FADD is required for DR4- and DR5-mediated apoptosis: lack of trail-induced apoptosis in FADD-deficient mouse embryonic fibroblasts.

TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) is a member of the tumor necrosis factor family that can kill a wide variety of tumor cells but not normal cells. TRAIL-induced apoptosis in humans is mediated by its receptors DR4 (TRAIL-R1) and DR5 (TRAIL-R2). What constitutes the signaling molecules downstream of these receptors, however, remains highly controversial. Using the FADD dominant negative molecule, several groups have reached different conclusions with respect to the role of FADD in TRAIL-induced apoptosis. More recently, using FADD-deficient (-/-) mouse embryonic fibroblasts, Yeh et al. (Yeh, W.-C., Pompa, J. L., McCurrach, M. E., Shu, H.-B., Elia, A. J., Shahinian, A., Ng, M., Wakeham, A., Khoo, W., Mitchell, K., El-Deiry, W. S., Lowe, S. W., Goeddel, D. V., and Mak, T. W. (1998) Science 279, 1954-1958) concluded that DR4 utilizes a FADD-independent apoptotic pathway. The latter experiment, however, involved transient overexpression, which often leads to nonspecific aggregation of death domain-containing receptors. To address this issue in a more physiological setting, we stably transfected mouse DR4/5, human DR4, or human DR5 into FADD(-/-) mouse embryonic fibroblast cells. We showed that FADD(-/-) MEF cells stably transfected with TRAIL receptors are resistant to TRAIL-mediated cell death. In contrast, TRAIL receptors stably transfected into heterozygous FADD(+/-) cells or FADD(-/-) cells reconstituted with a FADD retroviral construct are sensitive to the TRAIL cytotoxic effect. We conclude that FADD is required for DR4- and DR5-mediated apoptosis.

Nur77 transcription activity correlates with its apoptotic function in vivo.

Nur77 is a transcription factor that is induced to a high level during TCR-mediated apoptosis of thymocytes and T cell hybridomas. Expression of a dominant-negative mutant of Nur77 can inhibit TCR-mediated apoptosis, while constitutive expression of full-length Nur77 in thymocytes leads to massive apoptosis. Nur77 is similar to the steroid receptor family and consists of a transactivation, a DNA-binding and a C-terminal "ligand-binding" domain. In contrast to the other nuclear receptors, Nur77 activity does not appear to depend on any ligand. However, its C-terminal region can regulate its transactivation activity. A short C-terminal deletion results in a protein with only 15 - 20% activity while deletion of the entire C-terminal region increases its activity. To further study the role of Nur77 transcription in apoptosis, we have generated transgenic mice expressing Nur77 with a short C-terminal deletion or Nur77 without its entire C-terminal domain. Mice expressing the shorter deletion/transcriptionally less active mutant displayed a mild phenotype. However, mice with the larger deletion/more transcriptionally active mutant showed massive thymocyte apoptosis. These data suggest that Nur77 transcription correlates with its apoptotic function in vivo.

Failure patterns of cryopreserved vein grafts in liver transplantation.

Reports of early success with cryopreserved saphenous veins (CSV) as arterial conduits led us to develop cryopreserved iliac veins (CIV) as interposition grafts for portal vein reconstruction in living-related liver transplantation (LRLT). Despite encouraging short-term results, retrospective analysis of long-term cryopreserved vein graft performance in LRLT at our institution has revealed a high rate of late graft failures. Between July 1992 and JUly 1994, interposition grafts (CIV for portal vein interposition n=4, CSV for portal vein interposition n=3, and CSV for hepatic artery interposition n=2) were utilized in 7 LRLT. (Two transplanted organs had both CIV and CSV grafts.) Recipients included 5 children and two small adults (median: 3.5 years, range: 0.5--59 years). Posttransplant follow-up in excess of 36 months revealed portal vein (PV) and hepatic artery (HA) complications of cryopreserved grafts in each patient. PV complications included aneurysm (n=4) diagnosed at 28, 24, 18, and 1.5 mo, stricture (n=1) diagnosed at 11 mo, and thrombosis (n=1) diagnosed at 18 mo posttransplantation. All portal vein complications have been managed without retransplantation, but one (PV thrombosis) necessitated surgical shunt therapy. Each CSV hepatic artery interposition graft has been complicated by thrombosis (diagnosed at 11 days and 24 mo posttransplant) necessitating retransplantation. Based on these observations, we have adopted alternative strategies for HA and PV reconstruction. At present, 11 LRLT have been performed without cryopreserved vein conduits over 17 mo with no vascular complications. While this study does not permit statistical analysis, these results discourage the use cryopreserved iliac veins for portal interposition and cryopreserved saphenous veins for arterial interposition in liver transplantation.

Decreased mortality from technical failure improves results in pediatric liver transplantation.

BACKGROUND: Until recently, pediatric liver transplantation was associated with a high rate of technical failure, which contributed substantially to the overall prognosis. OBJECTIVE: To assess the impact of technical failure on outcome in pediatric liver transplantation. DESIGN AND SETTING: We retrospectively analyzed 90 pediatric transplant procedures in a university medical center. PATIENTS: Between February 1988 and December 1995, 80 children ( < 15 years old) received 90 transplants. Fifty-three percent (n = 42) were less than 2 years of age, 45% (n = 36) had cholestatic liver disease, 26% (n = 21) had metabolic errors, and 11% (n = 9) had fulminant hepatitis. INTERVENTION: Patients underwent grafting using previously reported techniques, including cadaveric whole (61% [n = 55]), reduced-size (17% [n = 15]), and living related (22% [n = 20]) liver transplantation. MAIN OUTCOME MEASURES: Patient and graft survival and selected surgical complications. Outcomes were compared before (group 1) and after (group 2) the introduction of living related transplantation in July 1992. RESULTS: In group 1, 32 patients received 36 grafts (4 retransplants [13%]), and in group 2, 48 patients received 54 grafts (6 retransplants [13%]). Six- and 12-month patient survival rates were 78% (n = 25) and 75% (n = 24), respectively, for group 1 and 98% (n = 47) and 94% (n = 45) for group 2. Of the 9 deaths in group 1, 6 occurred early as a consequence of surgical complications, while in group 2, all 5 deaths that occurred were caused by the consequences of immunosuppression (lymphoproliferative disease, n = 2; late infections, n = 3). CONCLUSIONS: These results suggest that mortality caused by surgical complications has been reduced by improvement in management in recent years. Living related grafts have supplemented the graft supply and may be associated with the improved overall results. Despite these advances, children receiving transplants continue to experience the consequences of imperfect immunosuppression.

Interaction between NF-kappa B- and serum response factor-binding elements activates an interleukin-2 receptor alpha-chain enhancer specifically in T lymphocytes.

We find that a short enhancer element containing the NF-kappa B binding site from the interleukin-2 receptor alpha-chain gene (IL-2R alpha) is preferentially activated in T cells. The IL-2R alpha enhancer binds NF-kappa B poorly and is only weakly activated by the NF-kappa B site alone. Serum response factor (SRF) binds to a site adjacent to the NF-kappa B site in the IL-2R enhancer, and both sites together have strong transcriptional activity specifically in T cells. Surprisingly, the levels of SRF constitutively expressed in T cells are consistently higher than in other cell types. Overexpression of SRF in B cells causes the IL-2R enhancer to function as well as it does in T cells, suggesting that the high level of SRF binding in T cells is functionally important.