- Email: [email protected]
662 CHARACTERIZATION OF PEPTIDES AND PROTEINS CAPTURED BY THE MARS SYSTEM IN PATIENTS WITH CHOLESTASIS AND RESISTANT PRURITUS
06: AUTOIMMUNE AND CHRONIC CHOLESTATIC LIVER DISEASES was significantly higher in patients with advanced fibrosis stage at presentation (9.0±14.9 pack-years) compared to patients without (2.9±7.0, p < 0.01). A trend for an association between passive smoke exposure and advanced fibrosis stage at presentation was also observed (RR, 1.7; p = 0.08). In logistic regression, smoking history remained associated with advanced fibrosis stage (OR, 2.01; 95% CI, 1.19−3.41) after adjusting for age, gender, alcohol intake, BMI and diabetes mellitus. Each pack-year of increase in smoking history was associated with a 6% increased likelihood of advanced fibrosis stage at presentation. Conclusion: This is the second study suggesting a deleterious effect of tobacco exposure on the histological progression of PBC. Altogether, these two studies strongly indicate that PBC patients should be encouraged to stop smoking as soon as they are diagnosed. References [1] Zein et al. Hepatology, 2006.
661 PERFORMANCE OF SURROGATE MARKERS OF HEPATIC FIBROSIS IN PRIMARY BILIARY CIRRHOSIS F. Ferrara1 , D. Caroli1 , S. Antoniazzi1 , A. Variola1 , N. Cazzagon1 , V. Baldo2 , A. Floreani1 . 1 Dept of Surgical and Gastroenterological Sci, University of Padova, 2 Dept of Hygiene and Public Health, University of Padova, Padova, Italy E-mail: annarosa.fl[email protected] Background: Liver biopsy is considered the gold standard to define staging in PBC. Several limitations, however, continue to hamper the ability for monitoring disease progression. Several composite indices have been validated in chronic viral hepatitis, but remain understudied in PBC. Aim: To assess the diagnostic validity of 3 simple non invasive tests (APRI, FIB-4 and Fibroindex) in identifying fibrosis or cirrhosis in PBC. Methods: 248 patients with PBC (233 females and 15 males, mean age 52±11 yrs) were consecutively enrolled. Performance of each marker was compared to histological staging evaluated according to Scheuer’s classification at time of liver biopsy (stage I, n = 40; stage II, n = 79; stage III, n = 86; stage IV, n = 43) and was expressed by sensitivity, specificity, positive and negative predictive values (PPV, NPV), and area under the ROC curve (AUROC). Optimised cut-offs of non invasive markers were obtained through the AUROC analysis. Results: See the table. The 3 tests showed a high sensitivity but a low specificity in predicting stage IV disease. Presence of cirrhosis could be excluded with >93% of NPV by a specific cut-off of APRI index. None of the tests was able to predict presence of stage IV due to low PPV. APRI test revealed the best performance for identifying cirrhosis (AUROC=0.719). All tests failed to reveal a satisfactory performance in predicting III−IV staging.
APRI
FIB-4
Fibroindex
Stage
III−IV
IV
III−IV
IV
III−IV
IV
Cut-off Sensitivity (%) Specificity (%) PPV (%) NPV (%) AUROC
0.628 61 49 58 55 0.595
0.645 81 50 26 93 0.719
0.205 58 50 56 52 0.560
0.205 72 50 23 89 0.647
1.190 62 50 58 55 0.591
1.215 77 50 24 91 0.691
Conclusions: APRI identified cirrhosis in PBC with the highest degree of performance. Simple score systems using categorized values might be useful in clinical practice; further studies are warranted to establish if the combination of different serum tests could predict with better accuracy fibrosis or cirrhosis in PBC.
S243
662 CHARACTERIZATION OF PEPTIDES AND PROTEINS CAPTURED BY THE MARS SYSTEM IN PATIENTS WITH CHOLESTASIS AND RESISTANT PRURITUS M. Gay1 , M. Gorga1,2 , M. Carrascal1 , A. Mas2 , J. Abi´an1 , A. Par´es2 . 1 CSIC/UAB Proteomics Laboratory, HBB-CSIC, Autonomous University of Barcelona, Bellaterra, 2 Liver Unit, Digestive Diseases Institute, CIBEREHD, IDIBAPS, Hospital Cl´ınic, Barcelona, Spain E-mail: [email protected] Albumin dialysis using the Molecular Adsorbent Recirculating System (MARS) is a new therapeutic approach for liver diseases. Besides the clinical effects, the procedure has the potential to allow the analysis of the molecules removed by albumin dialysis, which may be involved in the pathophysiology on liver diseases. In this study we analyze peptides and proteins absorbed into the MARS strong anionic exchange (SAX) cartridges as a result of the MARS treatment. We have assessed by shotgun proteomic approach the peptides and proteins absorbed into the MARS strong anionic exchange (SAX) cartridge after treatment of patients with cholestasis and resistant pruritus. Since commercial albumin used for dialysis already contains a number of other residual proteins, a control sample was prepared recirculating 20% albumin through a MARS SAX cartridge for six hours. Extracts from control and patient samples were digested with two different enzymes (trypsin and GluC) and the resulting peptides were analyzed by multidimensional liquid chromatography coupled to tandem mass spectrometry (MDLC-MS/MS). Fragmentation spectra were searched using the SEQUEST engine against the Human UniProt database and its reversed copy. The identified proteins have been further validated by western blot. More than 2000 peptide sequences corresponding to 146 proteins were identified in these samples. This collection included 43 different proteins that were detected only in the patient-derived samples, being absent in the control human albumin extracts. The characterization of five of these proteins -Neutrophil defensin, Secreted Ly-6/uPAR-related protein 1, Serum amyloid A, Fibrinogen alpha chain and Pancreatic prohormone-, as proteins captured by the dialysis procedure, has been confirmed by Multiple Reaction Monitoring MS/MS. Neutrophil defensin and Secreted Ly-6/uPAR-related protein 1 have been detected and confirmed by western blot. In conclusion, in patients with cholestasis and resistant pruritus treated with albumin dialysis, we have identified a number of proteins that are removed from the patient’s blood and adsorbed in the SAX cartridges. Moreover, we have also documented that commercial human albumin contains peptides and proteins other than albumin. 663 C-MET IN HEPATOCYTES IS PROTECTIVE AGAINST CHOLESTATIC LIVER INJURY AND FIBROSIS PROGRESSION VIA ANTI-APOPTOTIC PATHWAYS IN MICE A. Giebeler1 , C. Klein2 , M. Boekschoten3 , M. Borowiak4 , C. Birchmeier5 , M. Mueller3 , C. Trautwein1 , K. Streetz1 . 1 Department of Medicine III, RWTH University Hospital, Aachen, 2 Department of Angiogenesis and Cardiovascular Pathology, Max Dellbrueck-Center, Berlin, Germany; 3 Department of Nutrition, Metabolism & Genomics, University Wageningen, Wageningen, The Netherlands; 4 Department of Molecular and Cellular Biology, Howard Hughes Medical Institute, Cambridge, USA; 5 Department of Developmental Biology/Signal Transduction, Max Dellbrueck-Center, Berlin, Germany E-mail: [email protected] Background: The HGF/c-Met system is an important inducer of hepatocyte growth and proliferation. Here we aimed to define the exact role of c-Met using a model of chronic cholestatic liver injury and an array based gene-expression analysis. Methods: Hepatocyte specific conditional c-Met knockout mice (cMetDhepa ) were generated and used for gene-array analysis and bile-ductligation (BDL) experiments.
661 PERFORMANCE OF SURROGATE MARKERS OF HEPATIC FIBROSIS IN PRIMARY BILIARY CIRRHOSIS F. Ferrara1 , D. Caroli1 , S. Antoniazzi1 , A. Variola1 , N. Cazzagon1 , V. Baldo2 , A. Floreani1 . 1 Dept of Surgical and Gastroenterological Sci, University of Padova, 2 Dept of Hygiene and Public Health, University of Padova, Padova, Italy E-mail: annarosa.fl[email protected] Background: Liver biopsy is considered the gold standard to define staging in PBC. Several limitations, however, continue to hamper the ability for monitoring disease progression. Several composite indices have been validated in chronic viral hepatitis, but remain understudied in PBC. Aim: To assess the diagnostic validity of 3 simple non invasive tests (APRI, FIB-4 and Fibroindex) in identifying fibrosis or cirrhosis in PBC. Methods: 248 patients with PBC (233 females and 15 males, mean age 52±11 yrs) were consecutively enrolled. Performance of each marker was compared to histological staging evaluated according to Scheuer’s classification at time of liver biopsy (stage I, n = 40; stage II, n = 79; stage III, n = 86; stage IV, n = 43) and was expressed by sensitivity, specificity, positive and negative predictive values (PPV, NPV), and area under the ROC curve (AUROC). Optimised cut-offs of non invasive markers were obtained through the AUROC analysis. Results: See the table. The 3 tests showed a high sensitivity but a low specificity in predicting stage IV disease. Presence of cirrhosis could be excluded with >93% of NPV by a specific cut-off of APRI index. None of the tests was able to predict presence of stage IV due to low PPV. APRI test revealed the best performance for identifying cirrhosis (AUROC=0.719). All tests failed to reveal a satisfactory performance in predicting III−IV staging.
APRI
FIB-4
Fibroindex
Stage
III−IV
IV
III−IV
IV
III−IV
IV
Cut-off Sensitivity (%) Specificity (%) PPV (%) NPV (%) AUROC
0.628 61 49 58 55 0.595
0.645 81 50 26 93 0.719
0.205 58 50 56 52 0.560
0.205 72 50 23 89 0.647
1.190 62 50 58 55 0.591
1.215 77 50 24 91 0.691
Conclusions: APRI identified cirrhosis in PBC with the highest degree of performance. Simple score systems using categorized values might be useful in clinical practice; further studies are warranted to establish if the combination of different serum tests could predict with better accuracy fibrosis or cirrhosis in PBC.
S243
662 CHARACTERIZATION OF PEPTIDES AND PROTEINS CAPTURED BY THE MARS SYSTEM IN PATIENTS WITH CHOLESTASIS AND RESISTANT PRURITUS M. Gay1 , M. Gorga1,2 , M. Carrascal1 , A. Mas2 , J. Abi´an1 , A. Par´es2 . 1 CSIC/UAB Proteomics Laboratory, HBB-CSIC, Autonomous University of Barcelona, Bellaterra, 2 Liver Unit, Digestive Diseases Institute, CIBEREHD, IDIBAPS, Hospital Cl´ınic, Barcelona, Spain E-mail: [email protected] Albumin dialysis using the Molecular Adsorbent Recirculating System (MARS) is a new therapeutic approach for liver diseases. Besides the clinical effects, the procedure has the potential to allow the analysis of the molecules removed by albumin dialysis, which may be involved in the pathophysiology on liver diseases. In this study we analyze peptides and proteins absorbed into the MARS strong anionic exchange (SAX) cartridges as a result of the MARS treatment. We have assessed by shotgun proteomic approach the peptides and proteins absorbed into the MARS strong anionic exchange (SAX) cartridge after treatment of patients with cholestasis and resistant pruritus. Since commercial albumin used for dialysis already contains a number of other residual proteins, a control sample was prepared recirculating 20% albumin through a MARS SAX cartridge for six hours. Extracts from control and patient samples were digested with two different enzymes (trypsin and GluC) and the resulting peptides were analyzed by multidimensional liquid chromatography coupled to tandem mass spectrometry (MDLC-MS/MS). Fragmentation spectra were searched using the SEQUEST engine against the Human UniProt database and its reversed copy. The identified proteins have been further validated by western blot. More than 2000 peptide sequences corresponding to 146 proteins were identified in these samples. This collection included 43 different proteins that were detected only in the patient-derived samples, being absent in the control human albumin extracts. The characterization of five of these proteins -Neutrophil defensin, Secreted Ly-6/uPAR-related protein 1, Serum amyloid A, Fibrinogen alpha chain and Pancreatic prohormone-, as proteins captured by the dialysis procedure, has been confirmed by Multiple Reaction Monitoring MS/MS. Neutrophil defensin and Secreted Ly-6/uPAR-related protein 1 have been detected and confirmed by western blot. In conclusion, in patients with cholestasis and resistant pruritus treated with albumin dialysis, we have identified a number of proteins that are removed from the patient’s blood and adsorbed in the SAX cartridges. Moreover, we have also documented that commercial human albumin contains peptides and proteins other than albumin. 663 C-MET IN HEPATOCYTES IS PROTECTIVE AGAINST CHOLESTATIC LIVER INJURY AND FIBROSIS PROGRESSION VIA ANTI-APOPTOTIC PATHWAYS IN MICE A. Giebeler1 , C. Klein2 , M. Boekschoten3 , M. Borowiak4 , C. Birchmeier5 , M. Mueller3 , C. Trautwein1 , K. Streetz1 . 1 Department of Medicine III, RWTH University Hospital, Aachen, 2 Department of Angiogenesis and Cardiovascular Pathology, Max Dellbrueck-Center, Berlin, Germany; 3 Department of Nutrition, Metabolism & Genomics, University Wageningen, Wageningen, The Netherlands; 4 Department of Molecular and Cellular Biology, Howard Hughes Medical Institute, Cambridge, USA; 5 Department of Developmental Biology/Signal Transduction, Max Dellbrueck-Center, Berlin, Germany E-mail: [email protected] Background: The HGF/c-Met system is an important inducer of hepatocyte growth and proliferation. Here we aimed to define the exact role of c-Met using a model of chronic cholestatic liver injury and an array based gene-expression analysis. Methods: Hepatocyte specific conditional c-Met knockout mice (cMetDhepa ) were generated and used for gene-array analysis and bile-ductligation (BDL) experiments.