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133 Ros formation by reoxygenation and its suppression by melatonin: Optical measurements in the isolated perfused rat liver
Catego~ 1: Liver Transplantation~Surgery~Acute Liver Failure
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PEGYI_ATED INTERFERON (PEG-IFN) o~-2b + RIBAVIRIN (RB) IN THE TREATMENT OF POSTLIVER TRANSPLANT (LT) RECURRENT HEPATITIS C
S. Martini 1, B. Lavezzo 1, S. Saettone 1, A. Franchello 2, A. Smedile 1, D. Cocchis 2, V Ghisetti 3, E. David4, M. Salizzoni 2, M. Rizzetto 1 .
I Gastrohepatology Department, Molinette Hospital, Turin, Italy," 2Liver Transplant Surgery Department, Molinette Hospital, Turin, Italy; s Virology Department, Molinette Hospital, Turin, Italy," 4Pathology, Molinette Hospital, Turin, Italy After LT recurrent chronic hepatitis C develops in many patients (pts). We evaluated effectiveness, safety and tolerance of combination therapy (CT: Peg-IFN ct-2b + Rb) in LT with recurrent hepatitis C. Methods: 86 pts after LT, mean age 53y, M/F 68/18, genotype 1-4 82.6%, with histological recurrence HCV chronic hepatitis, mean grading 6/18 (2-16), mean staging 2/6 (1-4), were treated with Peg-IFN ~-2b 1 pg/kg/wk plus Rba 800 mg/die for 12 months (rod). 45 pts were naives, 41 pts were relapser or non responder (NR) to previous conventional IFN+Rb therapy. The median delay between LT and first antiviral therapy was 12 mo (2-114). We evaluated virological response (qualitative HCV-RNA negative) at the end of treamlent (ETVR) arid at 24 weeks of follow-up (SVR). Results:
55
Dipivoxil (ADV) could be useful to prevent HBV recurrence in presence of YMDD mutants. Patients a n d Methods: This pilot, not controlled trial analyzes 22 HBVcarriers who selected YMDD variant during preemptive LAM therapy before OLT. Before surgery, 13 patients had developed a significant virernic breakthrough (HBV DNA >105 copies?ml), during LAM therapy (~oup A, phenotypic mutation). Two o f them continued only LAM and 11 were treated with a double therapy (ADV and LAM) before OLT. In opposition, 9 patients selected YMDD mutant without a significant virernic breakthrough (< l0 s copies/ml) and continued LAM therapy without ADV addition (group B, genotypic mutation). Results: Patients were followed-up for a mean of 35 months after OLT. In group A (mean f-u 29 months), hepatitis B recurred in both patients transplanted with a high viral load (>105 copies/ml), prophylaxed with LAM and HBIG after OLT, but in none with HBV DNA ~<10s copies/ml, inducted by ADV therapy before OLT, treated long-term with a deuble or a triple prophylaxis after surgery. In group B (mean f-u 43 months) the HBV recurrence developed only in those 2 subjects who suspended HBIG post-surgery, arid in none of those treated with a double prophylaxis post-OLT. Conclusions: Waiting OLT list suspension and ADV addition are needed in patients of group A, but not in group B. Post OLT long-term combined prophylaxis with HBIG and LAM is effective in both groups and triple prophylaxis could be the surest option in case of phenotypic mutation.
Response
Naive
Retreatment
ETVR (45 pts) SVR (30 pts fup ) 6 mo) ETVR (41 pts) SVR (41 pts)
All genotypes
Genotype 1-4
Genotype 2-3
22/45 (49%) 10/30 (33%)
10/33 (30%) 3/22 (14%)
12/12 (100%) 7/8 (88%)
13/41 (32%) 7/41 (17%)
10/38 (26%) 5/38 (13%)
3/3 (100%) 2/3 (67%)
Therapy reduction 66/86 (77%): Duration of therapy
12/86 lots (14%) ~<3mo 36/86 lots (42%) (only 6 lots fifll dosage of drug)
Severe side effects: WBC <1500:12/86 pts (14%); Depression: 11/86 (13%); Liver rejection: 3/86 (3%); Liver toxicity (increased ALT and janndice): 7/86 (8%). Conclusions: 49% of naive pts achieved ETVR and 33% SVR with CT. 17% of retreated pts maintained SVR. Response was satisfactory in genotype 2 3 and still disappointing in genotype 1 4. In these difficult to treat pts, full dosage maintainenlent and duration of therapy are important. Tolerance is poor and severe side effects are frequent.
[13~ PROPHYLAXIS OF HEPATITIS B VIRUS RECURRENCE AFTER LIVER TRANSPLANTATION A. Marzano I , P. Lampertico 2, M Mazzaferro 3, S. Gaia I , M. Vigano 2, R. Romito 3, A. Pulvirenti 3 , A. Franchello 4, M. Colombo 2 , M. Salizzoni 4, M. Rizzetto I . 1Gast~venterology Department, Molinette Hospital, Turin,
Italy," 2Department of Gastroenterology and Endocrinology, IRCCS Maggiore Hospital," Unioersity of Milan, Milan, Italy," 3 Gastrointestinal Surgery and Lioer Transplantation Centre, National Cancer Institute, Milan, Italy," 4Liver Transplantation Centre, Molinette Hospital, Turin, Italy Background: The combination of Lamivndine (LAM) and Hepatitis B immunoglobulins (HBIG) reduces the risk of hepatitis B virus (HBV) recurrence after orthotopic liver transplantation (OLT), but it can be ineffective in patients with YMMD mutant selection. New drugs as Adefovir
~ROS FORMATION BY REOXYGENATION AND ITS SUPPRESSION BY MELATONIN: OPTICAL MEASUREMENTS IN THE ISOLATED PERFUSED RAT LIVER J. Messner, T. Thalharnrner, J. Gra£ Department of Pathophysiology, Medical University of Vienna, Vienna, Austria Background: Reactive oxygen species (ROS) play an important role in mediating ischemia-reperfitsion injury (IRI) in the liver. Aim: The aim of our study was to establish a real-time ROS detection system with the intracolhtlar sensor carboxy-dihydrochlorfluorescein (C-DCF) and to investigate the role of melatonin as a free radical scavenger using the isolated perfused rat liver. M e t h o d s : The isolated rat liver was perfused with Krebs-Henseleit buffer (37°C) containing C-DCF in form of its cell membrane permeant di-acetate ester (C-DCF-DA) that is split by intracellular esterases to C-DCE The sensor becomes fluorescent through oxidation with H202, which is formed by the activity of the superoxide dismutase and is a source of subsequent highly reactive compounds which generate oxidised C-DCF (ox C-DCF). Intracellular generation of ox C-DCF was followed by fibre optic measurement of liver surface fluorescence (Excitadon: 485 nm, Emission: 535nm), and by the appearance of ox C-DCF in bile and venous effluent perfusate. Simultaneously, the excitation/emission pair 360 nm/460nm was used to detect changes of reduced nicotinamide adenine dinucleotide (phosphate) [NAD(P)H]. Results: During control perfitsion with C-DCF-DA (1 ~tM), surface fluorescence of ox C-DCF and its release into perfusate and bile reached a constant level indicating a steady state of intracellular generation of ox DCF and of its simultaneous release. Perfusion under hypoxic conditions (equilibration of the perfusate with 95%N2/5% CO2) for 10 min resulted in an increase of NAD(P)H and a decrease of ox C-DCF production and, after reexygenation, NAD(P)H decreased with a simultaneous burst of ox C-DCF production. This response was not attenuated if the experimental manoeuvre was repeated on the same liver. Application of in pharmacological concentrations (100-500 ~tM) caused a dose-dependent reduction of the H202 formation. Conclusions: A real-time method to analyse ROS formation in the isolated perfused rat liver is presented and the antioxidant effect of melatonin is directly demonstrated.
[
•
PEGYI_ATED INTERFERON (PEG-IFN) o~-2b + RIBAVIRIN (RB) IN THE TREATMENT OF POSTLIVER TRANSPLANT (LT) RECURRENT HEPATITIS C
S. Martini 1, B. Lavezzo 1, S. Saettone 1, A. Franchello 2, A. Smedile 1, D. Cocchis 2, V Ghisetti 3, E. David4, M. Salizzoni 2, M. Rizzetto 1 .
I Gastrohepatology Department, Molinette Hospital, Turin, Italy," 2Liver Transplant Surgery Department, Molinette Hospital, Turin, Italy; s Virology Department, Molinette Hospital, Turin, Italy," 4Pathology, Molinette Hospital, Turin, Italy After LT recurrent chronic hepatitis C develops in many patients (pts). We evaluated effectiveness, safety and tolerance of combination therapy (CT: Peg-IFN ct-2b + Rb) in LT with recurrent hepatitis C. Methods: 86 pts after LT, mean age 53y, M/F 68/18, genotype 1-4 82.6%, with histological recurrence HCV chronic hepatitis, mean grading 6/18 (2-16), mean staging 2/6 (1-4), were treated with Peg-IFN ~-2b 1 pg/kg/wk plus Rba 800 mg/die for 12 months (rod). 45 pts were naives, 41 pts were relapser or non responder (NR) to previous conventional IFN+Rb therapy. The median delay between LT and first antiviral therapy was 12 mo (2-114). We evaluated virological response (qualitative HCV-RNA negative) at the end of treamlent (ETVR) arid at 24 weeks of follow-up (SVR). Results:
55
Dipivoxil (ADV) could be useful to prevent HBV recurrence in presence of YMDD mutants. Patients a n d Methods: This pilot, not controlled trial analyzes 22 HBVcarriers who selected YMDD variant during preemptive LAM therapy before OLT. Before surgery, 13 patients had developed a significant virernic breakthrough (HBV DNA >105 copies?ml), during LAM therapy (~oup A, phenotypic mutation). Two o f them continued only LAM and 11 were treated with a double therapy (ADV and LAM) before OLT. In opposition, 9 patients selected YMDD mutant without a significant virernic breakthrough (< l0 s copies/ml) and continued LAM therapy without ADV addition (group B, genotypic mutation). Results: Patients were followed-up for a mean of 35 months after OLT. In group A (mean f-u 29 months), hepatitis B recurred in both patients transplanted with a high viral load (>105 copies/ml), prophylaxed with LAM and HBIG after OLT, but in none with HBV DNA ~<10s copies/ml, inducted by ADV therapy before OLT, treated long-term with a deuble or a triple prophylaxis after surgery. In group B (mean f-u 43 months) the HBV recurrence developed only in those 2 subjects who suspended HBIG post-surgery, arid in none of those treated with a double prophylaxis post-OLT. Conclusions: Waiting OLT list suspension and ADV addition are needed in patients of group A, but not in group B. Post OLT long-term combined prophylaxis with HBIG and LAM is effective in both groups and triple prophylaxis could be the surest option in case of phenotypic mutation.
Response
Naive
Retreatment
ETVR (45 pts) SVR (30 pts fup ) 6 mo) ETVR (41 pts) SVR (41 pts)
All genotypes
Genotype 1-4
Genotype 2-3
22/45 (49%) 10/30 (33%)
10/33 (30%) 3/22 (14%)
12/12 (100%) 7/8 (88%)
13/41 (32%) 7/41 (17%)
10/38 (26%) 5/38 (13%)
3/3 (100%) 2/3 (67%)
Therapy reduction 66/86 (77%): Duration of therapy
12/86 lots (14%) ~<3mo 36/86 lots (42%) (only 6 lots fifll dosage of drug)
Severe side effects: WBC <1500:12/86 pts (14%); Depression: 11/86 (13%); Liver rejection: 3/86 (3%); Liver toxicity (increased ALT and janndice): 7/86 (8%). Conclusions: 49% of naive pts achieved ETVR and 33% SVR with CT. 17% of retreated pts maintained SVR. Response was satisfactory in genotype 2 3 and still disappointing in genotype 1 4. In these difficult to treat pts, full dosage maintainenlent and duration of therapy are important. Tolerance is poor and severe side effects are frequent.
[13~ PROPHYLAXIS OF HEPATITIS B VIRUS RECURRENCE AFTER LIVER TRANSPLANTATION A. Marzano I , P. Lampertico 2, M Mazzaferro 3, S. Gaia I , M. Vigano 2, R. Romito 3, A. Pulvirenti 3 , A. Franchello 4, M. Colombo 2 , M. Salizzoni 4, M. Rizzetto I . 1Gast~venterology Department, Molinette Hospital, Turin,
Italy," 2Department of Gastroenterology and Endocrinology, IRCCS Maggiore Hospital," Unioersity of Milan, Milan, Italy," 3 Gastrointestinal Surgery and Lioer Transplantation Centre, National Cancer Institute, Milan, Italy," 4Liver Transplantation Centre, Molinette Hospital, Turin, Italy Background: The combination of Lamivndine (LAM) and Hepatitis B immunoglobulins (HBIG) reduces the risk of hepatitis B virus (HBV) recurrence after orthotopic liver transplantation (OLT), but it can be ineffective in patients with YMMD mutant selection. New drugs as Adefovir
~ROS FORMATION BY REOXYGENATION AND ITS SUPPRESSION BY MELATONIN: OPTICAL MEASUREMENTS IN THE ISOLATED PERFUSED RAT LIVER J. Messner, T. Thalharnrner, J. Gra£ Department of Pathophysiology, Medical University of Vienna, Vienna, Austria Background: Reactive oxygen species (ROS) play an important role in mediating ischemia-reperfitsion injury (IRI) in the liver. Aim: The aim of our study was to establish a real-time ROS detection system with the intracolhtlar sensor carboxy-dihydrochlorfluorescein (C-DCF) and to investigate the role of melatonin as a free radical scavenger using the isolated perfused rat liver. M e t h o d s : The isolated rat liver was perfused with Krebs-Henseleit buffer (37°C) containing C-DCF in form of its cell membrane permeant di-acetate ester (C-DCF-DA) that is split by intracellular esterases to C-DCE The sensor becomes fluorescent through oxidation with H202, which is formed by the activity of the superoxide dismutase and is a source of subsequent highly reactive compounds which generate oxidised C-DCF (ox C-DCF). Intracellular generation of ox C-DCF was followed by fibre optic measurement of liver surface fluorescence (Excitadon: 485 nm, Emission: 535nm), and by the appearance of ox C-DCF in bile and venous effluent perfusate. Simultaneously, the excitation/emission pair 360 nm/460nm was used to detect changes of reduced nicotinamide adenine dinucleotide (phosphate) [NAD(P)H]. Results: During control perfitsion with C-DCF-DA (1 ~tM), surface fluorescence of ox C-DCF and its release into perfusate and bile reached a constant level indicating a steady state of intracellular generation of ox DCF and of its simultaneous release. Perfusion under hypoxic conditions (equilibration of the perfusate with 95%N2/5% CO2) for 10 min resulted in an increase of NAD(P)H and a decrease of ox C-DCF production and, after reexygenation, NAD(P)H decreased with a simultaneous burst of ox C-DCF production. This response was not attenuated if the experimental manoeuvre was repeated on the same liver. Application of in pharmacological concentrations (100-500 ~tM) caused a dose-dependent reduction of the H202 formation. Conclusions: A real-time method to analyse ROS formation in the isolated perfused rat liver is presented and the antioxidant effect of melatonin is directly demonstrated.