Pumpkin Center, NC, United States
Pumpkin Center, NC, United States

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Lakner A.M.,University of North Carolina at Charlotte | Lakner A.M.,Liver Biliary Pancreatic Center | Steuerwald N.M.,Liver Biliary Pancreatic Center | Walling T.L.,Carolinas Medical Center | And 11 more authors.
Hepatology | Year: 2012

Hepatic stellate cell (HSC) activation is a pivotal event in initiation and progression of hepatic fibrosis and a major contributor to collagen deposition driven by transforming growth factor beta (TGF-β). MicroRNAs (miRs), small noncoding RNAs modulating messenger RNA (mRNA) and protein expression, have emerged as key regulatory molecules in chronic liver disease. We investigated differentially expressed miRs in quiescent and activated HSCs to identify novel regulators of profibrotic TGF-β signaling. miR microarray analysis was performed on quiescent and activated rat HSCs. Members of the miR-17-92 cluster (19a, 19b, 92a) were significantly down-regulated in activated HSCs. Because miR 19b showed the highest fold-change of the cluster members, activated HSCs were transfected with miR 19b mimic or negative control and TGF-β signaling and HSC activation assessed. miR 19b expression was determined in fibrotic rat and human liver specimens. miR 19b mimic negatively regulated TGF-β signaling components demonstrated by decreased TGF-β receptor II (TGF-βRII) and SMAD3 expression. Computational prediction of miR 19b binding to the 3′ untranslated region of TGF-βRII was validated by luciferase reporter assay. Inhibition of TGF-β signaling by miR 19b was confirmed by decreased expression of type I collagen and by blocking TGF-β-induced expression of α1(I) and α2(I) procollagen mRNAs. miR 19b blunted the activated HSC phenotype by morphological assessment and decreased smooth muscle α-actin expression. Additionally, miR 19b expression was markedly diminished in fibrotic rat liver compared with normal liver; similarly, miR 19b expression was markedly down-regulated in fibrotic compared with normal human livers. Conclusion: miR 19b is a novel regulator of TGF-β signaling in HSCs, suggesting a potential therapeutic approach for hepatic fibrosis. © 2012 American Association for the Study of Liver Diseases.


Hwang S.-I.,Liver Biliary Pancreatic Center | Hwang S.-I.,Proteomics Laboratory for Clinical and Translational Research | Lee Y.-Y.,Proteomics Laboratory for Clinical and Translational Research | Park J.-O.,Proteomics Laboratory for Clinical and Translational Research | And 4 more authors.
Clinica Chimica Acta | Year: 2011

Background: The measurement of serum hepcidin, a peptide hormone that regulates iron metabolism, is clinically important to the understanding of iron homeostasis in health and disease. To date, the quantification of serum hepcidin levels by conventional immunological detection methods has proven problematic due to challenges in obtaining high quality antibodies which demonstrate good reproducibility. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF) has been employed recently for more sensitive quantification of hepcidin; however, this method has high background levels and therefore less than optimal specificity. Methods: In order to increase the specificity of the mass spectrometry based assay, we developed a robust, ultra-performance liquid-chromatography-tandem mass spectrometry (UPLC-MS/MS) protocol using multiple selected reaction monitoring (mSRM) for quantification of hepcidin levels in urine and serum of human subjects. With this assay, we assessed levels of hepcidin before and for up to 8. h after oral ingestion of ferrous sulfate in ten adult human subjects without known disease. Results: The linear response of hepcidin quantitation on each instrument was measured, and the correlation coefficients of these calibrations were r 2=0.9512±0.0202 (n=5) for urine and r 2=0.9709±0.0291 (n=5) for serum [r 2=mean±SD]. Compared to baseline, the levels of urinary hepcidin between 2-4h and 4-8h of both women and men showed significant increases with p<0.05 and p<0.001, respectively. The levels of serum hepcidin between 4h and 8h in both women and men showed significant increases, compared with baseline values, with both p<0.01. Interestingly, we also observed some degree of oscillation of levels, occurring at later time points. Conclusions: We have developed and validated a new method for measuring hepcidin concentrations in human serum and urine and used it to demonstrate early increases with iron supplement in both urinary and serum levels of hepcidin, which return to baseline levels, except in urine samples from men. © 2011 Elsevier B.V.


McKinney K.Q.,Proteomics Laboratory for Clinical and Translational Research | Lee Y.Y.,Proteomics Laboratory for Clinical and Translational Research | Choi H.S.,Proteomics Laboratory for Clinical and Translational Research | Groseclose G.,Liver Biliary Pancreatic Center | And 8 more authors.
Journal of Proteomics | Year: 2011

Pancreatic cancer (PC) is a highly aggressive disease that frequently remains undetected until it has progressed to an advanced, systemic stage. Successful treatment of PC is hindered by the lack of early detection. The application of proteomic analysis to PC combined with subcellular fractionation has introduced new possibilities in the field of biomarker discovery. We utilized matched pairs of pancreas tumor and non-tumor pancreas from patients undergoing tumor resection. The tissues were treated to obtain cellular protein fractions corresponding to cytosol, membrane, nucleus and cytoskeleton. The fractions were then separated by molecular weight and digested with trypsin, followed by liquid chromatography and tandem mass spectrometry. The spectra obtained were searched using Sequest engine and combined into a single analysis file to obtain a semi-quantitative number, spectral count, using Scaffold software. We identified 2393 unique proteins in non-tumor and cancer pancreas. Utilizing PLGEM statistical analysis we determined 104 proteins were significantly changed in cancer. From these, we further validated four secreted proteins that are up-regulated in cancer and have potential for development as minimally-invasive diagnostic markers. We conclude that subcellular fractionation followed by gel electrophoresis and tandem mass spectrometry is a powerful strategy for identification of differentially expressed proteins in pancreatic cancer. © 2010 Elsevier B.V.


O'Brien T.R.,U.S. National Cancer Institute | Pfeiffer R.M.,U.S. National Cancer Institute | Paquin A.,U.S. National Cancer Institute | Lang Kuhs K.A.,U.S. National Cancer Institute | And 10 more authors.
Journal of Hepatology | Year: 2015

Background & Aims Genetic polymorphisms within the interferon lambda (IFN-λ) region are strongly associated with hepatitis C virus (HCV) clearance; the IFNL4-ΔG/TT (rs368234815) polymorphism, which controls the generation of IFN-λ4 protein, is more strongly associated with HCV clearance than rs12979860 (the 'IL28B variant'). An IFNL3 3′ untranslated region polymorphism (rs4803217) has been proposed as a causal variant that may affect HCV clearance by altering IFNL3 mRNA stability. Methods We compared IFNL4-ΔG/TT and rs4803217 for association with response to pegylated-IFN-α/ribavirin in the VIRAHEP-C and HALT-C trials, and spontaneous HCV clearance in the ALIVE, UHS and WIHS studies. Genotyping was performed with TaqMan assays. We compared differences in mean reduction in HCV RNA levels by genotype and haplotype. For HCV clearance, we calculated p-values comparing c-statistics for IFNL4-ΔG/TT and rs4803217 genotypes by a bootstrap approach. Results Among European Americans, linkage disequilibrium between IFNL4-ΔG/TT and rs4803217 was strong (r2 = 0.89-0.99) and there were no significant differences between the variants. In African American (AA) individuals enrolled in VIRAHEP-C, HCV RNA at treatment day 28 was more strongly associated with IFNL4-ΔG/TT than rs4803217 (p = 0.003); the IFNL4-ΔG:rs4803217-G haplotype, which includes the putatively favorable IFNL3 allele, was actually associated with the poorest day 28 response (p = 0.03, comparison to IFNL4-ΔG:rs4803217-T haplotype). Among AA participants, associations were stronger for IFNL4-ΔG/TT than rs4803217 for undetectable HCV RNA at week 24 in Virahep-C (p = 0.03) and week 20 in HALT-C (p = 0.03), as well as for spontaneous HCV clearance (p = 0.048). Conclusion IFNL4-ΔG/TT is the primary IFN-λ region polymorphism for impaired HCV clearance. ©.


Steuerwald N.M.,Liver Biliary Pancreatic Center | Foureau D.M.,Liver Biliary Pancreatic Center | Norton H.J.,Liver Biliary Pancreatic Center | Zhou J.,Liver Biliary Pancreatic Center | And 15 more authors.
PLoS ONE | Year: 2013

Drug-induced liver injury (DILI) is the most common cause of acute liver failure in the United-States. The aim of the study was to describe serum immune profiles associated with acute DILI, to investigate whether there are profiles associated with clinical features or types of DILI and/or with prognosis, and to assess temporal changes in levels. Twenty-seven immune analytes were measured in the sera of 78 DILI subjects in the Drug-Induced Liver Injury Network (DILIN) and compared with 40 healthy controls. Immune analytes (14 cytokines, 7 chemokines and 6 growth factors) were measured by BioPlex multiplex ELISA at DILI onset and after 6 months. A modeling process utilizing immune principles was used to select a final set of variables among 27 immune analytes and several additional clinical lab values for prediction of early death (within 6 months of DILI onset). Nineteen of the 27 immune analytes were differentially expressed among healthy control, DILI onset and 6-month cohorts. Disparate patterns of immune responses, especially innate and adaptive cellular (mostly TH17) immunity were evident. Low values of four immune analytes (IL-9, IL-17, PDGF-bb and RANTES) and serum albumin are predictive of early death [PPV = 88% (95% CI, 65%-100%), NPV = 97% (95% CI, 93%-100%), accuracy = 96% (95% CI, 92%-100%)]. Conclusions: Acute DILI is associated with robust and varying immune responses. High levels of expression of cytokines associated with innate immunity are associated with a poor prognosis, whereas high levels of expression of adaptive cytokines are associated with good long-term prognosis and eventual recovery. Serum immune analyte profiles at DILI onset appear to be of prognostic, and perhaps, diagnostic significance. © 2013 Steuerwald et al.


Ghaziani T.,Beth Israel Deaconess Medical Center | Sendi H.,Carolinas HealthCare System | Shahraz S.,Brandeis University | Zamor P.,Carolinas HealthCare System | Bonkovsky H.L.,Liver Biliary Pancreatic Center
World Journal of Gastroenterology | Year: 2014

Hepatitis B virus (HBV) continues to be a major cause of morbidity and mortality worldwide. It is estimated that about 350 million people throughout the world are chronically infected with HBV. Some of these people will develop hepatic cirrhosis with decompensation and/or hepatocellular carcinoma. For such patients, liver transplantation may be the only hope for cure or real improvement in quality and quantity of life. Formerly, due to rapidity of recurrence of HBV infection after liver transplantation, usually rapidly progressive, liver transplantation was considered to be contraindicated. This changed dramatically following the demonstration that hepatitis B immune globulin (HBIG), could prevent recurrent HBV infection. HBIG has been the standard of care for the past two decades or so. Recently, with the advent of highly active inhibitors of the ribose nucleic acid polymerase of HBV (entecavir, tenofovir), there has been growing evidence that HBIG needs to be given for shorter lengths of time; indeed, it may no longer be necessary at all. In this review, we describe genetic variants of HBV and past, present, and future prophylaxis of HBV infection during and after liver transplantation. We have reviewed the extant medical literature on the subject of infection with the HBV, placing particular emphasis upon the prevention and treatment of recurrent HBV during and after liver transplantation. For the review, we searched PubMed for all papers on the subject of "hepatitis B virus AND liver transplantation". We describe some of the more clinically relevant and important genetic variations in the HBV. We also describe current practices at our medical centers, provide a summary and analysis of comparative costs for alternative strategies for prevention of recurrent HBV, and pose important still unanswered questions that are in need of answers during the next decade or two. We conclude that it is now rational and cost-effective to decrease and, perhaps, cease altogether, the routine use of HBIG during and following liver transplantation for HBV infection. Here we propose an individualized prophylaxis regimen, based on an integrated approach and risk-assessment. © 2014 Baishideng Publishing Group Inc. All rights reserved.


Lakner A.M.,University of North Carolina at Charlotte | Bonkovsky H.L.,University of North Carolina at Charlotte | Bonkovsky H.L.,Liver Biliary Pancreatic Center | Bonkovsky H.L.,University of North Carolina at Chapel Hill | And 3 more authors.
World Journal of Gastroenterology | Year: 2011

microRNAs (miRs) are small non-coding RNAs that regulate both mRNA and protein expression of target genes, which results in alterations in mRNA stability or translation inhibition. miRs influence at least one third of all human transcripts and are known regulators of various important cellular growth and differentiation factors. miRs have recently emerged as key regulatory molecules in chronic liver disease. This review details recent contributions to the field of miRs that influence liver development and the broad spectrum of disease, from non-alcoholic fatty liver disease to fibrosis/cirrhosis, with particular emphasis on hepatic stellate cells and potential use of miRs as therapeutic tools. © 2011 Baishideng.


Hou W.,Liver Biliary Pancreatic Center | Hou W.,University of North Carolina at Charlotte | Tian Q.,Liver Biliary Pancreatic Center | Zheng J.,Liver Biliary Pancreatic Center | And 5 more authors.
Hepatology | Year: 2010

Hepatitis C virus (HCV) directly induces oxidative stress and liver injury. Bach1, a basic leucine zipper mammalian transcriptional repressor, negatively regulates heme oxygenase 1 (HMOX1), a key cytoprotective enzyme that has antioxidant and anti-inflammatory activities. microRNAs (miRNAs) are small noncoding RNAs (≈22 nt) that are important regulators of gene expression. Whether and how miRNAs regulate Bach1 or HCV are largely unknown. The aims of this study were to determine whether miR-196 regulates Bach1, HMOX1, and/or HCV gene expression. HCV replicon cell lines (Con1 and 9-13) of the Con1 isolate and J6/JFH1-based HCV cell culture system were used in this study. The effects of miR-196 mimic on Bach1, HMOX1, and HCV RNA, and protein levels were measured by way of quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting, respectively. The Dual Glo Luciferase Assay System was used to determine reporter activities. miR-196 mimic significantly down-regulated Bach1 and up-regulated HMOX1 gene expression and inhibited HCV expression. Dual luciferase reporter assays demonstrated that transfection of miR-196 mimic resulted in a significant decrease in Bach1 3′-untranslated region (UTR)-dependent luciferase activity but not in mutant Bach1 3′-UTR - dependent luciferase activity. Moreover, there was no detectable effect of mutant miR-196 on Bach1 3′-UTR-dependent luciferase activity. Conclusion: miR-196 directly acts on the 3′-UTR of Bach1 messenger RNA and translationally represses the expression of this protein, and up-regulates HMOX1. miR-196 also inhibits HCV expression in HCV replicon cell lines (genotype 1b) and in J6/JFH1 (genotype 2a) HCV cell culture system. Thus, miR-196 plays a role in both HMOX1/Bach1 expression and the regulation of HCV expression in human hepatocytes. Overexpression of miR-196 holds promise as a potential novel strategy to prevent or ameliorate hepatitis C infection, and to protect against liver injury in chronic HCV infection. Copyright © 2009 by the American Association for the Study of Liver Diseases.


Tian Q.,Liver Biliary Pancreatic Center | Li T.,Liver Biliary Pancreatic Center | Hou W.,Liver Biliary Pancreatic Center | Zheng J.,Liver Biliary Pancreatic Center | And 6 more authors.
Journal of Biological Chemistry | Year: 2011

5-Aminolevulinic acid synthase (ALAS-1) is the first rate controlling enzyme that controls cellular heme biosynthesis. Negative feedback regulation of ALAS-1 by the end product heme is well documented and provides the foundation for heme treatment of acute porphyrias, a group of diseases caused by genetic defects in the heme biosynthesis pathway and exacerbated by controlled up-regulation of ALAS-1. Heme is known to affect ALAS-1 activity by repressing gene transcription, accelerating mRNA degradation, and impeding pre-ALAS-1 mitochondrial translocation. In the current study, we examined the effect of heme on the rate of mature ALAS-1 protein turnover in human cells and tissues and explored the mediator involved in this new regulatory mechanism. We found that heme and other metalloporphyrins such as CoPP and CrPP decreased mitochondrial ALAS-1 protein through proteolysis. This degradative effect cannot be emulated by iron or free protoporphyrin, two major chemical components of the heme ring, and is independent of oxidative stress. Down-regulating the activity of mitochondrial LONP1, an ATP-dependent protease that controls the selective turnover of mitochondrial matrix proteins, with potent inhibitors and specific siRNA diminished the negative effect of heme on mitochondrial ALAS-1. Therefore, our data support the existence of a conserved heme feedback regulatory mechanism that functions on the mature form of ALAS-1 protein through the activity of a mitochondrial proteolytic system. © 2011 by The American Society for Biochemistry and Molecular Biology, Inc.


Gong Y.,University of Manitoba | Wang G.,Liver Biliary Pancreatic Center | Yan J.,University of Manitoba | Chen Y.,University of Manitoba | Burczynski F.J.,University of Manitoba
BMC Gastroenterology | Year: 2014

Background: FABP1 has been reported to possess strong antioxidant properties. Upon successful transfection of the Chang cell line, which has undetectable FABP1 mRNA levels, with human FABP1 cDNA, the Chang cells were shown to express FABP1. Using the transfected and control (normal) Chang cells and subjecting them to oxidative stress, transfected cells were reported to be associated with enhanced cell viability. This study extends those observations by investigating the effect of FABP1 on acetaminophen (AAP)-induced hepatotoxicity. We hypothesized that presence of FABP1 would enhance cell viability compared to control cells (vector transfected cells).Methods: Following AAP treatment of Chang FABP1 transfected and control cells, cell viability, oxidative stress, and apoptosis were evaluated using lactate dehydrogenase (LDH) release, the fluorescent probe DCF, and Bax expression, respectively.Results: FABP1 cDNA transfected cells showed greater resistance against AAP toxicity than vector transfected cells. Significantly lower LDH levels (p < 0.05) were observed as were lower DCF fluorescence intensity (p < 0.05) in FABP1 cDNA transfected cells compared to vector transfected cells. FABP1 expression also attenuated the expression of Bax following AAP induced toxicity.Conclusion: FABP1 attenuated AAP-induced toxicity and may be considered a cytoprotective agent in this in vitro model of drug induced oxidative stress. © 2014 Gong et al.; licensee BioMed Central Ltd.

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