- Email: [email protected]
Effect of a selective agonist for the estrogen receptor β (Silymarin) upon colonic cell migration in normal mice
Abstracts ribavirin; however, abont 50% of relapsers may achieve a snstained response when retreated with Peg-IFN + ribavirin, in particnlar patients with favonrable genotypes show a high SVR similar to that fonnd among naive patients.
PA.142 INFLUENCE OF LIVER DISEASE STAGING ON SUSTAINED VIROLOGICAL RESPONSE IN HCV "FAVOURABLE" GENOTYPES TREATED WITH PEG-INTERFERON AND RIBAVIRIN: DATA FROM CLINICAL PRACTICE PL. Almasio, V. Di Marco, S. De Lisi, V. Calvarnso, F. D'Angelo, P Zizzo, A. !aca, A. Craxi
Unit ofGastroenterology and Hepatology, University ofPalermo, Palermo Background and aim: Data from registration trials have shown that patients infected by HCV 2 or 3 genotypes have a great benefit from combination therapy with PEG-IFN and ribavirin reaching a snstained viral response (SVR) in more than 90% of cases. However information is lacking in snbjects with advanced liver fibrosis treated in clinical practice setting. Aims: We evalnated the role of liver fibrosis on SVR in patients with "favonrable" genotypes. Material and methods: We analyzed 106 na'lve patients (mean age 49.1 ± 13.7 years, 68 males, 73 infected by genotype 2) that received at least one dose of PEG-IFN alfa-2a or alfa-2b pIns ribavirin according to gnide lines. They were classified in three gronps according to staging of liver disease: gronp 1 (patients with Fl-F2 METAVIR score), gronp 2 (patients with F3-F4 METAVIR score) and gronp 3 (patients with cirrhosis and oesophageal varices). Serum HCV-RNA negative by PeR 6 months after the end oftherapy was defined as SVR. Results: Overall 75 patients (70.7%) had a SVR. The rate of SVR according to HCV genotype and staging of liver disease is snmmarized in the table. No significant difference in the rate of SVR was observed in the 3 gronps and for genotype 2 or 3. At nnivariate analysis no other clinical variables was identified as significantly associated with LTR. Table 1
Genotype 2 Genotype 3
S105
strated no significant difference in snstained virological response (SVR) rate between peginterferon alpha-2b 1,5 mcglkg/week and 1 mcglkg/week in naive patients with chronic hepatitis C. In the attempt to optimise efficacy and resources, we designed a multi-centre observational protocol, treating chronic hepatitis C naive patients with peginterferon alpha-2b 1 mcglkg/week and ribavirin 800-l200mg, according to body weight, for 24 weeks in genotype 2 or 3 and for 48 weeks for genotype 1 or 4 infected patients Material and methods: 148 consecutive, unselected naive patients were enrolled between 200 1 and 2003 in five regional referring centers. 51.4% had genotype 1/4. None of the patients had clinical sign of cirrhosis. HCV-RNA was assessed at week 2, 4, 12, end of treatment and at the end of follow up Results: We had 6 dropout, two due to intolerance after few injections, 4 due to noncompliance with the treatment. 142 patients were adherent to the protocol and follow-up. No statistically significant differences were presents between the genotype gronps for age, sex, ALT levels. Overall, we achieve SVR in 59.4% of patients, with a 5.4% of relapseresponders (RRs) and a 35.2% of non-responders (NRs) (intention to treat). For genotype 1/4 (76 pts, 48 weeks treatment), we had 32.9% SVR, 7.9% RRs, 52.6% NRs and 6.6% DO. For genotype 2/3 (72 pts, 24 weeks treatment), we had 87.5% SVR, 2.8% RRs, 8.3% NRs and 1.4% DO. Week 2 and 4 HCV-RNA determinations did not prove to be reliable predictors of SVR. No major side effects, adverse events, dose rednction were reported, while a dose adjustment for anemia was necessary in 5% of the patients Conclusions: Peginterferon alpha-2b plus ribavirin combination treatment was well tolerated in onr group. The overall SVR is comparable with the published data. In genotype 1/4 our results are slightly below the best-pnblished results (32.9% vs 42%, Manns et al; peginterferon alpha-2b 1.5 mcglkg plus 800mg ribavirin), for genotype 2/3 we obtained identical resnlts (87.5% vs 82%, Manns et al. with a 48 weeks treatment). Onr results suggest that this combination therapy for 24 weeks can be considered the best option in genotype 2/3. With the same SVR ratio, we obtained to reduce significantly length of treatment, side effects, and the costs. Clearly, genotypes 1/4 need more aggressive schedules
PA.144 Group 1
Group 2
p
36/48 (75.0%) 18/24 (75.0%)
16/25 (64.0%) 5/9 (55.6%)
n.s. n.s.
Conclusions: The onr data confirms that the patients infected by genotypes 2 and 3 should be treated with antiviral drugs independently by staging of liver disease thus avoiding the need of liver biopsy in this clinical setting.
PA.143 PEGINTERFERON A-2B 1 MCGIKG DOSE PLUS RIBAVIRIN 800-1200MG FOR 24-48 WEEKS, ACCORDING TO GENOTYPE, IN NAIVE PATIENTS WITH CHRONIC HEPATITIS C M. Basso 1, A. Grasso 2, G. Percario 3, E. Azzola 4, S. Artioli 5 , N. Pelli 1, S. Bianchi 1 , F. Torre 1, A. Picciotto 1
lDepartment ofInternal Medicine, University of Genova, Genova ofInternal Medicine, Ospedale S Paolo, Savona 3 Department of Gastroenterology, Ospedale San Carlo, Genova 4Department of Gastroenterology, Ospedale Santa Corona, Pietra Ligure 5Department ofInfectious Diseases, Ospedale Civile SAndrea, !.JJ. Spezia 2Department
Background and aim: Dose finding stndy on monotherapy demon-
EFFECT OF A SELECTIVE AGONIST FOR THE ESTROGEN RECEPTOR P(SILYMARIN) UPON COLONIC CELL MIGRATION IN NORMAL MICE A. Di Leo*, M. Barone, M. Margiotta, A. Castellaneta, L. Troiani, L. Demarinis, S. Tanzi, R. Guido, A. Francavilla
Policlinico di Bari, Bari Background and aim: Estrogen receptor beta (ER~) is the predominant isoform of ER in the normal colon and decreases in human and experimental colon cancer althongh its mRNA levels remain unchanged suggesting a regulatory posttranscriptional mechanism (E. Foley et al. Cancer Res 60, 245, 2000). In a murine intestinal tumorigenesis model, Weyant et al.(Cancer Res 61, 2547, 20012) have shown that estrogens protect against tnmor formation and this prevention is associated with an increase of ER~ and cellular migration. The aim of this stndy was to demonstrate the effect of the silymarin (silymarin T, supplied by MADAUS srI, Italy) on cellular migration as well as ER~ protein and mRNA levels in normal mice colonic mucosa Material and methods: We used 36 male mice that were divided in 3 groups of 12 animals each and fed, for 30 days, on 3 different diet regimens: standard CK20 diet (gronp A), CK20 diet and silymarin 200 ppms (gronp B) and CK20 diet and silymarin 600 ppms (gronp C). Cellular migration was evaluated by using the method of Javid et al (Carcinogenesis 26, 587,2004). Briefly, BrdU was injected to all animals (0,75 mgl mouse) and 4 animals for each group were sacrificed at 24,48 and 72 honrs. BrdU nuclear incorporation was eval-
Abstracts
S106
uate by immunoistochemistIy (BrdU detection kit II, Roche, Milan). Cellular migratiou was evaluated by counting the BrdU marked cells and calculating the difference between the marked cells at 24 and 72 hours, expressed as percentage (ll. %). Statistical analysis of the data was performed by Wilcoxon test. ER~ was analyzed by RT-PCR and Western Blotting (anti ER~, Santa Cruz, California). Results: As shown in table, at 72 hours, 9%, 21 % and 40% of colonic marked cells disappeared in A, Band C groups respectively. At the same time points, ER~ mRNA was decreased while protein levels were increased in dose dependent manner.
sub-set of patients with less advanced MSI sporadic disease. In contrast, Pl6 methylation is associated with advanced disease, a feature largely explained by increasing prevalences of advanced stages in females with this epigenetic alteration.
Table 1. BrdU+ cells/lOO cells counted in 8 well oriented cripts
L. Polimeno .,1, B. Pesetti 1, E. Annoscia 1, G. Ingravallo 2,
Tunes
M. Valente 1, A. Amoruso 1, A. Francavilla 1
Group A Group B Group C
24 hrs
48 hes
72 hes
12.2±1.3 13.6±1.0 14.4±1.0
12.4±1.9 13.9±2.1 14.5± 0.6
11.1±1.l 1O.7±2.7 8.7±1.4
-9 -21 -40
Conclusions: Silymarin administration determines an increase of ER~ levels, probably by a post-transcriptional mechanism, and a concomitant increase of the cellular migration. Since these data suggest an anti tumor effect of silymarin, we will test its biological activity in an experimental model of intestinal tumorigenesis
PA.145 INFLUENCE OF GENDER AND OF P16 METHYLATION ON TUMOR STAGE OF COLORECTAL CANCERS WITH MICROSATELLITE INSTABILITY
L. Laghi', P. Bianchi, A.E. Randolph, M. Roncalli, A. Malesci Istituto Clinico Humanitns, Rozzano (MI) Background and aim: hMLHI and Pl6 promoter methylation occur frequently in colon cancers with microsatellite instability (MSI); while hMLH 1 methylation is associated with sporadic MSI, the features of MSI tumors associated with Pl6 methylation are less clear. To evaluate the correlation of hMLHI and Pl6 epigenetic alterations, as to demographics, inheritance, and pathological features of a series of MSI colo-rectal cancers (CRC). Material and methods: We studied hMLHI and Pl6 methylation in 88 (MIF, 50/38) MSI CRC, comprising 25 (MIF, 15/10) hereditary nonpolyposis CRC (Amsterdam Criteria), and 63 (MIF, 34/29) sporadic cases. Tumor were staged by AJCC criteria. Results: Out of 88 MSI CRC, 59 (67%) had methylation of hMLHI (MIF, 28/31; p=O.OI), and 45 (51 %) of Pl6 (MIF, 29/16; hMLHl vs Pl6 methylation in females, p=0.0004). Methylation of hMLHI only occurred in 21 (24%) CRC (MIF, 6/15; p=0.003) and of Pl6 only in 7 (8%; MIF, 7/0; p=O.OI). In 63 sporadic MSI CRC, 49 (77%) showed methylation of hMLHI (MIF 24/25), and 37 (59%) of Pl6 (MIF, 23/14), hMLHI being more frequently methylated than Pl6 in females (p=0.002). Upon univariate analysis, CRC stage 3-4 (29, 33%) were more frequent in males than in females (MIF 21/8; p=0.03), in CRC with methylated than in those with unmethylated Pl6 (21/45, vs 8/43; p=O.OO5), and in proximal than distal tumors (27/69, vs 2/19; p=0.02). In sporadic cases, only Pl6 status was significantly associated with advanced stages (4/26 in un-methylated vs 19/37 in methylated CRC; p=0.003). Upon multivariate analysis, proximal location and Pl6 methylation were independently associated with advanced tumor stage (p=0.04 and p=O.OI, respectively) in all cases, but only Pl6 in sporadic disease (p=0.003). Accordingly, in females, we observed 7 stage 3-4 cancers out of 16 with methylation of both Pl6 and hMLHl, as compared with 1 (7%) out of 15 with only hMLHI methylation (p=0.03). Conclusions: Prevalences of hMLHI methylated MSI CRC are high in old patients with sporadic disease, while Pl6 is more frequently methylated in stage 3-4 than in stage 1-2 cancers. The combination of female gender and hMLHl-only methylated cancers can identify a
PA.146 THE IN VIVO EFFECT OF AUGMENTER OF LIVER REGENERATION (ALR) ON SIGNAL TRANSDUCER AND ACTIVATOR OF TRANSCRIPTION·3 (STAT3) FACTOR
ISezione di Gastroenterologia edEndoscopia Digestiva, DErO, Universitii degli Studi, Bari 2Universitii di Bari, Bari Background and aim: STATs are a family of transcription factors activated by a wide variety of extracellular polypeptides including EGF, PDGF and IL-6. Recent studies have shown a Stat3 activation (Ser- and Tyr-phosphorylation) in the murine partial hepatectomy model (PH) that induces anti-apoptotic factors such as bcl-2, bcl-xL, attributing to Stat3 an anti-apoptotic role in liver regenerative process. Recently, Augmenter of Liver Regeneration (ALR), a hepatic growth factor, has been identified as a control factor of the apoptotic process that occurs after PH. The present work has been done to evaluate if ALR exerts its apoptotic effect through Stat3 activation. Material and methods: Stat3 expression and its activation were evaluated on livers from rAlrp-treated or not animals. Two sets of experiments have been performed. In the first, Stat3 was evaluated, by Western Blot, on hepatic cytosol from 35 intact mice, divided in 7 groups of 5 each. Three groups received, i.p., a single injection ofrAlrp (500 ng) and three groups a same dose of albumin (control). Five treated- and 5 controlmice were sacrificed at 3, 12 and 24 hrs each time. Five animals (time zero) were sacrificed after injection. In the second, Stat3 expression and its phosphorylation were evaluated by immunohistochemistIy techniques on liver sections from 70% PH rats. 35 male rats underwent PH and were divided in 7 groups of 5 animals each. Three groups (treated) were injected, i.p., with multiple doses of rAlrp (500 ng/rat/every 12 hrs) starting from the 18th hour after PH and sacrificed at 24, 36,48 hrs after surgery; three groups (control) received a similar dose of albumin following the same protocol. Five animals (time zero) were sacrificed after PH. Results: The first group of experiments show a statistically significant (p
PA.147 THE HSI,2A ENHANCER ALLELE 2 SHOWS AN INCREASED FREQUENCY IN PATIENTS WITH CROHN'S DISEASE D. Frezza, C. Petruzziello, M. Cretella, E. Calabrese, S. Onali, V. Giambra, C. Mattioli, F. Pallone, L. Biancone
Universitii "Tor Vergatn", Roma Background and aim: The genetics of Crohn's Disease (CD) is characterised by multiple susceptibility loci, genetic heterogeneity and incomplete penetrance. The 3' regulatory complex of the genes of the Ig heavy chain constant region encompass at least 3 enhancers: the central
PA.142 INFLUENCE OF LIVER DISEASE STAGING ON SUSTAINED VIROLOGICAL RESPONSE IN HCV "FAVOURABLE" GENOTYPES TREATED WITH PEG-INTERFERON AND RIBAVIRIN: DATA FROM CLINICAL PRACTICE PL. Almasio, V. Di Marco, S. De Lisi, V. Calvarnso, F. D'Angelo, P Zizzo, A. !aca, A. Craxi
Unit ofGastroenterology and Hepatology, University ofPalermo, Palermo Background and aim: Data from registration trials have shown that patients infected by HCV 2 or 3 genotypes have a great benefit from combination therapy with PEG-IFN and ribavirin reaching a snstained viral response (SVR) in more than 90% of cases. However information is lacking in snbjects with advanced liver fibrosis treated in clinical practice setting. Aims: We evalnated the role of liver fibrosis on SVR in patients with "favonrable" genotypes. Material and methods: We analyzed 106 na'lve patients (mean age 49.1 ± 13.7 years, 68 males, 73 infected by genotype 2) that received at least one dose of PEG-IFN alfa-2a or alfa-2b pIns ribavirin according to gnide lines. They were classified in three gronps according to staging of liver disease: gronp 1 (patients with Fl-F2 METAVIR score), gronp 2 (patients with F3-F4 METAVIR score) and gronp 3 (patients with cirrhosis and oesophageal varices). Serum HCV-RNA negative by PeR 6 months after the end oftherapy was defined as SVR. Results: Overall 75 patients (70.7%) had a SVR. The rate of SVR according to HCV genotype and staging of liver disease is snmmarized in the table. No significant difference in the rate of SVR was observed in the 3 gronps and for genotype 2 or 3. At nnivariate analysis no other clinical variables was identified as significantly associated with LTR. Table 1
Genotype 2 Genotype 3
S105
strated no significant difference in snstained virological response (SVR) rate between peginterferon alpha-2b 1,5 mcglkg/week and 1 mcglkg/week in naive patients with chronic hepatitis C. In the attempt to optimise efficacy and resources, we designed a multi-centre observational protocol, treating chronic hepatitis C naive patients with peginterferon alpha-2b 1 mcglkg/week and ribavirin 800-l200mg, according to body weight, for 24 weeks in genotype 2 or 3 and for 48 weeks for genotype 1 or 4 infected patients Material and methods: 148 consecutive, unselected naive patients were enrolled between 200 1 and 2003 in five regional referring centers. 51.4% had genotype 1/4. None of the patients had clinical sign of cirrhosis. HCV-RNA was assessed at week 2, 4, 12, end of treatment and at the end of follow up Results: We had 6 dropout, two due to intolerance after few injections, 4 due to noncompliance with the treatment. 142 patients were adherent to the protocol and follow-up. No statistically significant differences were presents between the genotype gronps for age, sex, ALT levels. Overall, we achieve SVR in 59.4% of patients, with a 5.4% of relapseresponders (RRs) and a 35.2% of non-responders (NRs) (intention to treat). For genotype 1/4 (76 pts, 48 weeks treatment), we had 32.9% SVR, 7.9% RRs, 52.6% NRs and 6.6% DO. For genotype 2/3 (72 pts, 24 weeks treatment), we had 87.5% SVR, 2.8% RRs, 8.3% NRs and 1.4% DO. Week 2 and 4 HCV-RNA determinations did not prove to be reliable predictors of SVR. No major side effects, adverse events, dose rednction were reported, while a dose adjustment for anemia was necessary in 5% of the patients Conclusions: Peginterferon alpha-2b plus ribavirin combination treatment was well tolerated in onr group. The overall SVR is comparable with the published data. In genotype 1/4 our results are slightly below the best-pnblished results (32.9% vs 42%, Manns et al; peginterferon alpha-2b 1.5 mcglkg plus 800mg ribavirin), for genotype 2/3 we obtained identical resnlts (87.5% vs 82%, Manns et al. with a 48 weeks treatment). Onr results suggest that this combination therapy for 24 weeks can be considered the best option in genotype 2/3. With the same SVR ratio, we obtained to reduce significantly length of treatment, side effects, and the costs. Clearly, genotypes 1/4 need more aggressive schedules
PA.144 Group 1
Group 2
p
36/48 (75.0%) 18/24 (75.0%)
16/25 (64.0%) 5/9 (55.6%)
n.s. n.s.
Conclusions: The onr data confirms that the patients infected by genotypes 2 and 3 should be treated with antiviral drugs independently by staging of liver disease thus avoiding the need of liver biopsy in this clinical setting.
PA.143 PEGINTERFERON A-2B 1 MCGIKG DOSE PLUS RIBAVIRIN 800-1200MG FOR 24-48 WEEKS, ACCORDING TO GENOTYPE, IN NAIVE PATIENTS WITH CHRONIC HEPATITIS C M. Basso 1, A. Grasso 2, G. Percario 3, E. Azzola 4, S. Artioli 5 , N. Pelli 1, S. Bianchi 1 , F. Torre 1, A. Picciotto 1
lDepartment ofInternal Medicine, University of Genova, Genova ofInternal Medicine, Ospedale S Paolo, Savona 3 Department of Gastroenterology, Ospedale San Carlo, Genova 4Department of Gastroenterology, Ospedale Santa Corona, Pietra Ligure 5Department ofInfectious Diseases, Ospedale Civile SAndrea, !.JJ. Spezia 2Department
Background and aim: Dose finding stndy on monotherapy demon-
EFFECT OF A SELECTIVE AGONIST FOR THE ESTROGEN RECEPTOR P(SILYMARIN) UPON COLONIC CELL MIGRATION IN NORMAL MICE A. Di Leo*, M. Barone, M. Margiotta, A. Castellaneta, L. Troiani, L. Demarinis, S. Tanzi, R. Guido, A. Francavilla
Policlinico di Bari, Bari Background and aim: Estrogen receptor beta (ER~) is the predominant isoform of ER in the normal colon and decreases in human and experimental colon cancer althongh its mRNA levels remain unchanged suggesting a regulatory posttranscriptional mechanism (E. Foley et al. Cancer Res 60, 245, 2000). In a murine intestinal tumorigenesis model, Weyant et al.(Cancer Res 61, 2547, 20012) have shown that estrogens protect against tnmor formation and this prevention is associated with an increase of ER~ and cellular migration. The aim of this stndy was to demonstrate the effect of the silymarin (silymarin T, supplied by MADAUS srI, Italy) on cellular migration as well as ER~ protein and mRNA levels in normal mice colonic mucosa Material and methods: We used 36 male mice that were divided in 3 groups of 12 animals each and fed, for 30 days, on 3 different diet regimens: standard CK20 diet (gronp A), CK20 diet and silymarin 200 ppms (gronp B) and CK20 diet and silymarin 600 ppms (gronp C). Cellular migration was evaluated by using the method of Javid et al (Carcinogenesis 26, 587,2004). Briefly, BrdU was injected to all animals (0,75 mgl mouse) and 4 animals for each group were sacrificed at 24,48 and 72 honrs. BrdU nuclear incorporation was eval-
Abstracts
S106
uate by immunoistochemistIy (BrdU detection kit II, Roche, Milan). Cellular migratiou was evaluated by counting the BrdU marked cells and calculating the difference between the marked cells at 24 and 72 hours, expressed as percentage (ll. %). Statistical analysis of the data was performed by Wilcoxon test. ER~ was analyzed by RT-PCR and Western Blotting (anti ER~, Santa Cruz, California). Results: As shown in table, at 72 hours, 9%, 21 % and 40% of colonic marked cells disappeared in A, Band C groups respectively. At the same time points, ER~ mRNA was decreased while protein levels were increased in dose dependent manner.
sub-set of patients with less advanced MSI sporadic disease. In contrast, Pl6 methylation is associated with advanced disease, a feature largely explained by increasing prevalences of advanced stages in females with this epigenetic alteration.
Table 1. BrdU+ cells/lOO cells counted in 8 well oriented cripts
L. Polimeno .,1, B. Pesetti 1, E. Annoscia 1, G. Ingravallo 2,
Tunes
M. Valente 1, A. Amoruso 1, A. Francavilla 1
Group A Group B Group C
24 hrs
48 hes
72 hes
12.2±1.3 13.6±1.0 14.4±1.0
12.4±1.9 13.9±2.1 14.5± 0.6
11.1±1.l 1O.7±2.7 8.7±1.4
-9 -21 -40
Conclusions: Silymarin administration determines an increase of ER~ levels, probably by a post-transcriptional mechanism, and a concomitant increase of the cellular migration. Since these data suggest an anti tumor effect of silymarin, we will test its biological activity in an experimental model of intestinal tumorigenesis
PA.145 INFLUENCE OF GENDER AND OF P16 METHYLATION ON TUMOR STAGE OF COLORECTAL CANCERS WITH MICROSATELLITE INSTABILITY
L. Laghi', P. Bianchi, A.E. Randolph, M. Roncalli, A. Malesci Istituto Clinico Humanitns, Rozzano (MI) Background and aim: hMLHI and Pl6 promoter methylation occur frequently in colon cancers with microsatellite instability (MSI); while hMLH 1 methylation is associated with sporadic MSI, the features of MSI tumors associated with Pl6 methylation are less clear. To evaluate the correlation of hMLHI and Pl6 epigenetic alterations, as to demographics, inheritance, and pathological features of a series of MSI colo-rectal cancers (CRC). Material and methods: We studied hMLHI and Pl6 methylation in 88 (MIF, 50/38) MSI CRC, comprising 25 (MIF, 15/10) hereditary nonpolyposis CRC (Amsterdam Criteria), and 63 (MIF, 34/29) sporadic cases. Tumor were staged by AJCC criteria. Results: Out of 88 MSI CRC, 59 (67%) had methylation of hMLHI (MIF, 28/31; p=O.OI), and 45 (51 %) of Pl6 (MIF, 29/16; hMLHl vs Pl6 methylation in females, p=0.0004). Methylation of hMLHI only occurred in 21 (24%) CRC (MIF, 6/15; p=0.003) and of Pl6 only in 7 (8%; MIF, 7/0; p=O.OI). In 63 sporadic MSI CRC, 49 (77%) showed methylation of hMLHI (MIF 24/25), and 37 (59%) of Pl6 (MIF, 23/14), hMLHI being more frequently methylated than Pl6 in females (p=0.002). Upon univariate analysis, CRC stage 3-4 (29, 33%) were more frequent in males than in females (MIF 21/8; p=0.03), in CRC with methylated than in those with unmethylated Pl6 (21/45, vs 8/43; p=O.OO5), and in proximal than distal tumors (27/69, vs 2/19; p=0.02). In sporadic cases, only Pl6 status was significantly associated with advanced stages (4/26 in un-methylated vs 19/37 in methylated CRC; p=0.003). Upon multivariate analysis, proximal location and Pl6 methylation were independently associated with advanced tumor stage (p=0.04 and p=O.OI, respectively) in all cases, but only Pl6 in sporadic disease (p=0.003). Accordingly, in females, we observed 7 stage 3-4 cancers out of 16 with methylation of both Pl6 and hMLHl, as compared with 1 (7%) out of 15 with only hMLHI methylation (p=0.03). Conclusions: Prevalences of hMLHI methylated MSI CRC are high in old patients with sporadic disease, while Pl6 is more frequently methylated in stage 3-4 than in stage 1-2 cancers. The combination of female gender and hMLHl-only methylated cancers can identify a
PA.146 THE IN VIVO EFFECT OF AUGMENTER OF LIVER REGENERATION (ALR) ON SIGNAL TRANSDUCER AND ACTIVATOR OF TRANSCRIPTION·3 (STAT3) FACTOR
ISezione di Gastroenterologia edEndoscopia Digestiva, DErO, Universitii degli Studi, Bari 2Universitii di Bari, Bari Background and aim: STATs are a family of transcription factors activated by a wide variety of extracellular polypeptides including EGF, PDGF and IL-6. Recent studies have shown a Stat3 activation (Ser- and Tyr-phosphorylation) in the murine partial hepatectomy model (PH) that induces anti-apoptotic factors such as bcl-2, bcl-xL, attributing to Stat3 an anti-apoptotic role in liver regenerative process. Recently, Augmenter of Liver Regeneration (ALR), a hepatic growth factor, has been identified as a control factor of the apoptotic process that occurs after PH. The present work has been done to evaluate if ALR exerts its apoptotic effect through Stat3 activation. Material and methods: Stat3 expression and its activation were evaluated on livers from rAlrp-treated or not animals. Two sets of experiments have been performed. In the first, Stat3 was evaluated, by Western Blot, on hepatic cytosol from 35 intact mice, divided in 7 groups of 5 each. Three groups received, i.p., a single injection ofrAlrp (500 ng) and three groups a same dose of albumin (control). Five treated- and 5 controlmice were sacrificed at 3, 12 and 24 hrs each time. Five animals (time zero) were sacrificed after injection. In the second, Stat3 expression and its phosphorylation were evaluated by immunohistochemistIy techniques on liver sections from 70% PH rats. 35 male rats underwent PH and were divided in 7 groups of 5 animals each. Three groups (treated) were injected, i.p., with multiple doses of rAlrp (500 ng/rat/every 12 hrs) starting from the 18th hour after PH and sacrificed at 24, 36,48 hrs after surgery; three groups (control) received a similar dose of albumin following the same protocol. Five animals (time zero) were sacrificed after PH. Results: The first group of experiments show a statistically significant (p
PA.147 THE HSI,2A ENHANCER ALLELE 2 SHOWS AN INCREASED FREQUENCY IN PATIENTS WITH CROHN'S DISEASE D. Frezza, C. Petruzziello, M. Cretella, E. Calabrese, S. Onali, V. Giambra, C. Mattioli, F. Pallone, L. Biancone
Universitii "Tor Vergatn", Roma Background and aim: The genetics of Crohn's Disease (CD) is characterised by multiple susceptibility loci, genetic heterogeneity and incomplete penetrance. The 3' regulatory complex of the genes of the Ig heavy chain constant region encompass at least 3 enhancers: the central