Regulation of ceramide generation during macrophage apoptosis by ASMase and de novo synthesis.

The survival of macrophages depends on the presence of specific cytokines that activate survival signaling events, as well as suppressing formation of apoptosis-inducing pathways. We have previously shown that macrophages deprived of macrophage colony stimulating factor (M-CSF) produce ceramide that contributes to apoptosis of these cells, a pathway that is suppressed by exposure to oxidized LDL. In this study we have examined macrophages derived from mice lacking acid sphingomyelinase (ASMase) to ask whether these events are altered due to the impaired ability of these cells to break down sphingomyelin and produce ceramide. We found that these cells do survive better than cells from wild type mice, but they still undergo cell death and some ceramide is formed. We show that the ceramide is being produced by a de novo synthetic pathway. Therefore, ceramide production in M-CSF-deprived macrophages arises from a combination of ASMase activity and de novo synthesis.

Response to endoscopic therapy for biliary anastomotic strictures in deceased versus living donor liver transplantation.

BACKGROUND: Endoscopic therapy has been successful in the management of biliary complications after both deceased donor liver transplantation (DDLT) and living donor liver transplantation (LDLT). LDLT is thought to be associated with higher rates of biliary complications, but there are few studies comparing the success of endoscopic management of anastomotic strictures between the two groups. This study aims to compare our experience in the endoscopic management of anastomotic strictures in DDLT versus LDLT. METHODS: This is a retrospective database review of all liver transplant patients undergoing endoscopic retrograde cholangiopancreatography (ERCP) after liver transplantation. The frequency of anastomotic stricture and the time to develop and to resolve anastomotic stricture were compared between DDLT and LDLT. The response of anastomotic stricture to endoscopic therapy was also analyzed. RESULTS: A total of 362 patients underwent liver transplantation between 2003 and 2011, with 125 requiring ERCP to manage biliary complications. Thirty-three (9.9%) cases of DDLT and 8 (27.6%) of LDLT (P=0.01) were found to have anastomotic stricture. When comparing DDLT and LDLT, there was no difference in the mean time to the development of anastomotic strictures (98+/-17 vs 172+/-65 days, P=0.11), likelihood of response to ERCP [22 (66.7%) vs 6 (75.0%), P=0.69], mean time to the resolution of anastomotic strictures (268+/-77 vs 125+/-37 days, P=0.34), and the number of ERCPs required to achieve resolution (3.9+/-0.4 vs 4.7+/-0.9, P=0.38). CONCLUSIONS: Endoscopic therapy is effective in the majority of biliary complications relating to liver transplantation. Anastomotic strictures occur more frequently in LDLT compared with DDLT, with equivalent endoscopic treatment response and outcomes for both groups.

First successful treatment of post-liver transplant hepatitis C fibrosing cholestatic hepatitis with boceprevir, peginterferon and ribavirin in a pre-transplant null responder.

Fibrosing cholestatic hepatitis (FCH) is a less common but well-recognized severe complication of recurrent hepatitis C virus (HCV) infection post-liver transplant. This condition is fatal without successful treatment and to date; post-transplant antiviral interferon-based antiviral therapy has been associated with guarded success. The new era of protease inhibitors in the treatment of chronic HCV infection may alter the dismal outcome of this condition. To date, however, the experience with protease inhibitors in this condition is unreported. We report a post-liver transplant recipient with HCV associated FCH treated successfully with boceprevir, peginteferon and ribavirin for severe FCH. The patient was young woman who was a null responder pre-transplant to peginterferon and ribavirin. The peak serum bilirubin 391 µmol/L normalized to 15 µmol/L by week 8 of therapy. The pre-treatment HCV viral load of > 78 million IU/mL, decreased to 78 IU/mL at week 8 of therapy and was undetectable by week 12 and at the end of 48 week of treatment. 12 weeks post treatment, the HCV viral load remains undetectable. Significant anemia and neutropenia were encountered. Tacrolimus dosage titrated to trough levels, required marked reduction to 0.5 mg three times weekly. Despite the suboptimal peginterferon and ribavirin dosing, limited by adverse effects, full boceprevir dosing was maintained, with resolution of liver dysfunction. Boceprevir was obtained on compassionate grounds from the manufacturer before its licensure in Canada and this was the first use of boceprevir in the world for post-transplant FCH.

Familial hypobetalipoproteinemia-induced nonalcoholic steatohepatitis.

Familial hypobetalipoproteinemia (FHBL) is a rare genetic disorder of lipid metabolism that is associated with abnormally low serum levels of low-density lipoprotein (LDL) cholesterol and apolipoprotein B. It is an autosomal co-dominant disorder, and depending on zygosity, the clinical manifestations may vary from none to neurological, endocrine, hematological or liver dysfunction. Nonalcoholic fatty liver disease is common in persons with FHBL, however progression to nonalcoholic steatohepatitis is unusual. We describe here a patient with a novel APOB mutation, V703I, which appears to contribute to the severity of the FHBL phenotype. He had liver enzyme abnormalities, increased echogenicity of the liver consistent with steatosis, very low LDL cholesterol at 0.24 mmol/l (normal 1.8-3.5 mmol/l) and an extremely low apolipoprotein B level of 0.16 g/l (normal 0.6-1.2 g/l). APOB gene sequencing revealed him to be a compound heterozygote with two mutations (R463W and V703I). APOB R463W has previously been reported to cause FHBL. Genetic sequencing of his first-degree relatives identified the APOB V703I mutation in his normolipidemic brother and father and the APOB R463W mutation in his mother and sister, both of whom have very low LDL cholesterol levels. These results suggest that the APOB V703I mutation alone does not cause the FHBL phenotype. However, it is possible that it has a contributory role to a more aggressive phenotype in the presence of APOB R463W.

Fluorescent adducts formed by reaction of oxidized unsaturated fatty acids with amines increase macrophage viability.

Macrophages are prominent components of human atherosclerotic lesions and they are believed to accelerate the progression and/or complications of both early and advanced atherosclerotic lesions. We and others have shown that oxidized low-density lipoprotein (oxLDL) induces growth and inhibits apoptosis in murine bone marrow-derived macrophages. In this study, we sought to characterize the oxidative modification of LDL that is responsible for this prosurvival effect. We found that both the modified lipid and the modified protein components of oxLDL can increase the viability of macrophages. The key modification appeared to involve derivatization of amino groups in apoB or in phosphatidylethanolamine by lipid peroxidation products. These reactive oxidation products were primarily unfragmented hydroperoxide- or endoperoxide-containing oxidation products of linoleic acid or arachidonic acid. LC-MS/MS studies showed that some of the arachidonic acid-derived lysine adducts were isolevuglandins that contain lactam and hydroxylactam rings. MS/MS analysis of linoleic acid autoxidation adducts was consistent with 5- or 6-membered nitrogen-containing heterocycles derived from unfragmented oxidation products. The amine modification by oxidation products generated a fluorescence pattern with an excitation maximum at 350nm and emission maximum at 430nm. This is very similar to the fluorescence spectrum of copper-oxidized LDL.

OxLDL-mediated survival of macrophages does not require LDL internalization or signalling by major pattern recognition receptors.

Macrophages play a key role in the pathogenesis of atherosclerosis, in part by destabilizing plaques. We and others have shown that low concentrations of oxidized LDL (oxLDL) inhibit macrophage apoptosis. As oxLDL is present in lesions, this may be a mechanism by which macrophage populations in the intima are expanded. We have previously shown that oxLDL activates prosurvival signalling pathways such as the phosphoinositide 3-kinase (PI3K) pathway in bone marrow derived macrophages (BMDMs). However, little is known about more upstream signalling events especially at the receptor level. The endocytic pattern recognition receptors (PRRs), scavenger receptor A (SR-A) and CD36, are the main receptors on macrophages for uptake of oxLDL and are therefore important in foam cell formation. The signalling PRRs such as toll-like receptor (TLR) 2 and 4 also bind some types of oxLDL. This study was done to determine if any of the known PRRs are required for the anti-apoptotic effects of oxLDL in BMDMs. To do this, we tested the effect of oxLDL on viability of BMDMs lacking both SR-A and CD36 or lacking TLR2, TLR4, CD14, FcγRIIb, or RAGE. Our results indicate that none of these receptors are essential for activating the oxLDL prosurvival pathway. Furthermore, we show that the anti-apoptotic effect is not dependent on the uptake of oxLDL.

Spontaneous clearance of hepatitis C infection post-liver transplant: A rare but real phenomenon? A case report and review of the literature.

Recurrent hepatitis C virus (HCV) infection after liver transplantation is a significant cause of morbidity, mortality and graft loss. Spontaneous clearance of recurrent HCV after liver transplant is a rarely reported phenomenon. We report a case of a 66-year-old woman who underwent liver transplantation for HCV cirrhosis (treatment- naive genotype 2) under immunosuppression with tacrolimus, mycophenolate mofetil (MMF), and short-term corticosteroids. The patient developed histologically proved severe cholestatic recurrence of HCV hepatitis. Immunosuppression was reduced to tacrolimus monotherapy because of cytopenia. She subsequently became RNA negative at week 44 post- transplant while on tacrolimus and MMF despite no antiviral therapy. A spontaneous sustained virologic clearance was confirmed with subsequent HCV nucleotide testing. Only a few similar cases have been reported in the literature with uninterrupted immunosuppression and subsequent spontaneous clearance. Our experience, and the few other published cases in the literature, suggests that spontaneous clearance of HCV after liver transplantation is a rare but real phenomenon. Better understanding of this phenomenon may help to manage recurrent HCV disease after transplantation.

Predictors of relapse to significant alcohol drinking after liver transplantation.

BACKGROUND: End-stage alcoholic liver disease is common, with many of these patients referred for liver transplantation (LT). Alcohol relapse after LT can have detrimental outcomes such as graft loss and can contribute to a negative public perception of LT. OBJECTIVE: To identify factors that predict the recurrence of harmful alcohol consumption after LT. METHODS: A total of 80 patients who underwent LT for alcoholic cirrhosis or had significant alcohol consumption in association with another primary liver disease, from July 1992 to June 2006 in British Columbia, were retrospectively evaluated by chart review. Several demographic-, psychosocial- and addiction-related variables were studied. Univariate and multivariate logistic regression analyses were used to test possible associations among the variables studied and a return to harmful drinking after LT. RESULTS: The relapse rate of harmful alcohol consumption post-liver transplant was 10%, with two patient deaths occurring directly as a result of alcohol relapse. Univariate analysis revealed relapse was significantly associated with pretransplant abstinence of less than six months (P=0.003), presence of psychiatric comorbidities (P=0.016), female sex (P=0.019) and increased personal stressors (P=0.044), while age at transplant of younger than 50 years approached significance (P=0.054). Multivariate logistic regression analysis revealed the following independent factors for relapse: pretransplant abstinence of less than six months (OR 77.07; standard error 1.743; P=0.013) and female sex (OR 18.80; standard error 1.451; P=0.043). CONCLUSION: The findings of the present study strongly support a required minimum of six months of abstinence before LT because duration of abstinence was found to be the strongest predictor of recidivism. Female sex, younger age at transplant and psychiatric comorbidities were also associated with relapse to harmful drinking.

Multimodal therapy for hepatocellular carcinoma: a complementary approach to liver transplantation.

OBJECTIVE: To evaluate the survival benefit of multimodal therapy for the treatment of HCC. BACKGROUND: Orthotopic liver transplantation (OLT) is considered the treatment of choice for selected patients with hepatocellular carcinoma (HCC). However, donor organ shortages and patients whose HCCs exceed OLT criteria require consideration of alternate therapeutic options such as hepatic resection, radiofrequency ablation (RFA), ethanol injection (EI), transarterial chemoembolization (TACE), and chemotherapy (CTX). This study was performed to evaluate the survival benefit of multimodal therapy for treatment of HCC as complementary therapy to OLT. METHODS: A retrospective review was conducted of HCC patients undergoing therapy following multidisciplinary review at our institution from 1996 . 2006 with a minimum of a 2 year patient follow-up. Data were available on 247/252 patients evaluated. Relevant factors at time of diagnosis included symptoms, hepatitis B (HBV) and C (HCV) status, antiviral therapy, Child-Pugh classification, portal vein patency, and TNM staging. Patients underwent primary treatment by hepatic resection, RFA, EI, TACE, CTX, or were observed (best medical management). Patients with persistent or recurrent disease following initial therapy were assessed for salvage therapy. Survival curves and pairwise multiple comparisons were calculated using standard statistical methods. RESULTS: Mean overall survival was 76.8 months. Pairwise comparisons revealed significant mean survival benefits with hepatic resection (93.2 months), RFA (66.2 months), and EI (81.1 months), compared with TACE (47.4 months), CTX (24.9 months), or observation (31.4 months). Shorter survival was associated with symptoms, portal vein thrombus, or Child-Pugh class B or C. HCV infection was associated with significantly shorter survival compared with HBV infection. Antiviral therapy was associated with significantly improved survival in chronic HBV and HCV patients only with earlier stage disease. CONCLUSION: Multimodal therapy is effective therapy for HCC and may be used as complementary treatment to OLT.

Sphingosine kinase regulates oxidized low density lipoprotein-mediated calcium oscillations and macrophage survival.

We recently reported that oxidized LDL (oxLDL) induces an oscillatory increase in intracellular calcium ([Ca(2+)](i)) levels in macrophages. Furthermore, we have shown that these [Ca(2+)](i) oscillations mediate oxLDL's ability to inhibit macrophage apoptosis in response to growth factor deprivation. However, the signal transduction pathways by which oxLDL induces [Ca(2+)](i) oscillations have not been elucidated. In this study, we show that these oscillations are mediated in part by intracellular mechanisms, as depleting extracellular Ca(2+) did not completely abolish the effect. Inhibiting sarco-endoplasmic reticulum ATPase (SERCA) completely blocked [Ca(2+)](i) oscillations, suggesting a role for Ca(2+) reuptake by the ER. The addition of oxLDL resulted in an almost immediate activation of sphingosine kinase (SK), which can increase sphingosine-1-phosphate (S1P) levels by phosphorylating sphingosine. Moreover, S1P was shown to be as effective as oxLDL in blocking macrophage apoptosis and producing [Ca(2+)](i) oscillations. This suggests that the mechanism in which oxLDL generates [Ca(2+)](i) oscillations may be 1) activation of SK, 2) SK-mediated increase in S1P levels, 3) S1P-mediated Ca(2+) release from intracellular stores, and 4) SERCA-mediated Ca(2+) reuptake back into the ER.

Late acute celiac and hepatic artery thrombosis with portal vein thrombosis resulting in hepatic infarction 12 years post orthotopic liver transplantation.

Hepatic artery thrombosis (HAT) is relatively infrequent, but possibly a devastating complication of orthotopic liver transplantation (OLT). It often requires urgent retransplantation. Two main forms of HAT are recognized as early and late HAT (diagnosis within or after 30 days following LT). Early HAT typically results in graft failure. Late HAT features biliary obstruction, cholangitis, and hepatic abscess formation. We report here the case of a patient of Wilson's disease who presented twelve years post-liver transplant symptoms typical of acute HAT and hepatic infarction. On diagnostic imaging, celiac axis and hepatic artery were thrombosed, resulting in ischemic necrosis of the left hepatic lobe. The resulting sepsis and transient hepatic insufficiency were managed conservatively, and repeat OLT was avoided. The patient remains stable more than one year later. To the best of our knowledge this case report is unique in the literature for the unusually long interval between OLT and late acute HAT, as well as celiac and portal vein occlusion. The acute presentation of sub massive hepatic necrosis is also uncharacteristic of late HAT and more typical of acute HAT. This report describes our experience in managing this and a literature review of the topic.

Parvovirus B19 induced hepatic failure in an adult requiring liver transplantation.

Parvovirus B19 induced acute hepatitis and hepatic failure have been previously reported, mainly in children. Very few cases of parvovirus induced hepatic failure have been reported in adults and fewer still have required liver transplantation. We report the case of a 55-year-old immunocompetent woman who developed fulminant hepatic failure after acute infection with Parvovirus B19 who subsequently underwent orthotopic liver transplantation. This is believed to be the first reported case in the literature in which an adult patient with fulminant hepatic failure associated with acute parvovirus B19 infection and without hematologic abnormalities has been identified prior to undergoing liver transplantation. This case suggests that Parvovirus B19 induced liver disease can affect adults, can occur in the absence of hematologic abnormalities and can be severe enough to require liver transplantation.

LOX-1 augments oxLDL uptake by lysoPC-stimulated murine macrophages but is not required for oxLDL clearance from plasma.

Oxidized LDL (oxLDL) promotes lipid accumulation as well as growth and survival signaling in macrophages. OxLDL uptake is mainly due to scavenger receptors SR-AI/II and CD36. However, other scavenger receptors such as lectin-like oxLDL receptor-1 (LOX-1) may also play a role. We used mice with targeted inactivation of the LOX-1 gene to define the role of this receptor in the uptake of oxLDL and in activation of survival pathways. There was no difference in uptake or degradation of 125I-oxLDL in unstimulated macrophages from wild-type and LOX-1 knockout mice and no difference in the rate of clearance of oxLDL from plasma in vivo. However, when expression of LOX-1 was induced with lysophosphatidylcholine, oxLDL uptake and degradation increased 2-fold in wild-type macrophages but did not change in LOX-1 knockout macrophages. Macrophages lacking LOX-1 showed the same stimulation of PKB phosphorylation and enhancement of survival by oxLDL as wild-type cells. These data show that LOX-1 does not alter the uptake of oxLDL in unstimulated macrophages and is not essential for the pro-survival effect of oxLDL in these cells. However, LOX-1 expression is highly inducible by lysophosphatidylcholine and pro-inflammatory cytokines, and if that occurred in macrophages within atheromas, LOX-1 could substantially increase oxLDL uptake by lesion macrophages.

Surgical morbidity in severely obese liver transplant recipients - a single Canadian Centre Experience.

The prevalence of obesity is increasing globally, with nearly half of a billion of the world's population now considered to be overweight or obese. Obesity and overweight patients are one of the major health issues in Canada, resulting in approximately 57,000 deaths related to obesity over the last 15 years. The effect of obesity on outcomes following liver transplantation remains largely unclear. To determine the effect of obesity on outcome we reviewed 167 liver transplants, performed at the Vancouver General Hospital, between February 1999 and October 2003. Severe obesity was defined as body mass index (BMI) > 35 kg/m2 and moderate obesity as BMI of 30 - 34 kg/m2. One hundred forty three transplants were performed in patients with a body mass index (BMI) < 30 kg/m2, 14 in patients with a BMI of 30 - 34 kg/m2, and 10 in patients with a BMI > 35 kg/m2. Non-weight related patient demographics were similar between the groups. A very high proportion of Hepatitic C patients (7/10) were observed in the severely obese group. In the early postoperative course severely obese patients had a higher rate of wound infection (20% vs. 4%, p = 0.0001) and wound dehiscence (40% vs. 1.2%, p = 0.0001). Within the first twelve postoperative months severely obese liver transplant recipients had a higher rate of ventral wound herniation (30% vs. 2.8%, p = 0.0001) when compared to obese or non-obese recipients. The one-year graft and patient survival were similar to non-obese patients. An increased BMI in liver transplant recipients in our centre did not increase the risk of early postoperative mortality, but did increase surgical complications, such as wound infection and wound dehiscence. The 1-year patient and graft survival however was indistinguishable from those of non-obese patients.

Oxidized LDL-mediated macrophage survival involves elongation factor-2 kinase.

OBJECTIVE: Macrophage survival and proliferation is believed to be a contributing factor in the development of early atherosclerotic lesions. Oxidized low density lipoprotein (oxLDL), a key mediator in the pathogenesis of this disease, has been shown to block apoptosis in macrophages deprived of growth factor. In this report, we investigate the mechanism of oxLDL-mediated macrophage survival. METHODS AND RESULTS: OxLDL, but not native LDL (nLDL), induces an immediate and oscillatory increase in intracellular calcium ([Ca(2+)](i)). We also show that the calcium/calmodulin dependent kinase, eukaryotic elongation factor-2 kinase (eEF2 kinase), is activated in response to oxLDL, an effect that can be blocked by inhibiting calcium mobilization. Furthermore, selective inhibition of eEF2 kinase reverses the prosurvival effect of oxLDL and results in cellular apoptosis. p38 MAP kinase, a negative regulator of eEF2 kinase, is activated on growth factor withdrawal, a response that can be inhibited by oxLDL. Finally, we show that oxLDL, by activating eEF2 kinase, phosphorylates and therefore inhibits eEF2, resulting in an overall decrease in protein synthesis. CONCLUSIONS: These results indicate a novel signaling pathway in which oxLDL can block macrophage apoptosis by mobilizing calcium and activating eEF2 kinase.

VEGF secretion by macrophages is stimulated by lipid and protein components of OxLDL via PI3-kinase and PKCzeta activation and is independent of OxLDL uptake.

Oxidized LDL (OxLDL) is thought to play a role in the pathogenesis of early as well as advanced stages of atherosclerosis. One possible mechanism involves local upregulation of pro-inflammatory cytokines such as vascular endothelial growth factor (VEGF). This study was done to define the mechanism by which OxLDL increases secretion of VEGF in macrophages. The murine leukemia-derived RAW 264.7 macrophage cell line as well as mouse peritoneal macrophages and human monocyte-derived macrophages were used in these studies. Cells were exposed to native low-density lipoprotein (LDL), acetylated LDL, and LDL that had been modified by oxidation with copper or ferrous ions or by exposure to auto-oxidation products of arachidonic acid for 16h, and VEGF was then assayed in medium. Pharmacological inhibitors of phosphatidylinositol 3-kinase (PI3K) or PKCzeta blocked VEGF secretion by OxLDL. Inhibitors of other protein kinase C (PKC) subtypes had no effect, and neither did inhibitors of mitogen activated protein kinase kinase (MAPKK). We found that LDL with oxidative modification of either its lipid or protein component can induce VEGF expression. Higher degrees of oxidation of LDL conferred higher potency to induce VEGF. Macrophages from mice lacking both scavenger receptors A (SR-A) and CD36 were fully responsive to OxLDL with regard to VEGF secretion. These macrophages show an 85% reduction in OxLDL uptake compared to macrophages from wild-type mice. Macrophages from mice lacking LOX-1 were also fully responsive to oxLDL with regard to VEGF secretion. We conclude that VEGF upregulation is mediated through PI3K and PKCzeta, and does not involve the above three scavenger receptors or require uptake of oxidized LDL.

Mycophenolate mofetil in liver transplant patients with calcineurin-inhibitor-induced renal impairment.

BACKGROUND: Calcineurin inhibitors (CNIs) provide effective immunosuppression after orthotopic liver transplantation (OLTx), but the associated nephrotoxicity can cause substantial morbidity and mortality among transplant patients. In this study, we retrospectively investigated the efficacy and safety of mycophenolate mofetil (MMF) in OLTx patients with CNI-induced renal impairment. PATIENTS & METHODS: A chart review was undertaken of all liver transplant recipients followed at the Vancouver General Hospital. Twenty-one (12 male) patients were converted to either MMF monotherapy (n = 18) or MMF with corticosteroids (n = 3) for CNI-induced renal dysfunction. Six were excluded because of other factors contributing to renal dysfunction. Mean time from OLTx to conversion was 11.3 years and mean age was 60. Non-parametric Wilcoxon's signed rank testing was used to determine whether there was a difference between the serum creatinine (SCr) before conversion, and 3 or 6 months after conversion. RESULTS: Median follow-up was 294 days, ranging from 35 to 1103 days. The median SCr was significantly reduced from 144 micromol/L before conversion to 129 micromol/L and 139 micromol/L at 3 and 6 months follow-up (p = 0.001 and 0.008, respectively). MMF was well tolerated. Only one patient (6.7%) had elevated liver enzymes and required addition of sirolimus while two (13.4%) experienced gastrointestinal intolerance. CONCLUSIONS: MMF appears to be safe for stable OLTx recipients with CNI-induced nephrotoxicity. Serious side effects were uncommon as only one patient required discontinuation of the medication. However, longer follow-up and larger study populations are needed in the future to better determine its efficacy and safety.