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Effects of cold preservation and rewarming on isolated rat hepatocyte amino acid transport systems A, ASC and N
April 1995
@ VARIANT CD44 ADHESION MOLECULES ARE EXPRESSED IN C H O L A N G I O C E L L U L A R C A R C I N O M A S BUT NOT IN HEPATOCELLULAR CARCINOMAS. C. Sergi*, G. Otto**, K.-H, Heider***, H.F. Otto*, W.J. Hofinana* Dept,s. of *Pathology & **Surgery, University of Heidelberg, Germany, ***Bender+Co, Vienna, Austria
Aims: Expression of the CD44 hyahironen receptor/lymph node endothelial receptor has been linked to tumor growth and metastases in both human and rodent cancers. CD44 can bind to exlraeellular malrix molecules such as fibronectin and hyaluronate and it could confer metastatic capability to tumor cells. In the present study, the expression of the CD44 adhesion molecule in the hepatecellular carcinoma (HCC) and in the chnlangiocellularcareinoma(CCC) was assayed. Met_hpds: Detection of CD44 adhesion molecule and its variants was carried out using an immnnoperoxidasetechnique on frozen sections from surgical specimens of 20 HCC and 12 CCC. A monoclonal antibody against the standard form of the CD44 (SFF-2 or CD44s) and four monoelonal antibodies against the splice variants or isoforms of the CD44 (VFF-8 or v5, VFF-14 or vl0, VFF-17 or v7+8, VFF-18 or v6) were used. The proportion of tumor cells positivel3) stained and the localization and intensity of any staining were recorded. To calculate the Significance of the observed differences the Fisher's exact test was utilized. Results: Eleven out of twelve cases (91,7%) of CCC expressed CD44 variants whereas standard CD44 was found in only four cases (33.3%). Expression of v5 was present in nine CCC, whilst v7+8 and v6 were expressed in three and two tumors, respectively. Eight of the twelve CCC (67%) had tumour cells positive for vl0. Only two out of twenty eases of HCC contained variants of CD44 but no standard CD44. The observed differences between the standard form of CD44 and ist variants in the two hepatic tamours studied were significant for CD44v5 (9 < 0.00001), CD44v10 (9 < 0.001), and CD44s (p < 0.05). Non significant were the differences observed for CD44v7+8 end CD44v6. Conclusions: These results indicate that expression of CD44 may play a role in CCC, but it may eventually be of any value in the assessment of prognosis of HCC. hi particularly, the expression of CD44 variants may regulate the binding of tamour cells to hyalurnnan in the poricellular or extracellular matrix and influence the biology of CCC.
AASLD
Al167
EFFECTS OF COLD PRESERVATION AND REWARMING ON ISOLATED RAT HEPATOCYTE AMINO ACID TRANSPORT SYSTEMS A, ASC AND N. H. Serra[, J.F. Landr6 and P. Haddad, Groupe de Recherche en Transport Membranaire, Universitd de Montrtal, Montr6al, Qu6bec, CANADA. Primary dysfunction or non-function of liver transplants accounts for 15% of graft failure and can be imputable to injury caused by cold preservation, which is thought to involve disturbances in liver cell volume and pH. We have recently shown that volume regulatory mechanisms of hepatocytes are significantly attenuated after 48 and 72 hours of cold preservation in University of Wisconsin (U.W.) solution (Hepatol. 20:193A, t994). Recovery from both swollen (RVD) and shrunken (RVI) states are signicanfly diminished. The aim of the present work was to study the impact of cold preservation and rewarming on isolated rat hepatocyte amino acid transport systems A, ASC and N. We have used a model of isolated rat hepatocytes, kept at 4°C in U.W. solution for up to 72 hrs and subsequently cultured at 37°C for 1-2 hrs. Coverslips with adhered cells were placed in a perfusion chamber on an inverted microscope, ccaminoisobutyric acid (AIB), proline and glutamine were used as specific substrates for sodium-dependent transport systems A, ASC and N respectively. Cells were subjected to a 10 rain administration of 10 mM of each amino acid. Accumulation Of mnino acids is known to be reflected by changes in cell volume which we measured by digital analysis of single cell images obtained in bright-field illumination. This approach was validated by the sodium dependency of amino acid-induced cell swelling and by the hormonal stimulation (glucagon 100 nM) of AIB transport (10 raM) (swelling rate increasing from 0.45 to 1.58 pL/min, p<0.005, n=8). We have tbund that A, ASC and N transport systems were differently affected by the period of cold preservation. Proline transport (system ASC) decreased significantly from 0.160~0.044 to 0.080_+0.014 pL/min within 24 hrs and stayed depressed thereafter (0.060!-_0.011 and 0.040+0.027 pL/min at 48 and 72 hrs, n=7, paired ANOVA, p<0.05). In contrast, glutamine and A1B transport were not significantly affected by prolonged cold storage. The results suggest that part of the injury caused to liver grafts by cold preservation and rewarming can be related to the harmful changes of volume induced by amino acids when circulation is re-established in the host and especially when parenteral nutrition is used after transplantation. Supported by the Canadian Liver Foundation and the Canadian MRC.
@ HEPATIC TISSUE ENGINEERING IN ANALOGY TO THE IN V I V O L I V E R 1K.-Fr. Sewing. 1N. Friihauf, 2E. Knop, 1K. Bork, 3G. Schuhmann, 1A. Bader lInstitut fiir Allgemeine Pharmakologie, 2Abt. fiir Elektronenmikroskopie und Zellbiologie, 3Abteilung for Klinische Chemie, Medizinische Hochschule Hannover, Germany All current hollow fiber, microcarrier, or fiat bed devices provide an architecture which does not wholly respect the in vivo liver organization. Cellspecific function, such as albumin secretion, ureagenesis and cytochrome P450 activity in these devices are low and/or unstable. Methods: A novel bioreactor was designed by reconstructing cell plates and capillary structures on top and underneath such plates (bilaminar membranes, BLM-bioreactor). Monolayer hepatocytes were enclosed within two layers of collagen. To assess the 02 requirements of hepatocytes in such a plate configuration, incubations in an atmosphere with 20% 02 and/or 10% 02 on a gas-permeable membrane were performed (n= 180). Superoxide dismutase expression was analysed by Northern blots. Transmission electron microspcopy was used to assess the morphology of the hepatocytes. Albumin secretion was analysed by ELISA. Bioreactor runs were consecutively performed at 20% 02 for the attachment phase and 10% for the following two weeks. Besides albumin secretion, urea formation, GLDH and AST release were assessed by enzymatic measurements. On line biotransformation of the antihypertensive drug urapidil was measured by HPLC analysis. Hydroxylated metabolites were identified by mass spectrometry. Results: Hepatocytes seeded at 20% 02 on gas-permeable membranes and cultured at 10% thereafter maintained albumin secretion at 5-6 ~g/h/106 cells. GLDH and AST leakage decreased with time from 16.2 + 5 to 3.9~0.7 pmol/min/x 1/106 Cells and from 31.8 + 3 to 13.9 + 4 pmol/min/x 1/106 cells respectively. Persistence of higher 02 tensions resulted in a depressed albumin secretion and cell death by two weeks as shown by TEM. Superoxidedismutase was induced initially. In the bioreactor albumin production was to 5-8 pg/cell/h. Ureagenesis was stable at 4-5 ng/cell/h and oxidative biotransformation of urapidil was 0.4 pg/cell/h and increasing with time. Conclusion: BLM-bioreactors reconstruct extracellular matrix geometry and sinusoidal analogues in between cell plates. They provide a geometry closer to the microenvironment of the in vivo liver than standard devices. Functional performance of BLM-bioreactors is stable. Depending upon the duration of culture cell-specific activity is up to 10 *--1000x higher for albumin secretion, oxidative biotransformation and ureagenesis if compared to other previously reported devices. In contrast to all other bioreactor designs 02 supply within the d e v i c e s close to uniform for each hepatocyte. 02 supply can be set to values corresponding to the mixture of arterial and venous blood in the in vivo liver. Supported by BMFT Biotechnologie 2000
REASONS FOR EXCLUSION OF PATIENTS IN TREATMENT OF NANB HEPATITIS. I . SHAFRAN, M . N . Apter, D.J. Sprung. Dept of Medicine, Florida Hospital, Orlando, FL The purpose of our i n v e s t i g a t i o n was to determine the cause f o r excluding patients in an interferon t r i a l f o r NANB/C h e p a t i t i s . Seventy patients were considered f o r study in a four year span and 52 w e r e excluded f o r a v a r i e t y of reasons. Eighteen patients were treated with a total response rate of 44%. A t o t a l of 52 patients h a v e been excluded f o r a v a r i e t y of reasons: (1}. Chronic p e r s i s t e n t h e p a t i t i s in 12 (17%); (2). Cirrhosis or decompensated l i v e r disease in 10 (14%); (3). Refusal or declining of therapy or l o s t to follow-up in 20 (28%); (4). Coexistent l i v e r disease in 8.6% or (5). Other diagnoses and/or malignancies in 7%. While most studies h a v e been published by academic i n s t i t u t i o n s , t h i s is a study of p r i v a t e practice patients in a four year span, looking at reasons f o r exclusion. O n l y 25% of our t o t a l p a t i e n t population was considered f o r treatment because of these exclusion reasons. Recent studies have shown a more favorable response to i n t e r f e r o n in those with low t i t e r s to h e p a t i t i s C virus (RNA assay) 1 , without much active histology and/or biochemical abnormalities. We believe t h a t including patients with chronic persistent hepatitis in future studies might improve the outcome r e s u l t s at the end of a six month t r i a l in such p a t i e n t s . This subset of patients would be expected to have a higher response rate and more favorable outcome. 1.
Hagiwara H, Hayashi N, Mita E, et a l . Q u a n t i t a t i v e analysis of h e p a t i t i s C virus in serum during i n t e r f e r o n alpha therapy. Gastroenterology 1993;104:877-83.
@ VARIANT CD44 ADHESION MOLECULES ARE EXPRESSED IN C H O L A N G I O C E L L U L A R C A R C I N O M A S BUT NOT IN HEPATOCELLULAR CARCINOMAS. C. Sergi*, G. Otto**, K.-H, Heider***, H.F. Otto*, W.J. Hofinana* Dept,s. of *Pathology & **Surgery, University of Heidelberg, Germany, ***Bender+Co, Vienna, Austria
Aims: Expression of the CD44 hyahironen receptor/lymph node endothelial receptor has been linked to tumor growth and metastases in both human and rodent cancers. CD44 can bind to exlraeellular malrix molecules such as fibronectin and hyaluronate and it could confer metastatic capability to tumor cells. In the present study, the expression of the CD44 adhesion molecule in the hepatecellular carcinoma (HCC) and in the chnlangiocellularcareinoma(CCC) was assayed. Met_hpds: Detection of CD44 adhesion molecule and its variants was carried out using an immnnoperoxidasetechnique on frozen sections from surgical specimens of 20 HCC and 12 CCC. A monoclonal antibody against the standard form of the CD44 (SFF-2 or CD44s) and four monoelonal antibodies against the splice variants or isoforms of the CD44 (VFF-8 or v5, VFF-14 or vl0, VFF-17 or v7+8, VFF-18 or v6) were used. The proportion of tumor cells positivel3) stained and the localization and intensity of any staining were recorded. To calculate the Significance of the observed differences the Fisher's exact test was utilized. Results: Eleven out of twelve cases (91,7%) of CCC expressed CD44 variants whereas standard CD44 was found in only four cases (33.3%). Expression of v5 was present in nine CCC, whilst v7+8 and v6 were expressed in three and two tumors, respectively. Eight of the twelve CCC (67%) had tumour cells positive for vl0. Only two out of twenty eases of HCC contained variants of CD44 but no standard CD44. The observed differences between the standard form of CD44 and ist variants in the two hepatic tamours studied were significant for CD44v5 (9 < 0.00001), CD44v10 (9 < 0.001), and CD44s (p < 0.05). Non significant were the differences observed for CD44v7+8 end CD44v6. Conclusions: These results indicate that expression of CD44 may play a role in CCC, but it may eventually be of any value in the assessment of prognosis of HCC. hi particularly, the expression of CD44 variants may regulate the binding of tamour cells to hyalurnnan in the poricellular or extracellular matrix and influence the biology of CCC.
AASLD
Al167
EFFECTS OF COLD PRESERVATION AND REWARMING ON ISOLATED RAT HEPATOCYTE AMINO ACID TRANSPORT SYSTEMS A, ASC AND N. H. Serra[, J.F. Landr6 and P. Haddad, Groupe de Recherche en Transport Membranaire, Universitd de Montrtal, Montr6al, Qu6bec, CANADA. Primary dysfunction or non-function of liver transplants accounts for 15% of graft failure and can be imputable to injury caused by cold preservation, which is thought to involve disturbances in liver cell volume and pH. We have recently shown that volume regulatory mechanisms of hepatocytes are significantly attenuated after 48 and 72 hours of cold preservation in University of Wisconsin (U.W.) solution (Hepatol. 20:193A, t994). Recovery from both swollen (RVD) and shrunken (RVI) states are signicanfly diminished. The aim of the present work was to study the impact of cold preservation and rewarming on isolated rat hepatocyte amino acid transport systems A, ASC and N. We have used a model of isolated rat hepatocytes, kept at 4°C in U.W. solution for up to 72 hrs and subsequently cultured at 37°C for 1-2 hrs. Coverslips with adhered cells were placed in a perfusion chamber on an inverted microscope, ccaminoisobutyric acid (AIB), proline and glutamine were used as specific substrates for sodium-dependent transport systems A, ASC and N respectively. Cells were subjected to a 10 rain administration of 10 mM of each amino acid. Accumulation Of mnino acids is known to be reflected by changes in cell volume which we measured by digital analysis of single cell images obtained in bright-field illumination. This approach was validated by the sodium dependency of amino acid-induced cell swelling and by the hormonal stimulation (glucagon 100 nM) of AIB transport (10 raM) (swelling rate increasing from 0.45 to 1.58 pL/min, p<0.005, n=8). We have tbund that A, ASC and N transport systems were differently affected by the period of cold preservation. Proline transport (system ASC) decreased significantly from 0.160~0.044 to 0.080_+0.014 pL/min within 24 hrs and stayed depressed thereafter (0.060!-_0.011 and 0.040+0.027 pL/min at 48 and 72 hrs, n=7, paired ANOVA, p<0.05). In contrast, glutamine and A1B transport were not significantly affected by prolonged cold storage. The results suggest that part of the injury caused to liver grafts by cold preservation and rewarming can be related to the harmful changes of volume induced by amino acids when circulation is re-established in the host and especially when parenteral nutrition is used after transplantation. Supported by the Canadian Liver Foundation and the Canadian MRC.
@ HEPATIC TISSUE ENGINEERING IN ANALOGY TO THE IN V I V O L I V E R 1K.-Fr. Sewing. 1N. Friihauf, 2E. Knop, 1K. Bork, 3G. Schuhmann, 1A. Bader lInstitut fiir Allgemeine Pharmakologie, 2Abt. fiir Elektronenmikroskopie und Zellbiologie, 3Abteilung for Klinische Chemie, Medizinische Hochschule Hannover, Germany All current hollow fiber, microcarrier, or fiat bed devices provide an architecture which does not wholly respect the in vivo liver organization. Cellspecific function, such as albumin secretion, ureagenesis and cytochrome P450 activity in these devices are low and/or unstable. Methods: A novel bioreactor was designed by reconstructing cell plates and capillary structures on top and underneath such plates (bilaminar membranes, BLM-bioreactor). Monolayer hepatocytes were enclosed within two layers of collagen. To assess the 02 requirements of hepatocytes in such a plate configuration, incubations in an atmosphere with 20% 02 and/or 10% 02 on a gas-permeable membrane were performed (n= 180). Superoxide dismutase expression was analysed by Northern blots. Transmission electron microspcopy was used to assess the morphology of the hepatocytes. Albumin secretion was analysed by ELISA. Bioreactor runs were consecutively performed at 20% 02 for the attachment phase and 10% for the following two weeks. Besides albumin secretion, urea formation, GLDH and AST release were assessed by enzymatic measurements. On line biotransformation of the antihypertensive drug urapidil was measured by HPLC analysis. Hydroxylated metabolites were identified by mass spectrometry. Results: Hepatocytes seeded at 20% 02 on gas-permeable membranes and cultured at 10% thereafter maintained albumin secretion at 5-6 ~g/h/106 cells. GLDH and AST leakage decreased with time from 16.2 + 5 to 3.9~0.7 pmol/min/x 1/106 Cells and from 31.8 + 3 to 13.9 + 4 pmol/min/x 1/106 cells respectively. Persistence of higher 02 tensions resulted in a depressed albumin secretion and cell death by two weeks as shown by TEM. Superoxidedismutase was induced initially. In the bioreactor albumin production was to 5-8 pg/cell/h. Ureagenesis was stable at 4-5 ng/cell/h and oxidative biotransformation of urapidil was 0.4 pg/cell/h and increasing with time. Conclusion: BLM-bioreactors reconstruct extracellular matrix geometry and sinusoidal analogues in between cell plates. They provide a geometry closer to the microenvironment of the in vivo liver than standard devices. Functional performance of BLM-bioreactors is stable. Depending upon the duration of culture cell-specific activity is up to 10 *--1000x higher for albumin secretion, oxidative biotransformation and ureagenesis if compared to other previously reported devices. In contrast to all other bioreactor designs 02 supply within the d e v i c e s close to uniform for each hepatocyte. 02 supply can be set to values corresponding to the mixture of arterial and venous blood in the in vivo liver. Supported by BMFT Biotechnologie 2000
REASONS FOR EXCLUSION OF PATIENTS IN TREATMENT OF NANB HEPATITIS. I . SHAFRAN, M . N . Apter, D.J. Sprung. Dept of Medicine, Florida Hospital, Orlando, FL The purpose of our i n v e s t i g a t i o n was to determine the cause f o r excluding patients in an interferon t r i a l f o r NANB/C h e p a t i t i s . Seventy patients were considered f o r study in a four year span and 52 w e r e excluded f o r a v a r i e t y of reasons. Eighteen patients were treated with a total response rate of 44%. A t o t a l of 52 patients h a v e been excluded f o r a v a r i e t y of reasons: (1}. Chronic p e r s i s t e n t h e p a t i t i s in 12 (17%); (2). Cirrhosis or decompensated l i v e r disease in 10 (14%); (3). Refusal or declining of therapy or l o s t to follow-up in 20 (28%); (4). Coexistent l i v e r disease in 8.6% or (5). Other diagnoses and/or malignancies in 7%. While most studies h a v e been published by academic i n s t i t u t i o n s , t h i s is a study of p r i v a t e practice patients in a four year span, looking at reasons f o r exclusion. O n l y 25% of our t o t a l p a t i e n t population was considered f o r treatment because of these exclusion reasons. Recent studies have shown a more favorable response to i n t e r f e r o n in those with low t i t e r s to h e p a t i t i s C virus (RNA assay) 1 , without much active histology and/or biochemical abnormalities. We believe t h a t including patients with chronic persistent hepatitis in future studies might improve the outcome r e s u l t s at the end of a six month t r i a l in such p a t i e n t s . This subset of patients would be expected to have a higher response rate and more favorable outcome. 1.
Hagiwara H, Hayashi N, Mita E, et a l . Q u a n t i t a t i v e analysis of h e p a t i t i s C virus in serum during i n t e r f e r o n alpha therapy. Gastroenterology 1993;104:877-83.