (PT-AML).

Acute myeloid leukemia following organ transplantation (PT-AML) is a rare event with only a few published cases in the literature. We present three patients who developed AML (FAB M1, M5, M4) after renal, double lung or liver transplantation. Molecular analysis detected a t(9;11) in one patient and documented the recipient origin of AML in a second patient. All patients were treated with chemotherapy. Immunosuppression was reduced to cyclosporin A (CsA) and prednisone in two patients and to prednisone alone in one patient. Two patients achieved a complete remission (CR), with a remission duration of 4.6 months in one patient, the other patient died from septicemia after 15.2 months in CR. One patient was refractory to chemotherapy and died from septicemia. This report together with the documented cases in the literature suggests that PT-AML (1) develops after a median interval of 5 years after transplantation with variable latency (range, <1-17 years); (2) is heterogeneous with respect to FAB classification; (3) shows chromosomal and molecular changes typical of therapy-related AML (t-AML: -7, +8, 11q23, inv16, t(15;17)); (4) standard chemotherapy is feasible after reduction of immunosuppression and produces a CR rate of 56% with a median remission duration of 4.6 months and an overall survival of 2.6 months; (5) the major complications are early death (25%), gram-negative septicemia, progressive disease or relapse. This review provides diagnostic and therapeutic experiences and guidelines for the management of this increasing group of post-transplant patients.

Prediction of graft dysfunction by analysis of liver biopsies after cold storage.

Failure of the hepatic allograft continues to be a serious life-threatening risk for the recipient. Because no effective method of extracorporeal support is available for these patients, early retransplantation is the only alternative that offers the potential for survival. The aim of this prospective analysis was to search for a predictor of primary non-function of hepatic allografts before reperfusion. From March to June 1993 we investigated 19 liver biopsies which were obtained during the preparation of the donor liver in the back table bath immediately before the implantation of the organ. All organs were preserved by UW solution. Biopsies were stored at -80 degrees C, the working-up process was started by dividing the biopsy into several portions for the determination of fat (petrol-ether extraction), water (weighing before thawing and after drying) and free amino acids (OPA-HPLC method). Graft function was categorized into three groups: (1) good function; (2) fair function; (3) primary non-function (PNF). In addition to known risk factors for delayed graft function such as a long stay of the donor in intensive care and a prolonged anhepatic period of the recipient, we were able to demonstrate that organs with malfunction had a higher fat and water content. Donor livers developing PNF showed a trend towards higher total and subdivided amino acids, which could be explained by the incapacity of the liver to utilize available substrates for gluconeogenesis.

Tumor recurrence after oLTX.

Although early survival following transplantation for primary hepatic cancer is excellent, previously reported high recurrence rates have generally discouraged liver replacement for this condition. The aim of this retrospective analysis was to examine the influence of risk factors on the development of early tumor recurrence. Between December 1982 and June 1995, 480 liver transplantations were performed at a single institution. Out of these, 103 patients had unresectable primary hepatic cancer (88 hepatocellular cancer; HCCA; 20%) and 15 had cholangiocellular cancer (CHCA; 4%). The influence of the following tumor-associated risk factors was assessed: tumor size, tumor distribution within the liver, grading, pseudocapsular formation, vascular invasion, lymph node metastasis, and cirrhotic alteration. The diagnosis of tumor recurrence was made using various radiological imaging techniques, reelevation of serum alphafetoprotein, or autopsy. For patient survival and disease-free period, data analysis was performed by the method of Kaplan-Meier. The Cox model was used for multivariate analysis; a P-value of less than 0.05 was considered to be significant. The mean age of the 103 patients was 54 years (range 15-63a). There were 22 female and 81 male patients. The follow-up period ranged between 4 and 108 months. Twenty-nine patients (50%) died during the follow-up period due to recurrence of disease. The survival rates of the 88 patients with HCCA were 57%, 34%, and 26% at 1, 3, and 5 years, respectively, after orthotopic liver transplantation (oLTX; follow-up 36 month). Of the 15 pts with CHCA the rates were 53%, 33%, and 33%, respectively, with a median follow-up of 60 months. The influence of the risk factors studied showed a significantly longer disease-free period for the following tumor characteristics: grading below or equal 2 (P = 0.009) and absence of vascular invasion (P = 0.04). Regarding a median survival rate of 2-4 months for patients with unresectable malignant liver tumors, these results confirmed the indication for oLTX, especially if the patient does not compete with someone on the waiting list for benign liver disease.

Immunological risk factors are solely responsible for primary non-function of renal allografts.

Primary non-function (PNF) of renal allografts has been attributed to various risk factors, among them immunological ones, as well as unfavourable preservation conditions. To investigate the impact of these risk factory on the occurrence of PNF, 1335 consecutive kidney transplants performed at a single centre over a 10-year period were analysed. All patients received immunosuppression based on cyclosporine. As the method of analysis a conditional stepwise logistic regression model was chosen, comparing each graft suffering PNF with its partner kidney retrieved from the same donor. Thus, all donor-related variables could be omitted from the analysis, as they are the same in every pair of grafts. Risk factors analysed included panel-reactive antibodies, number of pretransplant transfusions, pregnancies, number of prior transplants, cold and second warm ischaemia time, mismatches on HLA loci A, B and DR and recipient age. The overall incidence of PNF was 87 grafts (6.5%). One patient suffered immediate rejection due to transplantation of an ABO incompatible graft. This case was excluded from further analysis. PNF occurred three times in recipients of living related grafts, twice in recipients of en-bloc grafts and four times in grafts, in which the paired kidney was either not transplanted or shipped outside the Eurotransplant region, so that no paired graft was available for matched case-control analysis. Of the remaining 77 pairs, twice both organs of one donor failed immediately. The remaining 73 complete pairs were analysed. Two of the investigated risk factors have independently a significant impact on the occurrence of PNF. Increasing the number of pretransplant transfusions raises the relative risk of graft failure up to six fold (P=0.02), while a history of prior transplants bears a relative risk of 0.21E05 (P=0.005). Ischaemia has no significant impact on the occurrence of PNF. Our data strongly suggest that immunological rather than donor risk factors are responsible for the non-function of kidney grafts.

Reasons for 50% reduction in the number of organ donors within 2 years--opinion poll amongst all ICUs of a transplant centre.

To detect the reasons for a massive decrease in the annual number of organ donors and as a means of evaluating the effectiveness of our information programme, a questionnaire was designed and sent to all intensive care units (ICUs) in our catchment area. We wished to obtain information about medical, organizational and capacity problems and negative occurrences that had happend during past retrievals. Although 60% of the answers we reiceved (87% feedback rate) mentioned the additional workload involved in treating an organ donor (and 88% had serious problems because of the shortage of nurses), less than 16% remembered a "lost" donor because of capacity problems. Eighty-six percent recognized our efforts to support them in any respect and were satisfied with the amount of "service" provided by the transplantation (TX) centre. About 45% remembered negative occurrences. More than 85% of all replies asked for more and continuing information related to organ donation and transplantation. We think that the key to a successful TX programme is a system of active care for the ICU staff in all peripheral hospitals; repeated mailing of updated information brochures, annual lectures about new developments, letters of thanks after each reported donor (including information on the fate of the organs), visiting donor ICU's accompanied by successfully transplanted recipients, etc.... The downwards trend of donor rates in our area clearly shows that it takes more than a stable legal situation to ensure the necessary amount of donor organs, even a very successful TX centre has to work hard to maintain a certain standard of knowledge, information and motivation amongst the staff of the peripheral hospitals. Moreover, the high turnover rate of ICU personnel requires a steady "flow of information" and cooperation between the "transplant people" and their coworkers outside to guarantee a permanent state of awareness concerning organ donation and transplantation. In fact, awareness seems to be the key issue: the activity of sending out the questionnaires was enough to raise the number of reported donors from 72 (estimated in July) to 96 (31 December 1992).

A reliable and safe way of shortening cadaver kidney ischemia time: prenephrectomy tissue typing using donor lymph node cells.

The purpose of this study was to investigate the impact of prenephrectomy donor tissue typing on tissue typing quality and transplantation outcome in human kidney transplantation. We report on 680 consecutive kidney transplantations performed at the Vienna Transplantation Center from 1986 to June 1991. In 343 of them, HLA typing was performed using donor lymph node cells obtained in a small surgical procedure several hours before organ retrieval. The mean cold ischemia time (CIT) could be reduced to 17.7 h in these patients compared with 21.9 h in the control group (n = 337, conventional tissue typing using spleen lymphocytes obtained during the organ removal, P = 0.0001). There was a trend towards better initial and long-term function in the lymph node group; however, this did not reach statistical significance. The clarity of tissue typing results was significantly better when lymph nodes were used as the lymphocyte source. We conclude that prenephrectomy tissue typing is a feasable and inexpensive method of shortening CIT in renal transplantation and favors HLA typing, both likely to benefit transplantation outcome particularly within organ exchange programs.