- Email: [email protected]
Human Liver Progenitor Cell Expansion With Preservation of Differentiation Capability Into Functional Hepatocytes
Tu1686
purpura, his platelets improved to 59,000 within 12 days. His platelet count has remained stable since, off of any therapy Discussion: Although a rare occurrence, both Hepatitis B vaccination and infection have been implicated in the acute development of ITP. To our knowledge less than 10 cases of ITP associated with the hepatitis B vaccine have been reported in the literature, the majority of those in pediatric patients. ITP associated with the hepatitis B vaccine is usually self limiting. Disease duration of one to three months has been previously published. Platelet levels below 10,000 should be treated appropriately with steroids and IVIG to prevent spontaneous bleeding complications. Other vaccines have been found to be associated with the development of ITP including the Measles, Mumps, Rubella (MMR) vaccine, small pox, and diphtheria, tetanus, pertuss (DTP). Although the pathophysiology is not well understood, vaccine induction of antibody formation is likely responsible for this rare complication. Conclusion: ITP secondary to the hepatitis B vaccine occurs infrequently. As in our case, severe thrombocytopenia can ensue requiring hospitalization and prompt treatment. Recognizing and reporting this rare life threatening complication is imperative.
AASLD Abstracts
Liver-Specific mRNAs Are Present in Cell-Free Plasma in Man and May Serve as Novel Biomarkers for Drug-Induced Liver Injury Farzad Nowrouzzadeh, Barbara Wetmore, Paul H. Hayashi, Russell S. Thomas, Paul B. Watkins There is compelling need for improved biomarkers for diagnosis and management of druginduced liver injury (DILI). We have recently shown in rats that during injury by APAP or galactosamine, the liver sheds microvesicles containing liver-specific mRNAs that can be readily detected in cell-free plasma (Hepatology 52:2127,2010). Moreover, microarray analyses of these vesicles reveal marked differences in the transcript profiles from the two forms of DILI. If microvesicles are shed from the liver in humans during DILI, transcriptome analysis of plasma may aid in establishing the diagnosis and in identifying the culprit drug in patients with polypharmacy. The purpose of this study was to determine whether liverderived mRNAs are detectable and elevated in cell-free plasma obtained from patients experiencing DILI. Methods: We enrolled twelve patients hospitalized at a single tertiary facility for DILI and concern for fulminant liver failure. Entry criteria included suspected clinical diagnosis of DILI; age between 18-80, ALT exceeding 200 IU/L and a Hgb level ≥11 g/dL at enrollment. Blood samples were obtained daily until discharge or ALT <200 IU/L. Control blood samples were collected daily from healthy donors. Cell-free plasma was isolated from blood by serial centrifugation. Microvesicles were then isolated from plasma following serial centrifugation at 100,000xg. RNA was isolated from the microvesicles using the Qiagen RNeasy Micro kit and reverse transcribed by the High Capacity cDNA RT kit. The resulting cDNA was analyzed by quantitative real-time (qRT)-PCR targeting albumin, haptoglobin,and fibrinogen B β-polypeptide (Fgb) mRNAs. Results: Twelve hospitalized patients diagnosed with DILI were enrolled in the study for an average of 3 days. All patients recovered, and none required liver transplant. Except for one, all patients stopped the offending drug at least 48 hours before enrollment. Seven patients were enrolled with either intentional or accidental APAP toxicity. Liver-specific mRNAs (albumin, haploglobin and Fgb) were readily detectable in circulating plasma in both healthy individuals and patients diagnosed with DILI. Further, the levels of these mRNAs were increased ~10-40 fold in the DILI patients over levels detected in the healthy controls, displaying fold-increases that were comparable to, if not greater than, the fold increases observed in ALT and AST levels. Discussion: To our knowledge, this is the first description of liver-derived mRNAs in cellfree plasma obtained from humans. As in rodents, the liver mRNAs were detectable at low levels in healthy controls, suggesting physiological release from the liver of microvesicles. The marked rise in circulating levels of the mRNAs during DILI supports a new avenue for biomarker discovery. Future work will include analyzing the full transcriptome in these samples.
Tu1689 Human Liver Progenitor Cell Expansion With Preservation of Differentiation Capability Into Functional Hepatocytes Dustin T. Boyd, Melissa R. Miller, Woodring E. Wright, Jay D. Horton, Jerry W. Shay, Andres I. Roig A persisting problem in long-term human hepatocyte culture is the dedifferentiation of epithelial cells to a mesenchymal phenotype and progressive loss of hepatocytic functional markers. Thus, it has been extremely challenging to use long-term proliferating normal human hepatocytes as models to investigate normal and pathological processes. Here we describe a novel method to expand hepatoblast progenitors while preserving the capability of cells to differentiate into mature hepatocytes. Human fetal livers (13 to 16 week-old) were processed via standard procedures to isolate liver progenitor cells. When in primary culture on collagen 1 coated plates, cells maintained in serum-free medium supplemented with epithelial growth factor, insulin, holo-transferrin, dexamethasone, nicotinamide, bovine serum albumin, 2-mercaptoethanol, free fatty acids, copper sulfate, and selenious acid (referred to as basal medium) transition to a fibroblastic morphology and lose epithelial features within 7 days. Addition of the glycogen synthase kinase 3 beta inhibitor BIO (6bromoindirubin-3'-oxime) and bone morphogenic protein antagonist noggin to the basal medium promotes expansion of tight colonies containing blast-like cells (small cytoplasm, large nuclei, and prominent nucleoli), and prevents mesenchymal dedifferentiation. Addition of the weak TNF-alpha agonist, tweak, and the notch agonist, jagged 1, significantly increases cellular expansion and extends the viability of blast-like cell colonies for up to one month, respectively. In this growth factor defined media the cells are able to tolerate passage when trypsinized for 15 minutes at 4 degrees Celsius. After passage, cellular division can be sustained by adding hepatocyte growth factor (HGF) to cells in basal medium or growth factor defined medium. Although cells in these conditions express the hepatocyte progenitor cell marker cytokeratin 19, with time the cells dedifferentiate to fibroblasts, exhibit increased vimentin expression, and lack hepatocyte functional markers such as albumin. Continuous maintenance of cells in the growth factor defined medium supplemented with HGF and oncostatin M promotes expansion and transitions the cells to a mature hepatocyte morphology consisting of increased cytoplasm, prominent mitochondrial structures, and binucleated cells. These cells have significantly decreased cytokeratin 19 and vimentin, exhibit cytokeratin 18 (mature hepatocyte marker), produce albumin, and display PAS positive staining (representing glycogen synthesis) that can be depleted with glucagon administration. These initial findings may lead to the development of a new method to maintain human hepatocytes in long-term culture that retain differentiation capability.
Tu1687 Drug Induced Liver Injury in Inpatients During the Period 2004-2009 in a Single Center Enrique De la Fuente-Fernandez, Maria Trapero-Marugan, Ricardo Moreno-Otero Background & aims. Drug induced liver injury (DILI) is a frequent cause of liver test abnormalities. Almost any drugs may cause DILI. It is actually the most frequent reason for withdrawal of medications from the marketplace. The aim was to evaluate and to analyze the clinical characteristics and outcome in patients with DILI. Material & Methods. Patient records were identified by ICD-9 code 573.3 for inpatient during the period 2004-2009. Patients with others diagnosis (but 573.3 code) and those that did not meet the criteria for DILI, were excluded. Demographic and related to DILI episode variables were registered. Causality scales were used for the diagnosis. Results. From a total of 91.197 inpatients during the study period, 67 were for DILI (0.07%). Mean age was 58 years. 58.8% were male. The most of them were attended at the Gastroenterology department (49.3%). 40.1% had a previous liver pathology (HCV 13.4%, HBV 3%, alcoholic liver damage 13.4%, other causes 10.4%). 41 drugs were deemed to be related to DILI. Related with DILI, both, the antiinfective group was the more frequently involved, with 32.8%, and amoxicilin-clavunate the most frequent drug. 34.3% had extrahepatic manifestations. In 7.5% of cases hepatic biopsy was performed for the diagnosis. 9% were received steroids. 1.5% was derived to a reference center for liver transplantation. 10.4% of patients died. The patterns were 55.4% hepatocellular, 27% cholestasic, 18% mixed. Elevated bilirrubin on admission was significantly associated with death (p=0,027) Conclusions. DILI is a frequent cause of liver test abnormalities that is associated with a significant mortality. Antimicrobial agents accounted the largest proportion group. Increased bilirrubin on admission is associated with worst outcome.
Tu1690 Characterization of Mallory-Denk Bodies in a Mouse Model of Erythropoietic Protoporphyria Amika Singla, David Moons, Natasha T. Snider, Elizabeth R. Wagenmaker, Bishr Omary Background and Aims: Mallory-Denk bodies (MDBs) are hepatocyte inclusions most commonly seen in patients with steatohepatitis. MDBs consist primarily of keratins 8 and 18 (K8/K18), supplemented with ubiquitin (Ub), the polyubiquitin binding protein p62 and chaperones. K8 crosslinking by transglutaminase 2 (TG2) and K8/K18 overexpression (K8>K18) are essential for MDB formation. MDBs can be induced in mice by chronic feeding with porphyrinogenic compounds [3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) is the most commonly used]. In humans, porphyria does not induce MDBs, as evidenced by MDB absence in patients with erythropoietic protoporphyria (EPP). EPP is caused by mutations in ferrochelatase (fch) which converts protoporphyrin and iron to heme. Depending on the fch mutation, patients with EPP can develop significant liver disease which is phenocopied in the Fechm1Pas mutant (Met98Lys) mice (fch/fch) that have an inactivating fch mutation. fch/fch mice develop spontaneous MDBs but the molecular factors involved in their formation and how they relate to DDC-induced MDBs are not known. We tested the hypothesis that fch mutation creates a molecular milieu that mimics drug-induced mouse MDBs. Methods: MDB presence in male and female 13 and 20 week (wk) old wild-type (wt/wt), heterozygous (wt/fch) and homozygous (fch/fch) mice was determined histologically by hematoxylin & eosin (HE) staining. Keratin crosslinking and biochemical evidence of MDB formation was determined by blotting using antibodies to keratins, p62, ubiquitin and TG2. K8/K18 mRNA was quantified using qPCR. Results: Serologic evaluation showed increased levels of alkaline phosphatase, alanine aminotransferase and bile acids in 13 and 20 wk old fch/fch mice compared to wt/wt and wt/fch mice. In 13 wk old mice, MDBs were not evident histologically although biochemical assessment demonstrated alterations conducive to MDB formation, including increased TG2, K8 and K18. In 20 wk old fch/fch mice, MDBs were detected histologically in association with significant increases in K8, K18, TG2, Ub and p62 proteins, K8-ubiquitin crosslinks and K8>K18 mRNA. Also, a short course (3 wk) of DDC feeding (in contrast to the standard 12 wk) markedly induced MDB formation in male and female fch/fch and wt/fch mice compared to untreated fch/fch mice as evidenced by increased K8, K18 and Ub levels, increased K8 crosslinking, and increased number of MDBs quantified
Tu1688 Hepatitis B Vaccination Induced Idiopathic Thrombocytopenic Purpura Kimberley Ackerman, Dan C. Cohen, Richard A. Erickson Objective: 1. To present a case of Hepatitis B vaccine induced idiopathic thrombocytopenic purpura (ITP). 2. To discuss the prevalence and treatment of hepatitis B vaccine induced ITP. Background: A 69 year old male with a history of mild cirrhosis presumed to be from non-alcoholic steatohepatitis was admitted after routine bloodwork revealed a platelet count of 13,000. He had a history of chronic thrombocytopenia presumed to be secondary to his cirrhosis and mild splenomegaly; however, two weeks prior to admission, his platelet count was 92,000 and his lowest previous platelet count recorded was 53,000. After admission, the patient's platelets continued to drop despite platelet transfusions reaching a nadir of 7,000. Initial hematologic consultation suggested his thrombocytopenia was still due to his cirrhosis and splenic sequestration. Gastroenterology was asked to see him to for further recommendations. On careful review, it was noted that the patient had received the hepatitis B and A vaccine 2 weeks prior to admission (EXACT BRAND OF VACCINES) as routine care for his chronic liver disease. Immune thrombocytopenia purpura secondary to the hepatitis B vaccine was suspected. The patient was started on prednisone and intravenous immunoglobulin (IVIG) therapy. Confirming the diagnosis of immune thrombocytopenic
AASLD Abstracts
S-976
purpura, his platelets improved to 59,000 within 12 days. His platelet count has remained stable since, off of any therapy Discussion: Although a rare occurrence, both Hepatitis B vaccination and infection have been implicated in the acute development of ITP. To our knowledge less than 10 cases of ITP associated with the hepatitis B vaccine have been reported in the literature, the majority of those in pediatric patients. ITP associated with the hepatitis B vaccine is usually self limiting. Disease duration of one to three months has been previously published. Platelet levels below 10,000 should be treated appropriately with steroids and IVIG to prevent spontaneous bleeding complications. Other vaccines have been found to be associated with the development of ITP including the Measles, Mumps, Rubella (MMR) vaccine, small pox, and diphtheria, tetanus, pertuss (DTP). Although the pathophysiology is not well understood, vaccine induction of antibody formation is likely responsible for this rare complication. Conclusion: ITP secondary to the hepatitis B vaccine occurs infrequently. As in our case, severe thrombocytopenia can ensue requiring hospitalization and prompt treatment. Recognizing and reporting this rare life threatening complication is imperative.
AASLD Abstracts
Liver-Specific mRNAs Are Present in Cell-Free Plasma in Man and May Serve as Novel Biomarkers for Drug-Induced Liver Injury Farzad Nowrouzzadeh, Barbara Wetmore, Paul H. Hayashi, Russell S. Thomas, Paul B. Watkins There is compelling need for improved biomarkers for diagnosis and management of druginduced liver injury (DILI). We have recently shown in rats that during injury by APAP or galactosamine, the liver sheds microvesicles containing liver-specific mRNAs that can be readily detected in cell-free plasma (Hepatology 52:2127,2010). Moreover, microarray analyses of these vesicles reveal marked differences in the transcript profiles from the two forms of DILI. If microvesicles are shed from the liver in humans during DILI, transcriptome analysis of plasma may aid in establishing the diagnosis and in identifying the culprit drug in patients with polypharmacy. The purpose of this study was to determine whether liverderived mRNAs are detectable and elevated in cell-free plasma obtained from patients experiencing DILI. Methods: We enrolled twelve patients hospitalized at a single tertiary facility for DILI and concern for fulminant liver failure. Entry criteria included suspected clinical diagnosis of DILI; age between 18-80, ALT exceeding 200 IU/L and a Hgb level ≥11 g/dL at enrollment. Blood samples were obtained daily until discharge or ALT <200 IU/L. Control blood samples were collected daily from healthy donors. Cell-free plasma was isolated from blood by serial centrifugation. Microvesicles were then isolated from plasma following serial centrifugation at 100,000xg. RNA was isolated from the microvesicles using the Qiagen RNeasy Micro kit and reverse transcribed by the High Capacity cDNA RT kit. The resulting cDNA was analyzed by quantitative real-time (qRT)-PCR targeting albumin, haptoglobin,and fibrinogen B β-polypeptide (Fgb) mRNAs. Results: Twelve hospitalized patients diagnosed with DILI were enrolled in the study for an average of 3 days. All patients recovered, and none required liver transplant. Except for one, all patients stopped the offending drug at least 48 hours before enrollment. Seven patients were enrolled with either intentional or accidental APAP toxicity. Liver-specific mRNAs (albumin, haploglobin and Fgb) were readily detectable in circulating plasma in both healthy individuals and patients diagnosed with DILI. Further, the levels of these mRNAs were increased ~10-40 fold in the DILI patients over levels detected in the healthy controls, displaying fold-increases that were comparable to, if not greater than, the fold increases observed in ALT and AST levels. Discussion: To our knowledge, this is the first description of liver-derived mRNAs in cellfree plasma obtained from humans. As in rodents, the liver mRNAs were detectable at low levels in healthy controls, suggesting physiological release from the liver of microvesicles. The marked rise in circulating levels of the mRNAs during DILI supports a new avenue for biomarker discovery. Future work will include analyzing the full transcriptome in these samples.
Tu1689 Human Liver Progenitor Cell Expansion With Preservation of Differentiation Capability Into Functional Hepatocytes Dustin T. Boyd, Melissa R. Miller, Woodring E. Wright, Jay D. Horton, Jerry W. Shay, Andres I. Roig A persisting problem in long-term human hepatocyte culture is the dedifferentiation of epithelial cells to a mesenchymal phenotype and progressive loss of hepatocytic functional markers. Thus, it has been extremely challenging to use long-term proliferating normal human hepatocytes as models to investigate normal and pathological processes. Here we describe a novel method to expand hepatoblast progenitors while preserving the capability of cells to differentiate into mature hepatocytes. Human fetal livers (13 to 16 week-old) were processed via standard procedures to isolate liver progenitor cells. When in primary culture on collagen 1 coated plates, cells maintained in serum-free medium supplemented with epithelial growth factor, insulin, holo-transferrin, dexamethasone, nicotinamide, bovine serum albumin, 2-mercaptoethanol, free fatty acids, copper sulfate, and selenious acid (referred to as basal medium) transition to a fibroblastic morphology and lose epithelial features within 7 days. Addition of the glycogen synthase kinase 3 beta inhibitor BIO (6bromoindirubin-3'-oxime) and bone morphogenic protein antagonist noggin to the basal medium promotes expansion of tight colonies containing blast-like cells (small cytoplasm, large nuclei, and prominent nucleoli), and prevents mesenchymal dedifferentiation. Addition of the weak TNF-alpha agonist, tweak, and the notch agonist, jagged 1, significantly increases cellular expansion and extends the viability of blast-like cell colonies for up to one month, respectively. In this growth factor defined media the cells are able to tolerate passage when trypsinized for 15 minutes at 4 degrees Celsius. After passage, cellular division can be sustained by adding hepatocyte growth factor (HGF) to cells in basal medium or growth factor defined medium. Although cells in these conditions express the hepatocyte progenitor cell marker cytokeratin 19, with time the cells dedifferentiate to fibroblasts, exhibit increased vimentin expression, and lack hepatocyte functional markers such as albumin. Continuous maintenance of cells in the growth factor defined medium supplemented with HGF and oncostatin M promotes expansion and transitions the cells to a mature hepatocyte morphology consisting of increased cytoplasm, prominent mitochondrial structures, and binucleated cells. These cells have significantly decreased cytokeratin 19 and vimentin, exhibit cytokeratin 18 (mature hepatocyte marker), produce albumin, and display PAS positive staining (representing glycogen synthesis) that can be depleted with glucagon administration. These initial findings may lead to the development of a new method to maintain human hepatocytes in long-term culture that retain differentiation capability.
Tu1687 Drug Induced Liver Injury in Inpatients During the Period 2004-2009 in a Single Center Enrique De la Fuente-Fernandez, Maria Trapero-Marugan, Ricardo Moreno-Otero Background & aims. Drug induced liver injury (DILI) is a frequent cause of liver test abnormalities. Almost any drugs may cause DILI. It is actually the most frequent reason for withdrawal of medications from the marketplace. The aim was to evaluate and to analyze the clinical characteristics and outcome in patients with DILI. Material & Methods. Patient records were identified by ICD-9 code 573.3 for inpatient during the period 2004-2009. Patients with others diagnosis (but 573.3 code) and those that did not meet the criteria for DILI, were excluded. Demographic and related to DILI episode variables were registered. Causality scales were used for the diagnosis. Results. From a total of 91.197 inpatients during the study period, 67 were for DILI (0.07%). Mean age was 58 years. 58.8% were male. The most of them were attended at the Gastroenterology department (49.3%). 40.1% had a previous liver pathology (HCV 13.4%, HBV 3%, alcoholic liver damage 13.4%, other causes 10.4%). 41 drugs were deemed to be related to DILI. Related with DILI, both, the antiinfective group was the more frequently involved, with 32.8%, and amoxicilin-clavunate the most frequent drug. 34.3% had extrahepatic manifestations. In 7.5% of cases hepatic biopsy was performed for the diagnosis. 9% were received steroids. 1.5% was derived to a reference center for liver transplantation. 10.4% of patients died. The patterns were 55.4% hepatocellular, 27% cholestasic, 18% mixed. Elevated bilirrubin on admission was significantly associated with death (p=0,027) Conclusions. DILI is a frequent cause of liver test abnormalities that is associated with a significant mortality. Antimicrobial agents accounted the largest proportion group. Increased bilirrubin on admission is associated with worst outcome.
Tu1690 Characterization of Mallory-Denk Bodies in a Mouse Model of Erythropoietic Protoporphyria Amika Singla, David Moons, Natasha T. Snider, Elizabeth R. Wagenmaker, Bishr Omary Background and Aims: Mallory-Denk bodies (MDBs) are hepatocyte inclusions most commonly seen in patients with steatohepatitis. MDBs consist primarily of keratins 8 and 18 (K8/K18), supplemented with ubiquitin (Ub), the polyubiquitin binding protein p62 and chaperones. K8 crosslinking by transglutaminase 2 (TG2) and K8/K18 overexpression (K8>K18) are essential for MDB formation. MDBs can be induced in mice by chronic feeding with porphyrinogenic compounds [3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) is the most commonly used]. In humans, porphyria does not induce MDBs, as evidenced by MDB absence in patients with erythropoietic protoporphyria (EPP). EPP is caused by mutations in ferrochelatase (fch) which converts protoporphyrin and iron to heme. Depending on the fch mutation, patients with EPP can develop significant liver disease which is phenocopied in the Fechm1Pas mutant (Met98Lys) mice (fch/fch) that have an inactivating fch mutation. fch/fch mice develop spontaneous MDBs but the molecular factors involved in their formation and how they relate to DDC-induced MDBs are not known. We tested the hypothesis that fch mutation creates a molecular milieu that mimics drug-induced mouse MDBs. Methods: MDB presence in male and female 13 and 20 week (wk) old wild-type (wt/wt), heterozygous (wt/fch) and homozygous (fch/fch) mice was determined histologically by hematoxylin & eosin (HE) staining. Keratin crosslinking and biochemical evidence of MDB formation was determined by blotting using antibodies to keratins, p62, ubiquitin and TG2. K8/K18 mRNA was quantified using qPCR. Results: Serologic evaluation showed increased levels of alkaline phosphatase, alanine aminotransferase and bile acids in 13 and 20 wk old fch/fch mice compared to wt/wt and wt/fch mice. In 13 wk old mice, MDBs were not evident histologically although biochemical assessment demonstrated alterations conducive to MDB formation, including increased TG2, K8 and K18. In 20 wk old fch/fch mice, MDBs were detected histologically in association with significant increases in K8, K18, TG2, Ub and p62 proteins, K8-ubiquitin crosslinks and K8>K18 mRNA. Also, a short course (3 wk) of DDC feeding (in contrast to the standard 12 wk) markedly induced MDB formation in male and female fch/fch and wt/fch mice compared to untreated fch/fch mice as evidenced by increased K8, K18 and Ub levels, increased K8 crosslinking, and increased number of MDBs quantified
Tu1688 Hepatitis B Vaccination Induced Idiopathic Thrombocytopenic Purpura Kimberley Ackerman, Dan C. Cohen, Richard A. Erickson Objective: 1. To present a case of Hepatitis B vaccine induced idiopathic thrombocytopenic purpura (ITP). 2. To discuss the prevalence and treatment of hepatitis B vaccine induced ITP. Background: A 69 year old male with a history of mild cirrhosis presumed to be from non-alcoholic steatohepatitis was admitted after routine bloodwork revealed a platelet count of 13,000. He had a history of chronic thrombocytopenia presumed to be secondary to his cirrhosis and mild splenomegaly; however, two weeks prior to admission, his platelet count was 92,000 and his lowest previous platelet count recorded was 53,000. After admission, the patient's platelets continued to drop despite platelet transfusions reaching a nadir of 7,000. Initial hematologic consultation suggested his thrombocytopenia was still due to his cirrhosis and splenic sequestration. Gastroenterology was asked to see him to for further recommendations. On careful review, it was noted that the patient had received the hepatitis B and A vaccine 2 weeks prior to admission (EXACT BRAND OF VACCINES) as routine care for his chronic liver disease. Immune thrombocytopenia purpura secondary to the hepatitis B vaccine was suspected. The patient was started on prednisone and intravenous immunoglobulin (IVIG) therapy. Confirming the diagnosis of immune thrombocytopenic
AASLD Abstracts
S-976