Timothy Schmitt

Mechanisms of Acetaminophen-induced Cell Death in Primary Human Hepatocytes

UNLABELLED Acetaminophen (APAP) overdose is the most prevalent cause of drug-induced liver injury in western countries. Numerous studies have been conducted to investigate the mechanisms of injury after APAP overdose in various animal models; however, the importance of these mechanisms for humans remains unclear. Here we investigated APAP hepatotoxicity using freshly isolated primary human hepatocytes (PHH) from either donor livers or liver resections. PHH were exposed to 5mM, 10mM or 20mM APAP over a period of 48 h and multiple parameters were assessed. APAP dose-dependently induced significant hepatocyte necrosis starting from 24h, which correlated with the clinical onset of human liver injury after APAP overdose. Interestingly, cellular glutathione was depleted rapidly during the first 3h. APAP also resulted in early formation of APAP-protein adducts (measured in whole cell lysate and in mitochondria) and mitochondrial dysfunction, indicated by the loss of mitochondrial membrane potential after 12h. Furthermore, APAP time-dependently triggered c-Jun N-terminal kinase (JNK) activation in the cytosol and translocation of phospho-JNK to the mitochondria. Both co-treatment and post-treatment (3h) with the JNK inhibitor SP600125 reduced JNK activation and significantly attenuated cell death at 24h and 48h after APAP. The clinical antidote N-acetylcysteine offered almost complete protection even if administered 6h after APAP and a partial protection when given at 15 h. CONCLUSION These data highlight important mechanistic events in APAP toxicity in PHH and indicate a critical role of JNK in the progression of injury after APAP in humans. The JNK pathway may represent a therapeutic target in the clinic.

Bile acid-induced necrosis in primary human hepatocytes and in patients with obstructive cholestasis.

Accumulation of bile acids is a major mediator of cholestatic liver injury. Recent studies indicate bile acid composition between humans and rodents is dramatically different, as humans have a higher percent of glycine conjugated bile acids and increased chenodeoxycholate content, which increases the hydrophobicity index of bile acids. This increase may lead to direct toxicity that kills hepatocytes, and promotes inflammation. To address this issue, this study assessed how pathophysiological concentrations of bile acids measured in cholestatic patients affected primary human hepatocytes. Individual bile acid levels were determined in serum and bile by UPLC/QTOFMS in patients with extrahepatic cholestasis with, or without, concurrent increases in serum transaminases. Bile acid levels increased in serum of patients with liver injury, while biliary levels decreased, implicating infarction of the biliary tracts. To assess bile acid-induced toxicity in man, primary human hepatocytes were treated with relevant concentrations, derived from patient data, of the model bile acid glycochenodeoxycholic acid (GCDC). Treatment with GCDC resulted in necrosis with no increase in apoptotic parameters. This was recapitulated by treatment with biliary bile acid concentrations, but not serum concentrations. Marked elevations in serum full-length cytokeratin-18, high mobility group box 1 protein (HMGB1), and acetylated HMGB1 confirmed inflammatory necrosis in injured patients; only modest elevations in caspase-cleaved cytokeratin-18 were observed. These data suggest human hepatocytes are more resistant to human-relevant bile acids than rodent hepatocytes, and die through necrosis when exposed to bile acids. These mechanisms of cholestasis in humans are fundamentally different to mechanisms observed in rodent models.

Impact of stem cell marker expression on recurrence of TACE-treated hepatocellular carcinoma post liver transplantation

AbstractBackgroundLiver transplantation is the most effective therapy for cirrhosis-associated hepatocellular carcinoma (HCC) but its utility is limited by post-transplant tumor recurrence. Use of the Milan, size-based criteria, has reduced recurrence rate to less than 10% but many patients remain ineligible. Reduction of tumor size with local therapies has been used to “downstage” patients to allow them to qualify for transplantation, but the optimal criteria to predict tumor recurrence in these latter patients has not been established. The existence of a progenitor cell population, sometimes called cancer stem cells (CSCs), has been proposed to be one mechanism accounting for the chemotherapy resistance and recurrence of hepatocellular carcinoma. The aim of this study was to determine if transcatheter arterial chemoemolization (TACE) treated tumors have increased CSC marker expression and whether these markers could be used to predict tumor recurrence.MethodsFormalin fixed specimens were obtained from 39 HCC liver explants (23 with no treatment and 16 after TACE). Immunohistochemical staining was performed for EpCAM, CD44, CD90, and CD133. Staining for each marker was scored 0–3 by evaluating the number and intensity of positive tumor cells in 5 hpf of tumor in each specimen.ResultsTACE treated tumors displayed greater necrosis and fibrosis than non-TACE treated samples but there were no differences in morphology between the viable tumor cells of both groups. In TACE treated specimens, the staining of both EpCAM and CD133 was greater than in non-TACE specimens but CD44 and CD90 were the same. In the TACE group, the presence of high EpCAM staining was associated with tumor recurrence. Four of ten EpCAM high patients recurred while 0 of 6 EpCAM low patients recurred (P = 0.040). None of the other markers predicted recurrence.ConclusionHigh pre-transplant EpCAM staining predicted HCC recurrence. This suggests that the abundance of tumor cells with a CSC phenotype may be a critical factor in the likelihood of tumor recurrence in patients receiving liver transplantation after TACE.

Donor PNPLA3 rs738409 genotype affects fibrosis progression in liver transplantation for hepatitis C

The rs738409 G>C single nucleotide polymorphism occurring in the patatin‐like phospholipase 3 gene has been identified as a novel genetic marker for hepatic steatosis. Recent studies also associated rs738409 with fibrosis in hepatitis C (HCV). Therefore, we sought to determine the impact of donor and recipient rs738409 genotype on the progression of fibrosis after liver transplantation for HCV. This cohort study included 101 patients infected with HCV who underwent liver transplantation between January 2008, and June 2011. Donor and recipient rs738409 genotypes were determined from donor wedge biopsies and recipient explants. The time to Ishak stage 3 fibrosis, or HCV‐related mortality/graft loss was analyzed by the Cox model adjusting for HCV‐Donor Risk Index, warm ischemic time, pretransplant Model for Endstage Liver Disease (MELD) and viral load. The rs738409 CC variant was present in 56% of donors and 57% of recipients. The median follow‐up period was 620 days. A total of 39 patients developed the primary outcome of ≥stage 3 fibrosis or HCV‐related mortality/graft loss, the time to which differed by donor (P = 0.019) but not recipient (P = 0.89) genotype. In the multivariate model, donor GC or GG variants had 2.53 times the risk (95% confidence interval [CI] 1.25‐5.02, P = 0.008) compared to CC variants. In the alternative endpoint: stage 3 fibrosis or all‐cause mortality/graft loss, the effect of donor genotype was attenuated but remained significant at 1.98 (95% CI 1.11‐3.53). Conclusions: The rs738409 genotype is an important predictor of posttransplant outcome in HCV. Liver, and not adipocytes, is the site at which this effect occurs. Our finding may be useful in donor selection for liver transplantation with HCV, and may guide decisions regarding early antiviral treatment. (Hepatology 2014;59:453–460)

Fibrin deposition following bile duct injury limits fibrosis through an αMβ2-dependent mechanism

Coagulation cascade activation and fibrin deposits have been implicated or observed in diverse forms of liver damage. Given that fibrin amplifies pathological inflammation in several diseases through the integrin receptor αMβ2, we tested the hypothesis that disruption of the fibrin(ogen)-αMβ2 interaction in Fibγ(390-396A) mice would reduce hepatic inflammation and fibrosis in an experimental setting of chemical liver injury. Contrary to our hypothesis, α-naphthylisothiocyanate (ANIT)-induced liver fibrosis increased in Fibγ(390-396A) mice, whereas inflammatory cytokine expression and hepatic necrosis were similar to ANIT-challenged wild-type (WT) mice. Increased fibrosis in Fibγ(390-396A) mice appeared to be independent of coagulation factor 13 (FXIII) transglutaminase, as ANIT challenge in FXIII-deficient mice resulted in a distinct pathological phenotype characterized by increased hepatic necrosis. Rather, bile duct proliferation underpinned the increased fibrosis in ANIT-exposed Fibγ(390-396A) mice. The mechanism of fibrin-mediated fibrosis was linked to interferon (IFN)γ induction of inducible nitric oxide synthase (iNOS), a gene linked to bile duct hyperplasia and liver fibrosis. Expression of iNOS messenger RNA was significantly increased in livers of ANIT-exposed Fibγ(390-396A) mice. Fibrin(ogen)-αMβ2 interaction inhibited iNOS induction in macrophages stimulated with IFNγ in vitro and ANIT-challenged IFNγ-deficient mice had reduced iNOS induction, bile duct hyperplasia, and liver fibrosis. Further, ANIT-induced iNOS expression, liver fibrosis, and bile duct hyperplasia were significantly reduced in WT mice administered leukadherin-1, a small molecule that allosterically enhances αMβ2-dependent cell adhesion to fibrin. These studies characterize a novel mechanism whereby the fibrin(ogen)-integrin-αMβ2 interaction reduces biliary fibrosis and suggests a novel putative therapeutic target for this difficult-to-treat fibrotic disease.

Filling Defect on ERCP: Biliary Cystadenoma, a Rare Tumor.

Biliary cystadenomas are rare tumors of the bile ducts most commonly presenting as large right liver lobe lesions. These are usually slow-growing and mostly benign. They commonly present with abdominal pain. On physical exam an abdominal mass can be identified occasionally. Walls of biliary cystadenomas appear thicker than simple cysts, with soft tissue nodules and enhancing septations on CT or MRI. Radiographic images can vary with the amount of protein content in the fluid on CT or MRI. Due to the risk of malignant transformation, complete surgical resection is advised. Hereby, we describe a 37-year-old lady who presented to the outpatient clinic with bloating and abdominal discomfort with intermittent elevated liver enzymes and hyperbilirubinemia. Ultrasound of the liver and bile ducts followed by CT scan and magnetic resonance cholangiopancreatography confirmed the presence of biliary cystadenoma of the intra- and extrahepatic ducts. It was seen as a filling defect of the intra- and extrahepatic ducts (common hepatic duct) on endoscopic retrograde cholangiopancreatography. Involvement of the intra- and extrahepatic bile ducts simultaneously is a rare presentation of this tumor. She later on underwent exploratory laparotomy with extrahepatic bile duct resection, left hepatic lobe resection and reconstruction with hepaticojejunostomy. Pathology confirmed the presence of biliary cystadenoma with ovarian-like stroma. She had recovered uneventfully from the surgery when seen 2 weeks later in the clinic. Biliary cystadenoma is a rare, mostly benign neoplasm of the biliary tract that should be considered in the differential diagnosis of cystic lesions of the biliary tract.

Microcystin-LR induced liver injury in mice and in primary human hepatocytes is caused by oncotic necrosis.

ABSTRACT Microcystins are a group of toxins produced by freshwater cyanobacteria. Uptake of microcystin‐leucine arginine (MC‐LR) by organic anion transporting polypeptide 1B2 in hepatocytes results in inhibition of protein phosphatase 1A and 2A, and subsequent cell death. Studies performed in primary rat hepatocytes demonstrate prototypical apoptosis after MC‐LR exposure; however, no study has directly tested whether apoptosis is critically involved in vivo in the mouse, or in human hepatocytes. MC‐LR (120 &mgr;g/kg) was administered to C57BL/6J mice and cell death was evaluated by alanine aminotransferase (ALT) release, caspase‐3 activity in the liver, and histology. Mice exposed to MC‐LR had increases in plasma ALT values, and hemorrhage in the liver, but no increase in capase‐3 activity in the liver. Pre‐treatment with the pan‐caspase inhibitor z‐VAD‐fmk failed to protect against cell death measured by ALT, glutathione depletion, or hemorrhage. Administration of MC‐LR to primary human hepatocytes resulted in significant toxicity at concentrations between 5 nM and 1 &mgr;M. There were no elevated caspase‐3 activities and pretreatment with z‐VAD‐fmk failed to protect against cell death in human hepatocytes. MC‐LR treated human hepatocytes stained positive for propidium iodide, indicating membrane instability, a marker of necrosis. Of note, both increases in PI positive cells, and increases in lactate dehydrogenase release, occurred before the onset of complete actin filament collapse. In conclusion, apoptosis does not contribute to MC‐LR‐induced cell death in the in vivo mouse model or in primary human hepatocytes in vitro. Thus, targeting necrotic cell death mechanisms will be critical for preventing microcystin‐induced liver injury. HighlightsMicrocystin induced liver injury causes significant necrosis and hemorrhage in mice.Caspase inhibition fails to protect against microcystin induced liver injury in mice.There is limited caspase activation in human hepatocytes exposed to microcystin‐LR.Caspase inhibitors do not protect against microcystin in human hepatocytes.Cell death via necrosis precedes cytoskeletal disruption.

Late-onset renal vein thrombosis: A case report and review of the literature

INTRODUCTION Renal vein thrombosis, a rare complication of renal transplantation, often causes graft loss. Diagnosis includes ultrasound with Doppler, and it is often treated with anticoagulation or mechanical thrombectomy. Success is improved with early diagnosis and institution of treatment. PRESENTATION OF CASE We report here the case of a 29 year-old female with sudden development of very late-onset renal vein thrombosis after simultaneous kidney pancreas transplant. This resolved initially with thrombectomy, stenting and anticoagulation, but thrombosis recurred, necessitating operative intervention. Intraoperatively the renal vein was discovered to be compressed by a large ovarian cyst. DISCUSSION Compression of the renal vein by a lymphocele or hematoma is a known cause of thrombosis, but this is the first documented case of compression and thrombosis due to an ovarian cyst. CONCLUSION Early detection and treatment of renal vein thrombosis is paramount to restoring renal allograft function. Any woman of childbearing age may have thrombosis due to compression by an ovarian cyst, and screening for this possibility may improve long-term graft function in this population.

Functional coupling of ATP binding cassette transporter Abcb6 to Cytochrome P450 expression and activity in liver

Background: Physiological signals that negatively regulate CYP450s are not well understood. Results: Loss of Abcb6 in mice results in suppression of CYP450 activity. Conclusion: Suppression of P450 activity in Abcb6 deficiency may result from altered endogenous metabolites involved in maintaining homeostasis. Significance: Understanding metabolite alterations in Abcb6 deficiency should help understand the physiological signals and the mechanisms involved in negative regulation of P450s. Although endogenous mechanisms that negatively regulate cytochrome P450 (P450) monooxygenases in response to physiological and pathophysiological signals are not well understood, they are thought to result from alterations in the level of endogenous metabolites, involved in maintaining homeostasis. Here we show that homeostatic changes in hepatic metabolite profile in Abcb6 (mitochondrial ATP-binding cassette transporter B6) deficiency results in suppression of a specific subset of hepatic P450 activity. Abcb6 null mice are more susceptible to pentobarbital-induced sleep and zoxazolamine-induced paralysis, secondary to decreased expression and activity of Cyp3a11 and Cyp2b10. The knock-out mice also show decrease in both basal and xeno-inducible expression and activity of a subset of hepatic P450s that appear to be related to changes in hepatic metabolite profile. These data, together with the observation that liver extracts from Abcb6-deficient mice suppress P450 expression in human primary hepatocytes, suggest that this mouse model may provide an opportunity to understand the physiological signals and the mechanisms involved in negative regulation of P450s.

Donor-specific antibodies present at the time of kidney transplantation in immunologically unmodified patients increase the risk of acute rejection

BACKGROUND Human leukocyte antigens (HLA) class II donor-specific antibodies (DSAs) are associated with microcirculation inflammation, transplant glomerulopathy and ultimately graft loss. There is however no data on allograft outcomes in deceased donor kidney transplant recipients who have not received any desensitization prior to transplantation. METHODS We prospectively evaluated the association of HLA DR and DQ DSAs on rejection and short-term graft survival in patients who did not receive desensitization prior to transplantation. On the basis of their cumulative strength of HLA DR and/or DQ DSA, the patients were dichotomized into: 1) median fluorescence intensity (MFI)<1000 and 2) MFI≥1000. RESULTS In the two year study period, 50 consecutive patients with HLA DR and/or DQ sensitization were transplanted in our two centers. Post-transplantation, the incidence of acute rejection was significantly greater in the MFI≥1000 group (35%; 8/22) compared to the MFI<1000 group (7%; 2/28) (p<0.001). There were two graft losses, both in the MFI≥1000 group. CONCLUSION The strength of DR and/or DQ DSA at the time of renal transplantation influences the risk of rejection in non-desensitized recipients with HLA class II DSA.