Caspase inhibition by ZVAD-fmk reduces apoptotic death of cryopreserved porcine hepatocytes

GASTROENTEROLOGY Vol. 118, No.4

AI008 AASLD ABSTRACTS

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CASPASE INHIBITION BY ZVAD·FMK REDUCES APOPTOTIC DEATH OF CRYOPRESERVED PORCINE HEPATOCYTES. Scott L. Nyberg, Toshikazu Yagi, Joseph A. Hardin, Gregory 1. Gores, Mayo Clin, Rochester, MN.

HYPERLIPIDEMIC STATE AND CARDIOVASCULAR RISK IN COURSE OF PRIMARY BILIARY CIRRHOSIS. Matteo Longo, Andrea Crosignani, Pier Maria Battezzati, Pietro Invernizzi, Cristina Squarcia, Cristina Pipia, Livio Colombo, Alessandro Colombo, Sabina Dosio, Francesca Perego, Francesca Meda, Mauro Podda, Div of Internal Medicine, Sch of Medicine San Paolo, Milano, Italy. Chronic cholestatic liver diseases, such as primary biliary cirrhosis (PBC), are characterized by elevation of serum lipids levels. Hyperlipidemia is an estabilished risk factor for cardiovascular diseases (CVD). To asses if hyperlipidemia is associated with higher rate of cardiovascular (CV) events in PBC, we studied 402 PBC patients (mean follow-up of 62:±:53 months) with serial lipid level determinations and monitoring of cardiovascular events. Cardiovascular events in PBC were compared with data taken from a large longitudinal study involving about 400.000 Italian people. used to calculate the number of expected CV events. In addition. patients were stratified in three groups based on cholesterol levels and survival free of cardiovascular events was evaluated. The risk of CVD associated with cholesterol levels was adjusted for the presence of other, well known CV risk factors. At entry, a significant trend toward higher total cholesterol (p
Introduction: Cryopreserved porcine hepatocytes are used in bioartificial liver device. However, death of cryopreserved hepatocytes by apoptosis limits the effectiveness of these devices. Because apoptosis is mediated by caspase proteases, we determined if a capase inhibitor, ZVAD-fmk, improved the viability of porcine hepatocytes following cryopreservation. Methods: Freshly harvested hepatocytes were resuspended at 5xlO 6 cellsfmL in 1 mL of William's E medium supplemented with 10% fetal calf serum and 10% DMSO. Suspensions also received 60 1J.M (treated) ZVAD-fmk (Z-Valine-Alanine-Aspartate [OMe]-CH 2F) prior to controlled rate freezing and cryopreservation. Cryovials underwent rapid thawing on day 14; hepatocytes were entrapped in type I collagen gel. Gels were cultured in media for 24 hrs. Cell viability was quantified by vital staining with fluorescein diacetate:ethidium bromide [FDA:EB]) at t=O hr and 24 hr after gel entrapment. Apoptosis was quantified by the TUNEL assay and verified by electron microscopy. Significance was determined by paired t test: *p<0.05, **p
2407 Timepoinl Postlhaw ohrin gel 8hrin gel 24hrin gel

%Viability oIlMZVAD 60IlMZVAD 82±5'10 57±10% 44±11'10 39+6%

89±5'10 75±5'10" 64±8'10" 62±4'10"

%Apoplosis OIlMZVAD 60gMZVAD 14±5'10 25±11'10 30±10%

<5%' <5%*-

12±6'10'

2405 DIRECT EVIDENCE OF DE NOVO TISSUE ENGINEERED LIVER IN RATS TRANSPLANTED WITH A COLLAGEN·BASED POLYPROPYLENE COMPOSITE SCAFFOLD. Yukinobu Takimoto, Vivek Dixit, Marika H. Arthur. Gary Gitnick, UCLA Sch of Medicine, Los Angeles, CA. Tissue engineering, a multidisciplinary technology, can facilitate the creation of new tissue for organ replacement therapy. However, creation of de novo liver tissue using such methodologies has previously not been very successful. We present here the first evidence of de novo formation of liver tissue, as shown by the presence of putative liver stem cells, within a novel collagen-based polypropylene (C-PP) composite biomaterial, developed in our laboratory specifically for liver tissue engineering. Aim: To create de novo liver tissue in vivo. Methods: C-PP composite implants were formed in a cone-shaped configuration by injecting and freeze-drying a modified collagen sponge into a 500 1J.m polypropylene mesh. The collagen solution was agitated into a foam-like sponge, placed into the PP scaffold and frozen overnight at -20°C. The scaffolds were freeze-dried for 24 hours. Following a small mid-line laparotomy, the C-PP or control (empty) PP scaffolds were implanted into the liver by stabbing and securing them to the medial lobe. A total of 21 animals were divided into control and C-PP groups and sacrificed at 7, 14, and 21 days post implantation. PP scaffolds were retrieved from the animals and examined by H&E and immunohistochemistry: desmin, alphafetoprotein (AFP) and OV-6 (monoclonal antibody) stains. Results: Control animals showed PP scaffolds that appeared hollow and were encapsulated within a tight connective tissue capsule. In contrast, the C-PP scaffolds were not encapsulated as above, but showed a loose proliferative connective tissue infiltration within the C-PP scaffolds. H&E staining revealed extensive neovascularization and a loose network of fibroblast-like cells inside the C-PP scaffolds. Desmin stain revealed an extensive network of hepatic stellate, or Ito cells, inside the C-PP scaffolds. The putative oval cells that can differentiate into hepatocytes or billiary epithelial cells were detected by OV-6 stain after 14 and 21 days. AFP staining was also observed inside the C-PP scaffolds. Conclusions: (1) This is the first reported observation of the early stages of de novo liver tissue formation. (2) The C-PP composite scaffold is capable of supporting the development of de novo liver tissue growth in vivo. (3) Tissue engineered scaffolds can play an important role in studying the developmental biology of organogenesis in the adult animal.

PROSPECTIVE STUDY OF LIVER DYSFUNCTION IN PREG· NANCY IN SOUTH WALES, UK. Chin Lye Ch'ng, Jeremy G. Kingham, Margery Morgan, Singleton Hosp, Swansea, United Kingdom. BACKGROUND AND AIMS: Liver disease is uncommon in pregnancy but has serious consequences. Its frequency varies according to country and ethnic origin and is not documented in Wales. We have prospectively determined incidence, causes and outcome of liver dysfunction in pregnancy in an obstetric unit in South Wales, UK. METHODS: A central laboratory identified all abnormal liver tests from patients in ante-natal clinics and wards of an obstetric unit serving a population of 270,000. During the seven month study there were 2,150 deliveries. Abnormal liver tests were defined as bilirubin > 251J.mollL, aspartate transaminase (AST) > 40UIL or gamma GT > 35UIL. Patients were studied prospectively on receipt of blood results and medical advice was provided to obstetricians. Clinical course of mother and fetusfinfant was recorded. RESULTS: 63 patients had abnormal liver tests: elevation of AsT in 54 (range 41 - 4123), gamma GT in 23 (range 36 - 278) and bilirubin in 4 (range 26-155). Thrombocytopaenia was seen in 19 (range 23 - 148 x 1091L) and hyperuricaemia in 15 (range 0.41 - 0.63 p,mollL). Hepatobiliary ultrasound scan (USS) was abnormal in 5. Liver biopsy was not justifiable in any patient. Causes of liver dysfunction were determined by symptoms. clinical circumstances, laboratory tests and USS. There were 75 diagnoses amongst 63 patients: pre-eclampsia 35, intrahepatic cholestasis of pregnancy (lCP) 9, HELLP syndrome 9, sepsis 6, post Caesarian section 4, hyperemesis gravidarum 3, bile duct stones 2, post-partum haemorrhage 2, drug toxicity 2, acute fatty liver I, hepatic haematoma I and chronic hepatitis C 1. Elevation of AsT was the predominant abnormality in ICP. There were no maternal deaths but there was one intrauterine death at 30 weeks associated with pre-eclampsia. 20 patients required delivery by induction or emergency Caesarian section to improve hepatic function. II babies required admission to special care unit. CONCLUSIONS: Liver dysfunction was seen in 63 of2150 pregnancies (frequency I in 34) during a seven-month prospective study giving an annual incidence of 293 per 10,000 pregnancies. Transient but sometimes serious effects on maternal and infant morbidity were observed but there were no maternal deaths and only one stillbirth.

2408 URSODEOXYCHOLIC ACID IN THE TREATMENT OF INTRA· HEPATIC CHOLESTASIS OF PREGNANCY. A to·YEAR EXPE· RIENCE ON ITS EFFICACY, SAFETY AND THE PERINATAL OUTCOME. Rodrigo Zapata, Lorena Sandoval. Joaquin Palma, Jose Ribalta, Ismael Hernandez, Humberto Reyes, Univ of Chile Sch of Medicine. Santiago, Chile. Background: Intrahepatic cholestasis of pregnancy (ICP) is characterized by skin pruritus (SP) and biochemical cholestasis. The consequences are premature deliveries and perinatal morbimortality. Since our first report, in 1990, several case reports have coincided in that ursodeoxycholic acid (UDCA) ameliorates SP and improves serum liver tests, without adverse effects. The results on perinatal outcome (PO) are still unclear. Aim: To