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.

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.

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.

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.

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.

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.

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.

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.

Determination of ceramides and diglycerides by the diglyceride kinase assay.

This review will discuss the utilization of the diglyceride (DG) kinase assay as an analytical method that allows the simultaneous quantitation of DG and ceramide from cell and tissue samples. This enzymatic approach is sensitive, quantitative, and linear over a broad range (20 pmol to 20-25 nmol) for both classes of lipids. It is also practical in that it can be applied to crude lipid extracts and used to process many samples (up to 100) concurrently. However, it has become apparent that this assay has not been conducted optimally, primarily as a result of lack of adherence to its basic principles. The principles illustrated here are also useful to all enzymatic quantitative methods.

Proteolytic cleavage of phospholipase C-gamma1 during apoptosis in Molt-4 cells.

Apoptosis is a cell suicide mechanism that requires the activation of cellular death proteases for its induction. We examined whether the progress of apoptosis involves cleavage of phospholipase C-gamma1 (PLC-gamma1), which plays a pivotal role in mitogenic signaling pathway. Pretreatment of T leukemic Molt-4 cells with PLC inhibitors such as U-73122 or ET-18-OCH(3) potentiated etoposide-induced apoptosis in these cells. PLC-gamma1 was fragmented when Molt-4 cells were treated with several apoptotic stimuli such as etoposide, ceramides, and tumor necrosis factor alpha. Cleavage of PLC-gamma1 was blocked by overexpression of Bcl-2 and by specific inhibitors of caspases such as Z-DEVD-CH(2)F and YVAD-cmk. Purified caspase-3 and caspase-7, group II caspases, cleaved PLC-gamma1 in vitro and generated a cleavage product of the same size as that observed in vivo, suggesting that PLC-gamma1 is cleaved by group II caspases in vivo. From point mutagenesis studies, Ala-Glu-Pro-Asp(770) was identified to be a cleavage site within PLC-gamma1. Epidermal growth factor receptor (EGFR) -induced tyrosine phosphorylation of PLC-gamma1 resulted in resistance to cleavage by caspase-3 in vitro. Furthermore, cleaved PLC-gamma1 could not be tyrosine-phosphorylated by EGFR in vitro. In addition, tyrosine-phosphorylated PLC-gamma1 was not significantly cleaved during etoposide-induced apoptosis in Molt-4 cells. This suggests that the growth factor-induced tyrosine phosphorylation may suppress apoptosis-induced fragmentation of PLC-gamma1. We provide evidence for the biochemical relationship between PLC-gamma1-mediated signal pathway and apoptotic signal pathway, indicating that the defect of PLC-gamma1-mediated signaling pathway can facilitate an apoptotic progression.

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.

Serine palmitoyltransferase regulates de novo ceramide generation during etoposide-induced apoptosis.

The de novo pathway of sphingolipid synthesis has been identified recently as a novel means of generating ceramide during apoptosis. Furthermore, it has been suggested that the activation of dihydroceramide synthase is responsible for increased ceramide production through this pathway. In this study, accumulation of ceramide mass in Molt-4 human leukemia cells by the chemotherapy agent etoposide was found to occur primarily due to activation of the de novo pathway. However, when the cells were labeled with a substrate for dihydroceramide synthase in the presence of etoposide, there was no corresponding increase in labeled ceramide. Further investigation using a labeled substrate for serine palmitoyltransferase, the rate-limiting enzyme in the pathway, resulted in an accumulation of label in ceramide upon etoposide treatment. This result suggests that the activation of serine palmitoyltransferase is the event responsible for increased ceramide generation during de novo synthesis initiated by etoposide. Importantly, the ceramide generated from de novo synthesis appears to have a distinct function from that induced by sphingomyelinase action in that it is not involved in caspase-induced poly (ADP-ribose)polymerase proteolysis but does play a role in disrupting membrane integrity in this model system. These results implicate serine palmitoyltransferase as the enzyme controlling de novo ceramide synthesis during apoptosis and begin to define a unique function of ceramide generated from this pathway.

Ischemic preconditioning protects the mouse liver by inhibition of apoptosis through a caspase-dependent pathway.

A short period of ischemia and reperfusion, called ischemic preconditioning, protects various tissues against subsequent sustained ischemic insults. We previously showed that apoptosis of hepatocytes and sinusoidal endothelial cells is a critical mechanism of injury in the ischemic liver. Because caspases, calpains, and Bcl-2 have a pivotal role in the regulation of apoptosis, we hypothesized that ischemic preconditioning protects by inhibition of apoptosis through down-regulation of caspase and calpain activities and up-regulation of Bcl-2. A preconditioning period of 10 minutes of ischemia followed by 15 minutes of reperfusion maximally protected livers subjected to prolonged ischemia. After reperfusion, serum aspartate transaminase (AST) levels were reduced up to 3-fold in preconditioned animals. All animals subjected to 75 minutes of ischemia died, whereas all those who received ischemic preconditioning survived. Apoptosis of hepatocytes and sinusoidal endothelial cells, assessed by in situ TUNEL assay and DNA fragmentation by gel electrophoresis, was dramatically reduced with preconditioning. Caspase activity, measured by poly (adenosine diphosphate ribose) polymerase (PARP) proteolysis and a specific caspase-3 fluorometric assay, was inhibited by ischemic preconditioning. The antiapoptotic mechanism did not involve calpain-like activity or Bcl-2 expression because levels were similar in control and preconditioned livers. In conclusion, ischemic preconditioning confers dramatic protection against prolonged ischemia via inhibition of apoptosis through down-regulation of caspase 3 activity, independent of calpain-like activity or Bcl-2 expression.

Phosphatidic acid is a potent and selective inhibitor of protein phosphatase 1 and an inhibitor of ceramide-mediated responses.

In the present study, we report that phosphatidic acid (PA) functions as a novel, potent, and selective inhibitor of protein phosphatase 1 (PP1). The catalytic subunit of PP1alpha was inhibited by PA dose-dependently in a noncompetitive manner with a K(i) value of 80 nM. The inhibition by PA was specific to PP1 as PA failed to inhibit protein phosphatase 2A (PP2A) or PP2B. Furthermore, PA was the most effective and potent inhibitor of PP1 compared with other phospholipids. Because we recently showed that ceramides activated PP1, we next examined the effects of PA on ceramide stimulation of PP1. PA inhibited both basal and ceramide-stimulated PP1 activities, and ceramide showed potent and stereoselective activation of PP1 in the presence of PA. Next, the effects of PA on ceramide-induced responses were examined. Molt-4 cells took up PA dose- and time-dependently such that by 1 and 3 h, uptake of PA was 0.37 and 0. 65% of total PA added, respectively. PA at 30 microM and calyculin A at 10 nM (an inhibitor of PP1 and PP2A at low concentrations), but not okadaic acid at 10 nM (a PP2A inhibitor at low concentrations) prevented poly(ADP-ribose) polymerase proteolysis induced by C(6)-ceramide. Moreover, the combination of PA with okadaic acid prevented retinoblastoma gene product dephosphorylation induced by C(6)-ceramide. These data suggest that PA functions as a specific regulator of PP1 and may reverse or counteract those effects of ceramide that are mediated by PP1, such as apoptosis and retinoblastoma gene product dephosphorylation.