The Use of Donation After Cardiac Death Allografts Does Not Increase Recurrence of Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) recurrence in patients undergoing liver transplantation (LT) with donation after brain death (DBD) and donation after cardiac death (DCD) allografts has not previously been investigated. Rates and patterns of HCC recurrences were investigated in patients undergoing DBD (N = 1633) and DCD (N = 243) LT between 2003 and 2012. LT for HCC was identified in 397 patients (340 DBD and 57 DCD). No difference in tumor number (p = 0.26), tumor volume (p = 0.34) and serum alphafetoprotein (AFP) (p = 0.47) was seen between the groups. HCC recurrence was identified in 41 (12.1%) patients in the DBD group and 7 (12.3%) patients in the DCD group. There was no difference in recurrence-free survival (p = 0.29) or cumulative incidence of HCC recurrence (p = 0.91) between the groups. Liver allograft was the first site of recurrence in 22 (65%) patients in the DBD group and two (37%) patients in the DCD group (p = 0.39). LT for HCC with DBD and DCD allografts demonstrate no difference in the rate of HCC recurrence. Previously published differences in survival demonstrated between recipients with HCC receiving DBD and DCD allografts despite statistical adjustment can likely be explained by practice patterns not captured by variables contained in the SRTR database.

Avoiding stay in the intensive care unit after liver transplantation: a score to assign location of care.

Select liver transplantation (LT) recipients in our program are transferred from operating room to postanesthesia care unit for recovery and extubation with transfer to the ward, completely eliminating an intensive care unit (ICU) stay. Developing a reliable method to determine patients suitable for fast-tracking would be of practical benefit to centers considering this practice. The aim of this study was to create a fast-tracking probability score that could be used to predict successful assignment of care location after LT. Recipient, donor and operative characteristics were assessed for independent association with successful fast-tracking to create a probability score. Of the 1296 LT recipients who met inclusion criteria, 704 (54.3%) were successfully fast-tracked and 592 (45.7%) were directly admitted to the ICU after LT. Based on nine readily available variables at the time of LT, we created a scoring system that classified patients according to the likelihood of being successfully fast-tracked to the surgical ward, with an area under the curve (AUC) of 0.790 (95% CI: 0.765-0.816). This score was validated in an independent group of 372 LT with similar AUC. We describe a score that can be used to predict successful fast-tracking immediately after LT using readily available clinical variables.

Cytokine response modifier A (CrmA) inhibits ceramide formation in response to tumor necrosis factor (TNF)-alpha: CrmA and Bcl-2 target distinct components in the apoptotic pathway.

Proteases are now firmly established as major regulators of the "execution" phase of apoptosis. Here, we examine the role of proteases and their relationship to ceramide, a proposed mediator of apoptosis, in the tumor necrosis factor-alpha (TNF-alpha)-induced pathway of cell death. Ceramide induced activation of prICE, the protease that cleaves the death substrate poly(ADP-ribose) polymerase. Bcl-2 inhibited ceramide-induced death, but not ceramide generation. In contrast, Cytokine response modifier A (CrmA), a potent inhibitor of Interleukin-1 beta converting enzyme and related proteases, inhibited ceramide generation and prevented TNF-alpha-induced death. Exogenous ceramide could overcome the CrmA block to cell death, but not the Bcl-2 block. CrmA, however, did not inhibit the activation of nuclear factor (NF)-kappa B by TNF-alpha, demonstrating that other signaling functions of TNF-alpha remain intact and that ceramide does not play a role in the activation of NF-kappa B. These studies support a distinct role for proteases in the signaling/activation phase of apoptosis acting upstream of ceramide formation.

Proteasome inhibition causes apoptosis of normal human plasma cells preventing alloantibody production.

Antibody production by normal plasma cells (PCs) against human leukocyte antigens (HLA) can be a major barrier to successful transplantation. We tested four reagents with possible activity against PCs (rituximab, polyclonal rabbit antithymocyte globulin (rATG), intravenous immunoglobulin (IVIG) and the proteasome inhibitor, bortezomib) to determine their ability to cause apoptosis of human bone marrow-derived PCs and subsequently block IgG secretion in vitro. IVIG, rituximab and rATG all failed to cause apoptosis of PCs and neither rituximab nor rATG blocked antibody production. In contrast, bortezomib treatment led to PC apoptosis and thereby blocked anti-HLA and antitetanus IgG secretion in vitro. Two patients treated with bortezomib for humoral rejection after allogeneic kidney transplantation demonstrated a transient decrease in bone marrow PCs in vivo and persistent alterations in alloantibody specificities. Total IgG levels were unchanged. We conclude that proteasome activity is important for PC longevity and its inhibition may lead to new techniques of controlling antibody production in vivo.

Long-term management of the liver transplant patient: recommendations for the primary care doctor.

No official document has been published for primary care physicians regarding the management of liver transplant patients. With no official source of reference, primary care physicians often question their care of these patients. The following guidelines have been approved by the American Society of Transplantation and represent the position of the association. The data presented are based on formal review and analysis of published literature in the field and the clinical experience of the authors. These guidelines address drug interactions and side effects of immunosuppressive agents, allograft dysfunction, renal dysfunction, metabolic disorders, preventive medicine, malignancies, disability and productivity in the workforce, issues specific to pregnancy and sexual function, and pediatric patient concerns. These guidelines are intended to provide a bridge between transplant centers and primary care physicians in the long-term management of the liver transplant patient.

Agricultural Safety Education: Formative Assessment of a Curriculum Integration Strategy.

The purpose of this study was to assess an agricultural tractor and machinery safety curriculum for teacher training that focused on hands-on integration activities to assist with training youth in machinery safety skills. Teachers attended a single ten-hour summer training seminar hosted in Montana, South Dakota, or Utah during 2017. Teachers completed the National Tractor and Machinery Safe Operation (NSTMOP) exam to measure their existing knowledge prior to beginning the training. Upon seminar completion, teachers took an NSTMOP post-test to measure their knowledge gain of agricultural safety practices and hazard recognition associated with machinery and tractors. A total of 116 teachers completed the training. Fifty-three participants (45.7%) identified as female, and 63 (54.3%) identified as male. The average participant was 35 years old (SD = 11.3) and had 9.5 years of teaching experience (SD = 9.2). The average NSTMOP pre-test score was 35.2 out of 48 (SD = 3.3), and the average NSTMOP post-test score was 40.3 out of 48 (SD = 4.1). Participants' scores increased by ten percentage points. A paired-samples t-test was used to determine statistical significance. The difference between pre-test and post-test was significant (t(109) = 11.9, p < 0.001). Open responses indicated continuation of hands-on activities that focused on "how to teach" skills training that is relevant to the students. Teachers suggested developing new activities each year with a rotation of topics for upcoming seminars. Research is needed to determine the training's influence on the behaviors of young workers in agriculture.

Two novel assays of alloantibody-secreting cells demonstrating resistance to desensitization with IVIG and rATG.

Donor-specific alloantibody presents a major barrier to the successful transplantation of kidneys and hearts. However, the study of alloantibody production has been hampered by both an inadequate source of antibody-secreting cells (ASCs) and a paucity of assays to determine their function. We describe two new assays that allow for the determination of the frequency and specificities of allo-ASCs in humans using purified HLA as targets. These assays demonstrated allo-ASCs in the CD138(+) fraction of the bone marrow, but not in peripheral blood. Alloantibody specificities in these assays correlated well with those detected in the serum suggesting that bone marrow-derived ASCs are indeed a major source of alloantibody in vivo. However, ASCs for a specific HLA antigen were rare with an estimated frequency of only 1/2 x 10(6) marrow cells. Pretransplant treatment in vivo with multiple plasmaphereses and low-dose IVIG alone or in combination with rATG had no effect on ASC number or alloantibody production. These techniques allow for the study of allospecific ASCs and provide a method to test the potential efficacy of agents on alloantibody production in vivo.

Alloantibody levels and acute humoral rejection early after positive crossmatch kidney transplantation.

We examined the course of donor-specific alloantibody (DSA) levels early after transplant and their relationship with acute humoral rejection (AHR) in two groups of positive crossmatch (+XM) kidney transplant recipients: High DSA group-41 recipients with a baseline T- or B-cell flow crossmatch (TFXM, BFXM) channel shift >or=300 (molecules of equivalent soluble fluorochrome units (MESF) of approximately 19 300) who underwent pretransplant plasmapheresis (PP), and Low DSA group-29 recipients with a baseline channel shift <300 who did not undergo PP. The incidence of AHR was 39% (16/41) in the High DSA group and 31% (9/29) in the Low DSA group. Overall, mean DSA levels decreased by day 4 posttransplant and remained low in patients who did not develop AHR. By day 10, DSA levels increased in patients developing AHR with 92% (23/25) of patients with a BFXM >359 (MESF of approximately 34 000) developing AHR. The BFXM and the total DSA measured by single antigen beads correlated well across a wide spectrum suggesting that either could be used for monitoring. We conclude that AHR is associated with the development of High DSA levels posttransplant and protocols aimed at maintaining DSA at lower levels may decrease the incidence of AHR.

Bcl-2 acts upstream of the PARP protease and prevents its activation.

Apoptosis has recently been extensively studied and multiple factors have been implicated in its regulation. It remains unclear how these factors are ordered in the cell death pathway. Here we investigate the relationship between the inhibitor of apoptosis, bcl-2, and the PARP protease, prlCE/CPP32, recently implicated in apoptosis. Using PARP proteolysis as an indicator of the activation of the PARP protease, we find that the chemotherapeutic agent, etoposide, induces apoptosis and PARP proteolysis in Molt4 cells as early as 4 h with cell death lagging behind this event. In contrast, Molt4 cells that over-express bcl-2 show no PARP proteolysis or cell death. In order to determine if bcl-2 inhibits the PARP protease or its activation, we developed a cell-free system. Using this system with extracts from etoposide-treated cells and purified bovine PARP, we demonstrate that extracts from bcl-2 over-expressing cells cause little or no PARP proteolysis. Whereas, extracts from control vector cells contain an active PARP protease. This protease is inhibited by the tetrapeptide ICE-like protease inhibitor, YVAD-chloromethylketone. Interestingly, this protease is not inhibited by the addition of purified bcl-2 protein. These results rule out that bcl-2 directly inhibits the active protease or that it has an effect downstream of prlCE/CPP32 such as preventing access to the PARP substrate. These results also demonstrate a role of bcl-2 in interfering with an upstream signal required to activate the PARP protease and allow us to begin to order the components in the apoptotic pathway.

Ceramide and the regulation of apoptosis and the stress response.

The process of apoptosis or programmed cell death has been the subject of intense study due to the realization of its importance in a wide variety of biological and pathological conditions, including development, ischemia, cancer, and neurodegenerative disorders. Although a vast number of inducers of this process and several critical components have been identified, the signal transduction pathways regulating apoptosis are poorly understood. Recently, a pathway involving sphingolipid turnover and the production of the lipid mediator, ceramide, has emerged as a candidate regulator of apoptosis. This review provides a summary of the evidence implicating ceramide as a mediator of apoptosis and the stress response. (Trends Cardiovasc Med 1996;6:158-162).

Proteolytic cleavage of epidermal growth factor receptor by caspases.

Apoptotic proteases cleave and inactivate survival signaling molecules such as Akt/PKB, phospholipase C (PLC)-gamma1, and Bcl-2. We have found that treatment of A431 cells with tumor necrosis factor-alpha in the presence of cycloheximide resulted in the cleavage of epidermal growth factor receptor (EGFR) as well as the activation of caspase-3. Among various caspases, caspase-1, caspase-3 and caspase-7 were most potent in the cleavage of EGFR in vitro. Proteolytic cleavage of EGFR was inhibited by both YVAD-cmk and DEVD-fmk in vitro. We also investigated the effect of caspase-dependent cleavage of EGFR upon the mediation of signals to downstream signaling molecules such as PLC-gamma1. Cleavage of EGFR by caspase-3 significantly impaired the tyrosine phosphorylation of PLC-gamma1 in vitro. Given these results, we suggest that apoptotic protease specifically cleaves and inactivates EGFR, which plays crucial roles in anti-apoptotic signaling, to abrogate the activation of EGFR-dependent downstream survival signaling molecules.

Ceramide generation by two distinct pathways in tumor necrosis factor alpha-induced cell death.

Ceramide accumulation in the cell can occur from either hydrolysis of sphingomyelin or by de novo synthesis. In this study, we found that blocking de novo ceramide synthesis significantly inhibits ceramide accumulation and subsequent cell death in response to tumor necrosis factor alpha. When cells were pre-treated with glutathione, a proposed cellular regulator of neutral sphingomyelinase, inhibition of ceramide accumulation at early time points was achieved with attenuation of cell death. Inhibition of both pathways achieved near-complete inhibition of ceramide accumulation and cell death indicating that both pathways of ceramide generation are stimulated. This illustrates the complexity of ceramide generation in cytokine action.

The role of de novo ceramide synthesis in chemotherapy-induced apoptosis.

The de novo pathway of sphingolipid synthesis has been implicated as an alternative to sphingomyelinase activation in generating an apoptotic response through ceramide. A chemotherapy agent was used to activate this pathway in a human T-cell line in order to investigate the role of de novo ceramide synthesis in apoptosis. In data obtained from intact cell radiolabeling studies, it was observed that the first and rate-limiting enzyme in de novo synthesis, serine palmitoyltransferase, is activated and controls the production of ceramide through this pathway. Furthermore, using agents that selectively inhibit ceramide production by this pathway, partial protection from cell death was observed that was independent of caspase activation. These results reveal that serine palmitoyltransferase, an enzyme that controls sphingolipid synthesis for housekeeping functions, is activated during apoptosis and serves to mediate events in this process.