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Hepatocyte cell density regulates a minimal human and rat epidermal growth factor transcriptional regulatory region
April 1995
SUCCESSFUL ENDOSCOPIC THERAPY OF BLEEDING GASTRIC VARICES WITH ENBUCRILATE (HISTOACRYL) P Mosca, I Lorenzini, U Freddara, G Novelli, T Cilluffo, MT Candelaresi, E Peruzzi, F Tarsetti, R Galeazzi.
Divisione di Gastroentero/ogia, Ospeda/e Umberto /, Ancona, Italy. Endoscopic therapy of bleeding gastric vadces with sclerosants has been flawed by the high complication rate. The efficacy of cyanoacrylate glue has been already reported, but concerns have been raised about its safety for patients and endoscopes as well. We have treated with enbucrilate injection 11 pts (6 men, mean age 66.6 Ys, ranging from 40 to 82) who had bleeding gastric varices secondary to liver cirrhosis (n=10, Child-Pugh's B=6, C=4) or portal thrombosis (n=l)• Isolated gastric varices were present in 6 pts, 2 of which had their esophageal varices previously eradicated by sclerotherapy. Endoscopic sclerotherapy was subsequently offered to the remaining 5 pts with concomitant esophageal varices. Nine pts were treated within 48 Hs from the index bleeding episode. Enbucrilate was diluted 1:1 with lipiodol and injected intravariceally in an amount sufficient to completely fill and harden the varix. A single treatment was required in 6 pts, 2 or 3 treatments were needed in the remaining 5 pts; a median volume of 3 ml was injected with a range from 2 to 6 ml. No pt died after a median follow-up of 9 months (range 3 to 17). An early rebieeding, requiring transfusions, was observed in one pt before complete obliteration of the vadces was achieved (i.e. between the 2nd and 3rd treatment); 2 further pts rebled after 11 and 12 months from a relapsing fundal varix and from G-E junction varices respectively; both have been successfully retreated with enbucrilate. Ulcerations at the site of partial enbucrilate expulsion were regularly observed after the treatment, the glue was eventually extruded between 2 and 4 months after the injection with disappearance of the varix; these ulcers were symptomless and never bled. No distant embolization of the glue or other side effects nor any damage to the scopes were observed. These preliminary results show that glue injection is an effective therapeutic option for gastric varices. It may be the best treatment in pts with advanced age Or stage of liver disease and should be compared to other treatment modalities in RCTs.
• HEPATOCYTE CELL DENSITY REGULATES A MINIMAL HUMAN A N D R A T E P I D E R M A L G R O W T H F A C T O R TRANSCRIPTIONAL REGULATORY REGION: B. Mullhauvt. E. Fodor, A. Feren, A. Jones: Cell Biology, VA Medical Center and Dept. of Medicine, and Liver Center, University of California, San Francisco, CA 94143 Epidermal growth factor (EGF) is a complete and potent mitogen for hepatocyte cell replication in vivo and in vitro. Recently we have shown that EGF transcripts encoding the mature EGF peptide are constitutively expressed in rodent liver and up-regulated over 10-fold in the immediate early period (15 minutes) following 70% hepatcctomy. To identify elements controlling EGF gene transcription, we have isolated and sequenced over 1.5 kB of human and rat chromosomal DNA 5' to EGF Exon I. Alignment reveals an extraordinarily high degree of similarity exceeding 90% in the first 600 bp upstream of the transcriptional initiation site identified by primer extension analysis. Sequence analysis reveals a conserved TITAAA element at -30, an HLH binding site at -70 overlapping an AP 1 motif, and at -110 a high affinity HNF 3 binding site. To identify a minimal EGF transcriptional regulatory unit functional in hepatocytes, we have subcloned large regions of the human and rat sequence into the luciferase vector pGL2 and tested activity of successive E~<0 III deletions after efficient transient transfection to hepatocyte mono-layer cultures. Our, results show that a -250 bp fragment is sufficient to drive high levels of luciferase production and coincides with DNA regions exhibiting maximal sequence similarity. To determine if cell proliferation influences EGF transcription, we have carried similar analysis on hepatocytes plated and maintained at different densities. Our results show a full length and minimal human and rat EGF transcriptional control ~equence are similarly negatively regulated at high cell densities that exhibit low proliferation evidenced by a 92% reduction in DNA synthesis measured as 3HThymidine incorporation (Table, below). LacZ expression from coWansfccted pCMVb-Gal did not correlate with cell density: :Cell~i~ 3H~Thymidin~ H~ EGF5!~U~ Rat EGF5'~Luc
prol~ se results il .s a major determining factor in the relative levels of EGF gene expression. Since EGF will rapidly induce hepatocyte replication, our data point to the likelihood that EGF transcriptional activity may be autoregulated in hepatic tissue.
AASLD Al127
@REGULATION OF THE NA+fl-AUROCHOLATE COTRANSPORT POLYPEPTIDE (ntcp) BY CYCLIC AMP: ROLE FOR TRANSLOCATION AND PHOSPHORYLATION. S. M u k h o p a d h a y a y , M. Ananthanarayanan, B. Stieger, P.J. Meier, F.J. Suchy, MS. Anwer. Dept. of Veterinary Medicine, "i'ufts University, N. Grafton, MA, Dept. of Pediatrics, Yale University School of Medicine, New Haven, CT, Divn. of Clinical Pharmacology and Toxicology, University Hospital, Z~rich, Switzerland Cyclic AMP (cAMP) increases the maximal rate of Na+fTaurocholate (Na+fTC) cotransport within 15 min. This rapid effect is mediated via protein kinase A and potentiated by Ca++/ ealmodulin kinase (JBC 268:17734,1993). Whether this effect of cAMP was mediated via translocation and/or phosphorylation of the ntcp was studied in the present study using two different antibodies (from ZOrich and New Haven). Results obtained with both antibodies were similar. Studies were conducted in hepatocytes and plasma membranes isolated from cAMP (and/or okadaie acid)-treated hepatocytes, cAMP increased Na*/TC uptake by 70% and increased by 50% the amount of ntcp in the plasma membrane. Okadaic acid (1O0nM), an inhibitor of protein phosphatase 1 and 2.A, inhibited cAMP-stimulated (but not the basal) uptake of TC. Okadaic acid inhibited cAMP-induced increases in the amount of ntcp in the plasma membrane. Plasma membranes isolated from hepatocytes incubated with ~=PO4 followed by immunopreciptation, SDS-PAGE and autoradiography revealed that ntcp is a phosphoprotein. Cyclic AMP decreased and okadaic acid increased phosphorylation of plasma membrane ntep. However, okadaic acid failed to increase phosphorylation of ntcp in the presence of cAMP. Cyclohexamide failed to inhibit cAMP-stimulation of TC uptake, indicating that the stimulation did not involve new protein synthesis. These results indicate that 1) ntcp is a phosphoprotein, 2) cAMP may increase the maximal rate of Na+/TC uptake by translocating the ntcp to the plasma membrane and 3) protein phosphatase 1 and/or 2A may be involved in cAMP-induced stimulation of Na+/TC uptake.
@ THYMOSINe~ TREATMENTOF CHRONICHEPATITIS B: A MULTICENTER, RANDOMIZED, PLACEBO-CONTROLLED DOUBLE BLIND STUDY. MG Mutchnick, KL Lindsay, ER Schiff, GD Cummings, HD Appelman. Wayne State Univ., Detroit, MI; Univ. of S. Cal., L.A., CA; Univ. of Miami, Miami, FL; Univ. of Michigan, Ann Arbor, MI. Previous small clinical studies suggest that thymosin ~I (Tel) may be effective in the treatment of chronic hepatitis B (CHB). We describe the results of ~ multicenter, placebo controlled, double-blind study of 99 patients with serum HBV DNA and HBeAg positive CHB All patients were HBsAg(+) for at least 6 months mth perslstent ALT elevation. Following a 3 month screening period, 50 patients were randomized to receive TeI (1.6mg, S.C. 2x/week) and 49 to receive placebo (mannitol, NaPO4; S.C. 2x/week) for 6 months and followed at month intervals for 6 months• Thirty eight Te~ treated patients and 32 patients given placebo were followed after the lyr s t u d y for 23±7 and 22±7(SD) months, respectively. Two patients were removed from the study after randomization but prior to treatment. Results are presented for 49 patients in the TeI group and 48 patients in the placebo group. There were no differences in ALT values, HBV DNA levels and histological findings between the two groups at inclusion. A complete response (CR) to treatment was defined as a sustained serum HBV DNA(-) status (2 negative results at least 3 months apart) during the lyr study with negative HBV DNA and HBeAgat 12 months. A delayed response (DR) was determined as a sustained HBV DNA(-) status achieved after 12 months with a negative HBV DNA and HBeAg at last assessment. An incomplete response (IR) wag characterized as a sustained HBV DNA(-) status during the study or post study period with continued presence of HBeAg. The response to treatment was as follows: Group (n) CR(%) DR(%) IR(%) Te (49) 7 (14%) 5 (10%) 0 (0%) Pl~cebo (48) 2 (4~) 4 (8%) 2 (4~) p value 0.084 ns ns A total of 12 (25%) patients treated with Te~ and 6 (13%) patients given placebo (p
SUCCESSFUL ENDOSCOPIC THERAPY OF BLEEDING GASTRIC VARICES WITH ENBUCRILATE (HISTOACRYL) P Mosca, I Lorenzini, U Freddara, G Novelli, T Cilluffo, MT Candelaresi, E Peruzzi, F Tarsetti, R Galeazzi.
Divisione di Gastroentero/ogia, Ospeda/e Umberto /, Ancona, Italy. Endoscopic therapy of bleeding gastric vadces with sclerosants has been flawed by the high complication rate. The efficacy of cyanoacrylate glue has been already reported, but concerns have been raised about its safety for patients and endoscopes as well. We have treated with enbucrilate injection 11 pts (6 men, mean age 66.6 Ys, ranging from 40 to 82) who had bleeding gastric varices secondary to liver cirrhosis (n=10, Child-Pugh's B=6, C=4) or portal thrombosis (n=l)• Isolated gastric varices were present in 6 pts, 2 of which had their esophageal varices previously eradicated by sclerotherapy. Endoscopic sclerotherapy was subsequently offered to the remaining 5 pts with concomitant esophageal varices. Nine pts were treated within 48 Hs from the index bleeding episode. Enbucrilate was diluted 1:1 with lipiodol and injected intravariceally in an amount sufficient to completely fill and harden the varix. A single treatment was required in 6 pts, 2 or 3 treatments were needed in the remaining 5 pts; a median volume of 3 ml was injected with a range from 2 to 6 ml. No pt died after a median follow-up of 9 months (range 3 to 17). An early rebieeding, requiring transfusions, was observed in one pt before complete obliteration of the vadces was achieved (i.e. between the 2nd and 3rd treatment); 2 further pts rebled after 11 and 12 months from a relapsing fundal varix and from G-E junction varices respectively; both have been successfully retreated with enbucrilate. Ulcerations at the site of partial enbucrilate expulsion were regularly observed after the treatment, the glue was eventually extruded between 2 and 4 months after the injection with disappearance of the varix; these ulcers were symptomless and never bled. No distant embolization of the glue or other side effects nor any damage to the scopes were observed. These preliminary results show that glue injection is an effective therapeutic option for gastric varices. It may be the best treatment in pts with advanced age Or stage of liver disease and should be compared to other treatment modalities in RCTs.
• HEPATOCYTE CELL DENSITY REGULATES A MINIMAL HUMAN A N D R A T E P I D E R M A L G R O W T H F A C T O R TRANSCRIPTIONAL REGULATORY REGION: B. Mullhauvt. E. Fodor, A. Feren, A. Jones: Cell Biology, VA Medical Center and Dept. of Medicine, and Liver Center, University of California, San Francisco, CA 94143 Epidermal growth factor (EGF) is a complete and potent mitogen for hepatocyte cell replication in vivo and in vitro. Recently we have shown that EGF transcripts encoding the mature EGF peptide are constitutively expressed in rodent liver and up-regulated over 10-fold in the immediate early period (15 minutes) following 70% hepatcctomy. To identify elements controlling EGF gene transcription, we have isolated and sequenced over 1.5 kB of human and rat chromosomal DNA 5' to EGF Exon I. Alignment reveals an extraordinarily high degree of similarity exceeding 90% in the first 600 bp upstream of the transcriptional initiation site identified by primer extension analysis. Sequence analysis reveals a conserved TITAAA element at -30, an HLH binding site at -70 overlapping an AP 1 motif, and at -110 a high affinity HNF 3 binding site. To identify a minimal EGF transcriptional regulatory unit functional in hepatocytes, we have subcloned large regions of the human and rat sequence into the luciferase vector pGL2 and tested activity of successive E~<0 III deletions after efficient transient transfection to hepatocyte mono-layer cultures. Our, results show that a -250 bp fragment is sufficient to drive high levels of luciferase production and coincides with DNA regions exhibiting maximal sequence similarity. To determine if cell proliferation influences EGF transcription, we have carried similar analysis on hepatocytes plated and maintained at different densities. Our results show a full length and minimal human and rat EGF transcriptional control ~equence are similarly negatively regulated at high cell densities that exhibit low proliferation evidenced by a 92% reduction in DNA synthesis measured as 3HThymidine incorporation (Table, below). LacZ expression from coWansfccted pCMVb-Gal did not correlate with cell density: :Cell~i~ 3H~Thymidin~ H~ EGF5!~U~ Rat EGF5'~Luc
prol~ se results il .s a major determining factor in the relative levels of EGF gene expression. Since EGF will rapidly induce hepatocyte replication, our data point to the likelihood that EGF transcriptional activity may be autoregulated in hepatic tissue.
AASLD Al127
@REGULATION OF THE NA+fl-AUROCHOLATE COTRANSPORT POLYPEPTIDE (ntcp) BY CYCLIC AMP: ROLE FOR TRANSLOCATION AND PHOSPHORYLATION. S. M u k h o p a d h a y a y , M. Ananthanarayanan, B. Stieger, P.J. Meier, F.J. Suchy, MS. Anwer. Dept. of Veterinary Medicine, "i'ufts University, N. Grafton, MA, Dept. of Pediatrics, Yale University School of Medicine, New Haven, CT, Divn. of Clinical Pharmacology and Toxicology, University Hospital, Z~rich, Switzerland Cyclic AMP (cAMP) increases the maximal rate of Na+fTaurocholate (Na+fTC) cotransport within 15 min. This rapid effect is mediated via protein kinase A and potentiated by Ca++/ ealmodulin kinase (JBC 268:17734,1993). Whether this effect of cAMP was mediated via translocation and/or phosphorylation of the ntcp was studied in the present study using two different antibodies (from ZOrich and New Haven). Results obtained with both antibodies were similar. Studies were conducted in hepatocytes and plasma membranes isolated from cAMP (and/or okadaie acid)-treated hepatocytes, cAMP increased Na*/TC uptake by 70% and increased by 50% the amount of ntcp in the plasma membrane. Okadaic acid (1O0nM), an inhibitor of protein phosphatase 1 and 2.A, inhibited cAMP-stimulated (but not the basal) uptake of TC. Okadaic acid inhibited cAMP-induced increases in the amount of ntcp in the plasma membrane. Plasma membranes isolated from hepatocytes incubated with ~=PO4 followed by immunopreciptation, SDS-PAGE and autoradiography revealed that ntcp is a phosphoprotein. Cyclic AMP decreased and okadaic acid increased phosphorylation of plasma membrane ntep. However, okadaic acid failed to increase phosphorylation of ntcp in the presence of cAMP. Cyclohexamide failed to inhibit cAMP-stimulation of TC uptake, indicating that the stimulation did not involve new protein synthesis. These results indicate that 1) ntcp is a phosphoprotein, 2) cAMP may increase the maximal rate of Na+/TC uptake by translocating the ntcp to the plasma membrane and 3) protein phosphatase 1 and/or 2A may be involved in cAMP-induced stimulation of Na+/TC uptake.
@ THYMOSINe~ TREATMENTOF CHRONICHEPATITIS B: A MULTICENTER, RANDOMIZED, PLACEBO-CONTROLLED DOUBLE BLIND STUDY. MG Mutchnick, KL Lindsay, ER Schiff, GD Cummings, HD Appelman. Wayne State Univ., Detroit, MI; Univ. of S. Cal., L.A., CA; Univ. of Miami, Miami, FL; Univ. of Michigan, Ann Arbor, MI. Previous small clinical studies suggest that thymosin ~I (Tel) may be effective in the treatment of chronic hepatitis B (CHB). We describe the results of ~ multicenter, placebo controlled, double-blind study of 99 patients with serum HBV DNA and HBeAg positive CHB All patients were HBsAg(+) for at least 6 months mth perslstent ALT elevation. Following a 3 month screening period, 50 patients were randomized to receive TeI (1.6mg, S.C. 2x/week) and 49 to receive placebo (mannitol, NaPO4; S.C. 2x/week) for 6 months and followed at month intervals for 6 months• Thirty eight Te~ treated patients and 32 patients given placebo were followed after the lyr s t u d y for 23±7 and 22±7(SD) months, respectively. Two patients were removed from the study after randomization but prior to treatment. Results are presented for 49 patients in the TeI group and 48 patients in the placebo group. There were no differences in ALT values, HBV DNA levels and histological findings between the two groups at inclusion. A complete response (CR) to treatment was defined as a sustained serum HBV DNA(-) status (2 negative results at least 3 months apart) during the lyr study with negative HBV DNA and HBeAgat 12 months. A delayed response (DR) was determined as a sustained HBV DNA(-) status achieved after 12 months with a negative HBV DNA and HBeAg at last assessment. An incomplete response (IR) wag characterized as a sustained HBV DNA(-) status during the study or post study period with continued presence of HBeAg. The response to treatment was as follows: Group (n) CR(%) DR(%) IR(%) Te (49) 7 (14%) 5 (10%) 0 (0%) Pl~cebo (48) 2 (4~) 4 (8%) 2 (4~) p value 0.084 ns ns A total of 12 (25%) patients treated with Te~ and 6 (13%) patients given placebo (p