A comparison of sirolimus vs. calcineurin inhibitor-based immunosuppressive therapies in liver transplantation.

BACKGROUND: Sirolimus is a potent immunosuppressive agent whose role in liver transplantation has not been well-described. AIM: To evaluate the efficacy and side-effects of sirolimus-based immunosuppression in liver transplant patients. METHODS: Retrospective analysis of 185 patients who underwent orthotopic liver transplantation. Patients were divided into three groups: group SA, sirolimus alone (n = 28); group SC, sirolimus with calcineurin inhibitors (n =56) and group CNI, calcineurin inhibitors without sirolimus (n = 101). RESULTS: One-year patient and graft survival rates were 86.5% and 82.1% in group SA, 94.6% and 92.9% in group SC, and 83.2% and 75.2% in group CNI (P = N.S.). The rates of acute cellular rejection at 12 months were comparable among the three groups. At the time of transplantation, serum creatinine levels were significantly higher in group SA, but mean creatinine among the three groups at 1 month was similar. More patients in group SA required dialysis before orthotopic liver transplantation (group SA, 25%; group SC, 9%; group CNI, 5%; P = 0.008), but at 1 year, post-orthotopic liver transplantation dialysis rates were similar. CONCLUSIONS: Sirolimus given alone or in conjunction with calcineurin inhibitors appears to be an effective primary immunosuppressant regimen for orthotopic liver transplantation patients. Further studies to evaluate the efficacy and side-effect profile of sirolimus in liver transplant patients are warranted.

Analysis of causes of death in liver transplant recipients who survived more than 3 years.

Few studies have examined causes of death in long-term survivors of orthotopic liver transplantation (OLT). We reviewed causes of death among 299 adult liver transplant recipients who survived more than 3 years after OLT at 2 centers. Thirty-eight of the 299 patients subsequently died. Nonhepatic causes accounted for 22 of 38 late deaths (58%). Death caused by malignancies occurred in 9 patients between 3.3 and 8.0 years after OLT. Eight patients died of cardiovascular complications. The 6 patients who died of myocardial infarction had risk factors for coronary artery disease. Hepatic failure caused by recurrent liver disease or chronic rejection accounted for 16 of 38 late deaths (42%). These 16 patients were younger than patients who died of nonhepatic complications (mean ages, 50.7 v 62.1 years; P =.001). However, the mean interval between OLT and death was similar among patients who died of nonhepatic versus hepatic causes. Nine patients had recurrent liver disease leading to death, and 8 of 9 patients had recurrent chronic hepatitis C virus (HCV) infection. Chronic rejection resulting in graft failure and death occurred in 7 patients. In summary, de novo malignancies and cardiovascular complications accounted for more than half the late deaths. Patients who died of nonhepatic causes were significantly older than patients who died of hepatic causes. Chronic rejection and recurrent HCV infection accounted for the majority of hepatic causes of death. With longer follow-up, graft failure resulting from recurrent HCV infection will become the major cause of death in late survivors.

A 10-year experience of liver transplantation for hepatitis C: analysis of factors determining outcome in over 500 patients.

OBJECTIVE: To determine the factors affecting the outcome of orthotopic liver transplantation (OLT) for end-stage liver disease caused by hepatitis C virus (HCV) and to identify models that predict patient and graft survival. SUMMARY BACKGROUND DATA: The national epidemic of HCV infection has become the leading cause of hepatic failure that requires OLT. Rapidly increasing demands for OLT and depleted donor organ pools mandate appropriate selection of patients and donors. Such selection should be guided by a better understanding of the factors that influence the outcome of OLT. METHODS: The authors conducted a retrospective review of 510 patients who underwent OLT for HCV during the past decade. Seven donor, 10 recipient, and 2 operative variables that may affect outcome were dichotomized at the median for univariate screening. Factors that achieved a probability value less than 0.2 or that were thought to be relevant were entered into a stepdown Cox proportional hazard regression model. RESULTS: Overall patient and graft survival rates at 1, 5, and 10 years were 84%, 68%, and 60% and 73%, 56%, and 49%, respectively. Overall median time to HCV recurrence was 34 months after transplantation. Neither HCV recurrence nor HCV-positive donor status significantly decreased patient and graft survival rates by Kaplan-Meier analysis. However, use of HCV-positive donors reduced the median time of recurrence to 22.9 months compared with 35.7 months after transplantation of HCV-negative livers. Stratification of patients into five subgroups, based on time of recurrence, revealed that early HCV recurrence was associated with significantly increased rates of patient death and graft loss. Donor, recipient, and operative variables that may affect OLT outcome were analyzed. On univariate analysis, recipient age, serum creatinine, donor length of hospital stay, donor female gender, United Network for Organ Sharing (UNOS) status of recipient, and presence of hepatocellular cancer affected the outcome of OLT. Elevation of pretransplant HCV RNA was associated with an increased risk of graft loss. Of 15 variables considered by multivariate Cox regression analysis, recipient age, UNOS status, donor gender, and log creatinine were simultaneous significant predictors for patient survival. Simultaneously significant factors for graft failure included log creatinine, log alanine transaminase, log aspartate transaminase, UNOS status, donor gender, and warm ischemia time. These variables were therefore entered into prognostic models for patient and graft survival. CONCLUSION: The earlier the recurrence of HCV, the greater the impact on patient and graft survival. The use of HCV-positive donors may accelerate HCV recurrence, and they should be used judiciously. Patient survival at the time of transplantation is predicted by donor gender, UNOS status, serum creatinine, and recipient age. Graft survival is affected by donor gender, warm ischemia time, and pretransplant patient condition. The authors' current survival prognostic models require further multicenter validation.

Ground discarded tires remove naphthalene, toluene, and mercury from water.

Ground discarded tires adsorb naphthalene, toluene, and mercury ions (Hg2+) from aqueous solutions. Their sorption properties and kinetics were determined by batch equilibration techniques at 20 degrees C. The isotherms were linear for naphthalene and toluene and their sorption coefficients were about 1340 and 255 (ml/g), respectively. Sorption of the organic compounds by the ground rubber particles was relatively fast (within 30 min). However, the mercury isotherms were non-linear, and its sorption was slow as compared to the sorption of the organics. The rubber particles had a strong affinity for Hg2+. These results show that ground discarded tires are effective in removing organic compounds and Hg2+ from wastewater and other contaminated environments. In addition it would be a useful, environmentally friendly use of discarded tires (one tire per year per capita is discarded in the United States).

Perforated duodenal ulcer: an alternative therapeutic plan.

An alternative plan for the treatment of a perforated duodenal ulcer is proposed. We will focus on the now-recognized role of Helicobacter pylori in the genesis of the majority of duodenal ulcers and on the high rate of success of therapy with a combination of antibiotics and a proton-pump inhibitor or histamine2 blocker in treatment of such ulcers. Knowledge that half the cases of perforated duodenal ulcer may have securely sealed spontaneously at the time of presentation is incorporated in the therapeutic plan. Patients with a perforated duodenal ulcer who have already been evaluated for H pylori and are not infected or, if infected, have received appropriate therapy should undergo an ulcer-definitive operation if they are suitable surgical candidates. Most authorities recommend surgical closure of the perforation and a parietal cell vagotomy. The remaining patients should have a gastroduodenogram with water-soluble contrast medium. If the perforation is sealed, the patient can be treated nonsurgically. If the perforation is leaking, secure surgical closure of the perforation is necessary. Following recovery from the immediate consequences of the perforation, evaluation for H pylori should be conducted. If the patient is infected, combined medical therapy is recommended. If the patient is not infected, Zollinger-Ellison syndrome should be ruled out and medical therapy is recommended if the ulcer has not been treated previously. Elective ulcer-definitive surgery should be considered for the occasional uninfected patient who has already received appropriate medical therapy for the ulcer.

Weight-based heparin dosing: clinical response and resource utilization.

The objective of this study was to assess a weight-based heparin (WBH) nomogram (80-U/kg bolus, 18-U/kg-per-hour initial infusion) and determine its clinical performance and impact on resource utilization. All patients treated with heparin for venous thromboembolism or unstable angina during a 15-week study period were included in this retrospective, chart-review study. Three groups were identified: patients treated with WBH, patients whose regimen deviated from the weight-based nomogram (DEV), and matched historical controls (HCs). In patients receiving heparin for more than 24 hours, those treated with WBH achieved threshold activated partial thromboplastin time (aPTT) levels significantly faster than did HC or DEV patients. However, 42% of WBH-treated patients were found to have initial supratherapeutic responses. Logistic regression analysis identified age > or =67 years, prior warfarin therapy within 7 days of heparin, and high initial infusion rate as predictive of a supratherapeutic aPTT response; smoking was predictive of a subtherapeutic response. Bleeding events were not significantly different between groups. An infusion rate of 15 U/kg per hour was found to closely approximate our population's actual heparin infusion requirement. Resource utilization was significantly different between the WBH and HC groups in terms of nursing interventions at 48 to 72 hours. We concluded that WBH rapidly drives patients' aPTT response above the therapeutic threshold for heparin; however, prudent adjustment of the initial infusion rate is necessary to avoid a supratherapeutic aPTT response. Our data support a nomogram with an initial infusion rate of 15 U/kg per hour.

Donor origin of a posttransplant liver allograft malignancy identified by fluorescence in situ hybridization for the Y chromosome and DNA genotyping.

Posttransplantation malignancy in the allograft is a rare complication of orthotopic liver transplantation. In the described case, an abnormal T-tube cholangiogram, performed 6 months after orthotopic liver transplantation between a male donor and a female recipient, prompted needle liver biopsy. A moderately differentiated adenocarcinoma was found. Fluorescence in situ hybridization for the Y chromosome indicated male origin of malignancy. Donor-related disease was confirmed by comparative DNA analysis of genomic sequences from the donor liver, associated tumor, and recipient peripheral blood. Results of these investigations qualified the recipient for a second liver transplant.

Characterization of Cbl tyrosine phosphorylation and a Cbl-Syk complex in RBL-2H3 cells.

Tyrosine phosphorylation of the Cbl protooncogene has been shown to occur after engagement of a number of different receptors on hematopoietic cells. However, the mechanisms by which these receptors induce Cbl tyrosine phosphorylation are poorly understood. Here we demonstrate that engagement of the high affinity IgE receptor (Fc epsilon R1) leads to the tyrosine phosphorylation of Cbl and analyze how this occurs. We show that at least part of Fc epsilon R1-induced Cbl tyrosine phosphorylation is mediated by the Syk tyrosine kinase, and that the Syk-dependent tyrosine phosphorylation of Cbl occurs mainly distal to the Cbl proline-rich region within the COOH-terminal 250 amino acids. Furthermore, we show by coprecipitation that Cbl is present in a complex with Syk before receptor engagement, that the proline-rich region of Cbl and a region of Syk comprised of the two SH2 domains and intradomain linker are required for formation of the complex, and that little or no tyrosine-phosphorylated Cbl is detected in complex with Syk. Overexpression of truncation mutants of Cbl capable of binding Syk has the effect of blocking tyrosine phosphorylation of endogenous Cbl. These results define a potentially important intramolecular interaction in mast cells and suggest a complex function for Cbl in intracellular signaling pathways.

Regulation of the association of p120cbl with Grb2 in Jurkat T cells.

The c-cbl protooncogene product (p120(cbl)) is a known substrate of multiple tyrosine kinases. It is found in complexes with critical signal transduction molecules, including the linker protein Grb2. Here, we demonstrate using an immobilized Grb2-binding peptide that the Grb2-p120(cbl) complex dissociates in vivo following engagement of the T-cell antigen receptor in Jurkat T-cells. The early kinetics of this dissociation correlate with the known time course of tyrosine phosphorylation of p120(cbl) and other substrates. This dissociation persists in vivo even when p120(cbl) becomes dephosphorylated to basal levels. However, this decreased association is not observed in protein overlay assays on nitrocellulose membranes in which a Grb2 fusion protein is used to detect p120(cbl) from stimulated or unstimulated cells. These data suggest that the tyrosine phosphorylation of p120(cbl) does not completely account for the regulation of its association with Grb2. Additionally, we used truncation mutations of p120(cbl) to map the p120(cbl)-Grb2 interaction to amino acids 481-528 of p120(cbl); this interaction is stronger in longer constructs that include additional proline-rich motifs. The in vivo regulation of the Grb2-p120(cbl) complex further supports the idea of a significant role for p120(cbl) in receptor-mediated signaling pathways.

The protein product of the c-cbl protooncogene is the 120-kDa tyrosine-phosphorylated protein in Jurkat cells activated via the T cell antigen receptor.

Tyrosine phosphorylation of multiple cellular proteins is a critical event in T cell receptor (TCR)-mediated activation. This pathway has also been implicated in cellular transformation in multiple systems. The viral oncogene v-cbl is the transforming gene of a murine retrovirus that induces pre-B cell lymphomas and myelogenous leukemias. The product of its cellular homolog, p120cbl, is a 120-kDa cytoplasmic protein that is non-transforming when overexpressed. Here we show that the 120-kDa protein tyrosine phosphorylated in Jurkat T cells upon TCR engagement is p120cbl. Following stimulation through the TCR, this tyrosine phosphorylation is rapid and reversible. Tyrosine-phosphorylated p120cbl binds to glutathione S-transferase fusion proteins generated from SH2 domains of the Fyn, Lck, and Blk protein tyrosine kinases, GTPase-activating protein and phospholipase C gamma. The p120cbl from unactivated and activated cells also binds to full-length glutathione S-transferase-Grb2 and the Grb2 N-terminal SH3 domain, but not to the Grb2 C-terminal SH3 domain. Additionally, p120cbl binds to SH3 domains of Fyn and Lck, but not Blk. These data expand our knowledge of protein tyrosine kinase signaling pathways in T cells by identifying a prominent tyrosine kinase substrate. This protein, the product of the cellular homolog of a transforming oncogene, can interact with several known signaling molecules.

Restoration of CD2-mediated signaling by a chimeric membrane protein including the cytoplasmic sequence of CD45.

We showed previously that the TCR and CD2 fail to couple efficiently with their signal transduction machinery in J45.01, a CD45-deficient variant of the Jurkat T-cell line. Transfection into J45.01 of a cDNA encoding a chimeric membrane protein containing the cytoplasmic sequence of CD45 and extracellular and transmembrane sequences derived from the A2 allele of MHC class I rescues proximal signaling events after TCR stimulation. In this report, we describe rescue of CD2-mediated signaling and evaluate further the characteristics of TCR signaling in J45.01 after expression of the chimeric protein. Cells expressing the chimeric molecule demonstrate TCR- and CD2-mediated increases in PTK activity and PI turnover. Stimulation of the TCR and CD2 on the transfected cells also results in the expression of CD69 on the cell surface, a more distal signaling event. Although these measures of signal transduction via the TCR and CD2 are restored in the transfected cells, the magnitude of the responses are less than those seen in the wild-type Jurkat cells. These findings demonstrate that the cytoplasmic domain of CD45, expressed as a chimeric membrane protein, is sufficient for mediating signal transduction through CD2 as well as through the TCR complex. In addition, these results suggest that the extracellular and/or transmembrane domains of CD45 may contribute to the efficiency of signal transduction.

CD45 and the immune response.

CD45 is a major transmembrane glycoprotein expressed on all nucleated hematopoietic cells. Eight isoforms of CD45 are distributed through the immune system according to cell type and degree of cellular differentiation. Heterogeneity among the isoforms is found entirely in the extracellular domain, arising from the differential splicing of up to four exons of a single gene. The control of isoform expression suggests that the extracellular domain may participate in protein-protein interactions with isoform-specific ligands. The intracellular domain of CD45 is large (approximately 700 amino acids), identical for all isoforms, and highly conserved across species. Two nonidentical intracellular sequences of about 240 amino acids that are homologous with a tyrosine phosphatase consensus sequence have been identified. Studies with purified CD45 have shown that all isoforms possess enzymatic activity in in vitro assays. In several T and B cell lines and in natural killer cells, it appears that CD45 is required for optimal signal transduction after stimulation through a number of surface receptors. Although an in vivo substrate has not been identified conclusively, one model suggests that CD45 functions to dephosphorylate a negative-regulatory tyrosine residue on one or more protein tyrosine kinases involved in receptor-mediated second messenger formation. In T cells, the src family kinases, lck and fyn, are candidates for this regulated kinase. In this review, some of the structural and functional aspects of CD45 and its role in signal transduction in the immune system are discussed.

Rescue of signaling by a chimeric protein containing the cytoplasmic domain of CD45.

Surface expression of the CD45 tyrosine phosphatase is essential for the T cell antigen receptor (TCR) to couple optimally with its second messenger pathways. CD45 may be required to dephosphorylate a TCR-activated protein tyrosine kinase, which then transduces an activation signal from the TCR. A chimeric molecule that contained extracellular and transmembrane sequences from an allele of a major histocompatibility class I molecule and cytoplasmic sequences of CD45 restored TCR signaling in a CD45-deficient mutant T cell line. Thus, expression of the complex extracellular domain of CD45 is not required for the TCR to couple to its signaling machinery.

Insulin and glucose-dependent regulation of the glucose transport system in the rat L6 skeletal muscle cell line.

Differentiated rat L6 skeletal muscle cell cultures maintained in glucose-deficient medium containing 25 mM xylose displayed a rapid, reversible, time- and concentration-dependent 3-5-fold increase in glucose transport activity. Glucose deprivation in the continuous presence of insulin (24 h) resulted in an overall 9-10-fold stimulation of glucose transport activity. In contrast, acute (30 min) and chronic (24 h) insulin treatment of L6 cells maintained in high glucose (25 mM)-containing medium resulted in a 1.5- and 4-fold induction of glucose transport activity, respectively. Acute glucose deprivation and/or insulin treatment had no significant effect on the total amount of glucose transporter protein, whereas the long-term insulin- and glucose-dependent regulation of glucose transport activity directly correlated with an increase in the cellular expression of the glucose transporter protein. In situ hybridization of the L6 cells demonstrated a 3-, 4-, and 6-fold increase in glucose transporter mRNA induced by glucose deprivation, insulin, and glucose deprivation plus insulin treatments, respectively. Similarly, Northern blot analysis of total RNA isolated from glucose-deprived, insulin, and glucose-deprived plus insulin-treated cells resulted in a 4-, 3-, and 9-fold induction of glucose transporter mRNA, respectively. The continuous presence of insulin in the medium, either in the presence or absence of glucose, resulted in a transient alteration of the glucose transporter mRNA. The relative amount of the glucose transporter mRNA was maximally increased at 6-12 h which subsequently returned to the basal steady-state level within 48 h. These data demonstrate a role for insulin and glucose in the overall regulation of glucose transporter gene expression which may account for the alteration of glucose transporter activity of muscle tissue observed in pathophysiological states such as type II diabetes mellitus.

Glucose-dependent regulation of glucose transport activity, protein, and mRNA in primary cultures of rat brain glial cells.

D-Glucose deprivation of primary rat brain glial cell cultures, by incubation with 25 mM D-fructose for 24 h, resulted in a 4-5-fold induction of D-glucose transport activity. In contrast, 24-h D-glucose starvation of primary rat brain neuronal cultures had only a marginal effect (1.5-2-fold) on D-glucose transport activity. Northern blot analysis of total cellular RNA demonstrated that under these conditions the rat brain glial cells specifically increased the steady-state level of the D-glucose transporter mRNA 4-6-fold, whereas Northern blot analysis of the neuronal cell cultures revealed no significant alteration in the amount of D-glucose transporter mRNA by D-glucose deprivation. These findings demonstrated that the D-glucose-dependent regulation of the D-glucose transporter system occurred in a brain cell type-specific manner. The ED50 for the D-glucose starvation increase in the D-glucose transporter mRNA, in the glial cell cultures, occurred at approximately 3.5 mM D-glucose with maximal effect at 0.5 mM D-glucose. Readdition of D-glucose to the starved cell cultures reversed the increase in the D-glucose transporter mRNA levels and D-glucose transport activity to control values within 24 h. The increase in the D-glucose transporter mRNA was relatively rapid with half-maximal stimulation at approximately 2 h and maximal induction by 6-12 h of D-glucose deprivation. A similar time course was also observed for the starvation-induced increase in D-glucose transport activity and D-glucose transporter protein, as determined by Western blot analysis. These results document that, in rat brain glial cells, D-glucose transport activity, protein, and mRNA are regulated by the extracellular D-glucose concentration. Further, this suggests a potential role for hyperglycemia in the down-regulation of the D-glucose transport system in vivo.

N-hydroxysulfosuccinimido active esters and the L-(+)-lactate transport protein in rabbit erythrocytes.

Esters of N-hydroxysulfosuccinimide strongly inhibit L-(+)-lactate transport in rabbit erythrocytes, probably by acylating amino groups on the transport protein. Lactate transport studies using bis(sulfosuccinimido) suberate (BS3), bis(sulfosuccinimido) adipate (BS2A), bis(sulfosuccinimido) dithiobis(propionate), and a variety of monocarboxylate esters suggest that an exofacial amino group of the lactate transport protein is essential for lactate transport. Also, reductive methylation studies show that even when positive charge is preserved in modified amino groups, the transport is strongly inhibited. At pH less than 6, band 3 mediated inorganic anion transport is enhanced in BS3-treated cells, while at pH greater than 6, it is inhibited. BS3-induced inhibition of L-(+)-lactate transport does not have this pH dependence. BS3 reduces the labeling of a 40-50-kDa membrane polypeptide (band R) by tritiated 4,4'-diisothiocyanato-2,2-dihydrostilbenedisulfonate ([3H]H2DIDS) and by tritiated bis(sulfosuccinimido) adipate ([3H]BS2A). Tritiated sulfosuccinimido acetate (S2[3H]acetate) also labels band R, over a range of concentrations where lactate transport is inhibited in a dose-dependent manner by S2 acetate. BS3 is a known impermeant protein cross-linker. S2 acetate permeates rabbit red cell membranes by an H2DIDS-inhibitable mechanism. BS3 cross-links the proteolytic fragments of rabbit band 3 produced by extracellular chymotrypsin. These labeling experiments support an association between band R and specific monocarboxylate transport.