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TIMP-1 is expressed by non parenchymal cells in experimental liver injury; implications for matrix remodelling in progressive hepatic fibrosis
April 1995
AASLD
A1089
• BILE FLOW (BF), BILE ACID-INDEPENDENT BILE FLOW (BAIF), AND BILE ACID-DEPENDENT BILE FLOW (BADF) FOLLOWING O R T H O T O P I C R A T LIVER T R A N S P L A N T A T I O N . H. Imamura A. Brault, P.M. Huet, CRC A.-Viallet, U. de Montrtal, Montrtal, Canada. Bile formation is an active process which depends on 2 main hepatocyte transport mechanisms responsible for a BAIF and a BADF. It has been reported that, following human liver transplantation, BAIF recovery is delayed while BADF appears to be even higher than expected. We evaluated these parameters using isolated-perfused rat livers following Lewis-to-Lewis liver transplantation after 24-hr preservation in UW solution (4°C). In our laboratory, this preservation results in a 40 % survival rate, all non-survivors dying within the first 24 hr. Five groups of 8 rats (A-E) were studied. In group A, livers were perfused under baseline conditions; in group B, perfusions were done immediately after the preservation; in groups C, D and E, liver transplantation was performed after the preservation, then livers were perfused in situ 4 hr (C), 24 hr (D), and 4 days (E) after surgery. Perfusions were performed for 40 min using Krebsalbumin with erythrocytes (20%) and taurocholate (TC,21.4p_M) with a tracer dose of ~4C labeled TC. BAIF and BADF were calculated using linear regression between bile salt output (BSO) and BF, as the y-intercept and from the slope, respectively. BF was moderately but non significantly affected by cold preservation alone (- 18% group B vs A; p=NS) but decreased markedly 4 hr following transplantation (-64% vs A; p<0.001). BF showed signs of recovery in survivors (group D:-37% p<0.01), and remained altered until 4 days after transplantation (group E:-41% p<0.01). To the contrary, BAIF was altered by cold ischemia itself (-86% group B vs A;p<0.001), and it did not recover up to the 4 ~ day after surgery in survivors (group D:-81% and group E:-80%; p<0.01 vs A). Paradoxically, BADF increased following cold ischemia (+ 300% group B vs A; p<0.001), and increased even more after surgery in survivors (group D:+450% and group E:+450%; p<0.001 vs A). BAIF and BADF could not be calculated in group C, no regression being obtained between BS and BSO (probably because of the combination of survivors and non-survivors). The present data show that, BAIF is markedly altered after liver transplantation following prolonged cold ischemia in UW and does not recover rapidly. By contrast, BADF is already increased following cold preservation and further increases after transplantation as found in humans. The hypercholeretic effect of bile salts following transplantation remains to be further explored.
• DETECTION OF HBV-DNA BY PCR IN SERUM AND LIVER DURING THE FOLLOW LIP OF HBsAg POSITIVE TRANSPLANTED PATIENTS RECEIVING HEPATITIS B IMMUNOGLOBULIN PROPHYLAXIS
• A U T O I M M U N E C H O L A N G I T I S : AN I T A L I A N EXPERIENCE. P. Invernizzi A. Crosignani, P.M. Battezzati, G. De Valle, *G Covini, M, Zuin and M Podda. Institute of Internal Medicine, School of Medicine Ospedale San Paolo, and *Institute of Internal Medicine I, University of Milan, Italy.
e TIMP-1 IS EXPRESSED BY NON PARENCHYMAL CELLS IN EXPERIMENTAL LIVER INJURY; IMPLICATIONS FOR MATRIX REMODELLING IN PROGRESSIVE HEPATIC FIBROSIS. J P Iredale, N Clark, R C Benyon, R Alcolado, W F Ferris, M J P Arthur. University Medicine, University of Southampton.
Recently, some authors have described a subgroup of patients with clinical and histological diagnosis of PBC, who tested negative for antimithocondrial (AMA) and positive for antinuclear antibodies (ANA). They have suggested the existence of a new syndrome distinct from PBC, defined as "autoimmune cholangitis". However, in different geographic areas several features of autoimmune chronic liver disease may vary and no data coming from large series of patients from Southern Europe are available so far. We evaluated the prevalence of AMA by immunofluorescence assay in a series of 297 Italian PBC patients who consecutively presented at our center from June 1974 to June 1994 (89% women; mean age±SD, 54±11 yrs). We have also compared the main biochemical, serological and clinical features between the AMA +ve and AMA -ve groups at presentation and, in addition, we have retrospectively evaluated the clinical outcome of the disease during follow up. Twenty-four out of our 297 patients (8%) tested negative for AMA. There were no significant differences between AMA +ve and AMA -ve patients with respect to age, serum alkaline phosphatase, ALT, total bilirubin, albumin, prothrombin time (PT), total and HDL cholesterol, triglycerides and immunoglobulins (IgG and IgA). We only found a trend towards significantly higher values of IgM in AMA +re patients (591±554 vs 409±241; p=0.052). ANA were present in 71% of AMA -ve and in 31% of AMA +ve patients (p <0.001), while anti-smooth muscle antibodies tested positive in 37% of AMA -ve and in 9% of AMA +ve patients (p
P. Inturri P.Burra, P.Pontisso, R.M.Jemmolo, L.Rossaro, A.Graziotto, M.ORuvolatto, S.Faginali, U.Tedeschi, A.Alberti, R.Naccarato Gastroenterology Unit, Clinical Medicine Institute, Internal Medicine Institute, Surgery Department, University of Padua, Italy Liver transplantation (OLT) in FIBsAg positive patients is associated with high risk of reinfection and disease recurrence in the new graft. For these reasons, hepatitis B remains a controversial indication for OLT. However it has also been reported low rate of HBV recurrence in HBsAg positive/HBV-DNA negative patients who were given long-term (> 6 months) passive tmmunoprophylaxis after liver transplant The aim of the present study was to evaluate the rate of HBV reinfection after OLT in patients selected on the basis of undetectable serum HBVDNA, assessed by PCR, using primers from the S gene sequence. Thirty-six consecutive candidates for OLT were studied, 16 tmderwent OLT, Five out of 16 were transplanted before the selection/ mununopruphylaxis protocol was started. Four of them survived aiter surgery and reinfection of the graft occurred in three leading to death in the first, to reOLT in the second and to CAH in the third case. Subsequently, 11 FIBsAg positive / FIBV-DNA negative patients prior O L T were then followed up, all receiving passive unmunoprophylaxis (10.000 IU anti-HBs lg iv daily during the first 10 postoperative days, 10.000 IU iv weekly for 4 wks, 1500 IU irn weekly afterwards). Patients were studied at 3, 6 and 12 months after OLT. All of them were anti-FIBs positive/ HBV-DNA negative in serum at 3 and 6 months. At 6 months the presence of HBV-DNA was evaluated in liver biopsy of 10/11 patients and was andetectable in all cases, excluding a latent state of the virus in the liver. Seven patients reached 12 months of follow up, 6 of them are still I-IBVDNA negative in serum, whereas one patient is HBsAg /HBV-DNA positive; interestingly this patient has developed anti-HCV antibodies at the same time. In conclusion, our results demonstrate that proper selection of HBV patients and lmmunoprophylaxis with high dose of Ig are asseciated with no recurrence of HBV infection at 6 months and low rate of recarrence at 12 months after OLT.
Liver fibrosis results from the relative imbalance between matrix synthesis and degradation. We have previously described release of the potent collagenase inhibitor, Tissue inhibitor of metalloproteinase-1 (TIMP-1) by culture activated hepatic lipocytes. In these studies we have examined TIMP-1 expression relative to interstitial collagenase (IC) and procollagen-1 in culture activated lipocytes, experimental hepatic fibrosis and in individual liver cell fractions following acute CCI 4 intoxication. Total RNA from rat hepatic lipocytes, obtained by pronase/collagenase perfusion of normal rat liver, was subjected to Northern analysis. TIMP-1 expression was found to be upregulated with activation by culture on plastic as defined by procollagen-1 expression. In contrast, IC mRNA was expressed in early culture ( < 4 days) becoming undetectable in more activated cells (7-21 days). Expression of TIMP-1, interstitial collagenase, and procollagen-I mRNAs were studied by ribonuclease protection analysis of total liver RNA from rat models of parenchymal (IP CC14 2 x weekly) and biliary injury (bile duct ligation). TIMP-1 mRNA was demonstrated to rise in acute CC14 liver injury (24-72 hours post first injection), then decrease to control levels in early fibrosis (1 week post first injection), before becoming persistently elevated to at least 4 x control values during progressive fibrosis (4 weeks post first injection). TIMP-1 expression was also enhanced relative to control at 6, 24 hours and 3 days after bile duct ligation. Moreover, expression of T1MP-1 predated procollagen-1 in both models. In contrast, IC mRNA levels remained similar to control values after IP CC14 and bile duct ligation throughout both models of injury. To determine the cellular source(s) of TIMP-I during liver injury in vivo; total cellular RNA was extracted from freshly isolated hepatocytes, lipocytes and Kupffer cells prepared from rat livers 48 hours after IP CC14. By Northern analysis, TIMP-1 transcripts were observed in RNA from lipocytes and Kupffer cells only. Taken together, these data indicate that TIMP-1 expression by hepatic lipocytes may promote progression of liver fibrosis by preventing remodelling of secreted coUagen-1 during liver injury in vivo.
AASLD
A1089
• BILE FLOW (BF), BILE ACID-INDEPENDENT BILE FLOW (BAIF), AND BILE ACID-DEPENDENT BILE FLOW (BADF) FOLLOWING O R T H O T O P I C R A T LIVER T R A N S P L A N T A T I O N . H. Imamura A. Brault, P.M. Huet, CRC A.-Viallet, U. de Montrtal, Montrtal, Canada. Bile formation is an active process which depends on 2 main hepatocyte transport mechanisms responsible for a BAIF and a BADF. It has been reported that, following human liver transplantation, BAIF recovery is delayed while BADF appears to be even higher than expected. We evaluated these parameters using isolated-perfused rat livers following Lewis-to-Lewis liver transplantation after 24-hr preservation in UW solution (4°C). In our laboratory, this preservation results in a 40 % survival rate, all non-survivors dying within the first 24 hr. Five groups of 8 rats (A-E) were studied. In group A, livers were perfused under baseline conditions; in group B, perfusions were done immediately after the preservation; in groups C, D and E, liver transplantation was performed after the preservation, then livers were perfused in situ 4 hr (C), 24 hr (D), and 4 days (E) after surgery. Perfusions were performed for 40 min using Krebsalbumin with erythrocytes (20%) and taurocholate (TC,21.4p_M) with a tracer dose of ~4C labeled TC. BAIF and BADF were calculated using linear regression between bile salt output (BSO) and BF, as the y-intercept and from the slope, respectively. BF was moderately but non significantly affected by cold preservation alone (- 18% group B vs A; p=NS) but decreased markedly 4 hr following transplantation (-64% vs A; p<0.001). BF showed signs of recovery in survivors (group D:-37% p<0.01), and remained altered until 4 days after transplantation (group E:-41% p<0.01). To the contrary, BAIF was altered by cold ischemia itself (-86% group B vs A;p<0.001), and it did not recover up to the 4 ~ day after surgery in survivors (group D:-81% and group E:-80%; p<0.01 vs A). Paradoxically, BADF increased following cold ischemia (+ 300% group B vs A; p<0.001), and increased even more after surgery in survivors (group D:+450% and group E:+450%; p<0.001 vs A). BAIF and BADF could not be calculated in group C, no regression being obtained between BS and BSO (probably because of the combination of survivors and non-survivors). The present data show that, BAIF is markedly altered after liver transplantation following prolonged cold ischemia in UW and does not recover rapidly. By contrast, BADF is already increased following cold preservation and further increases after transplantation as found in humans. The hypercholeretic effect of bile salts following transplantation remains to be further explored.
• DETECTION OF HBV-DNA BY PCR IN SERUM AND LIVER DURING THE FOLLOW LIP OF HBsAg POSITIVE TRANSPLANTED PATIENTS RECEIVING HEPATITIS B IMMUNOGLOBULIN PROPHYLAXIS
• A U T O I M M U N E C H O L A N G I T I S : AN I T A L I A N EXPERIENCE. P. Invernizzi A. Crosignani, P.M. Battezzati, G. De Valle, *G Covini, M, Zuin and M Podda. Institute of Internal Medicine, School of Medicine Ospedale San Paolo, and *Institute of Internal Medicine I, University of Milan, Italy.
e TIMP-1 IS EXPRESSED BY NON PARENCHYMAL CELLS IN EXPERIMENTAL LIVER INJURY; IMPLICATIONS FOR MATRIX REMODELLING IN PROGRESSIVE HEPATIC FIBROSIS. J P Iredale, N Clark, R C Benyon, R Alcolado, W F Ferris, M J P Arthur. University Medicine, University of Southampton.
Recently, some authors have described a subgroup of patients with clinical and histological diagnosis of PBC, who tested negative for antimithocondrial (AMA) and positive for antinuclear antibodies (ANA). They have suggested the existence of a new syndrome distinct from PBC, defined as "autoimmune cholangitis". However, in different geographic areas several features of autoimmune chronic liver disease may vary and no data coming from large series of patients from Southern Europe are available so far. We evaluated the prevalence of AMA by immunofluorescence assay in a series of 297 Italian PBC patients who consecutively presented at our center from June 1974 to June 1994 (89% women; mean age±SD, 54±11 yrs). We have also compared the main biochemical, serological and clinical features between the AMA +ve and AMA -ve groups at presentation and, in addition, we have retrospectively evaluated the clinical outcome of the disease during follow up. Twenty-four out of our 297 patients (8%) tested negative for AMA. There were no significant differences between AMA +ve and AMA -ve patients with respect to age, serum alkaline phosphatase, ALT, total bilirubin, albumin, prothrombin time (PT), total and HDL cholesterol, triglycerides and immunoglobulins (IgG and IgA). We only found a trend towards significantly higher values of IgM in AMA +re patients (591±554 vs 409±241; p=0.052). ANA were present in 71% of AMA -ve and in 31% of AMA +ve patients (p <0.001), while anti-smooth muscle antibodies tested positive in 37% of AMA -ve and in 9% of AMA +ve patients (p
P. Inturri P.Burra, P.Pontisso, R.M.Jemmolo, L.Rossaro, A.Graziotto, M.ORuvolatto, S.Faginali, U.Tedeschi, A.Alberti, R.Naccarato Gastroenterology Unit, Clinical Medicine Institute, Internal Medicine Institute, Surgery Department, University of Padua, Italy Liver transplantation (OLT) in FIBsAg positive patients is associated with high risk of reinfection and disease recurrence in the new graft. For these reasons, hepatitis B remains a controversial indication for OLT. However it has also been reported low rate of HBV recurrence in HBsAg positive/HBV-DNA negative patients who were given long-term (> 6 months) passive tmmunoprophylaxis after liver transplant The aim of the present study was to evaluate the rate of HBV reinfection after OLT in patients selected on the basis of undetectable serum HBVDNA, assessed by PCR, using primers from the S gene sequence. Thirty-six consecutive candidates for OLT were studied, 16 tmderwent OLT, Five out of 16 were transplanted before the selection/ mununopruphylaxis protocol was started. Four of them survived aiter surgery and reinfection of the graft occurred in three leading to death in the first, to reOLT in the second and to CAH in the third case. Subsequently, 11 FIBsAg positive / FIBV-DNA negative patients prior O L T were then followed up, all receiving passive unmunoprophylaxis (10.000 IU anti-HBs lg iv daily during the first 10 postoperative days, 10.000 IU iv weekly for 4 wks, 1500 IU irn weekly afterwards). Patients were studied at 3, 6 and 12 months after OLT. All of them were anti-FIBs positive/ HBV-DNA negative in serum at 3 and 6 months. At 6 months the presence of HBV-DNA was evaluated in liver biopsy of 10/11 patients and was andetectable in all cases, excluding a latent state of the virus in the liver. Seven patients reached 12 months of follow up, 6 of them are still I-IBVDNA negative in serum, whereas one patient is HBsAg /HBV-DNA positive; interestingly this patient has developed anti-HCV antibodies at the same time. In conclusion, our results demonstrate that proper selection of HBV patients and lmmunoprophylaxis with high dose of Ig are asseciated with no recurrence of HBV infection at 6 months and low rate of recarrence at 12 months after OLT.
Liver fibrosis results from the relative imbalance between matrix synthesis and degradation. We have previously described release of the potent collagenase inhibitor, Tissue inhibitor of metalloproteinase-1 (TIMP-1) by culture activated hepatic lipocytes. In these studies we have examined TIMP-1 expression relative to interstitial collagenase (IC) and procollagen-1 in culture activated lipocytes, experimental hepatic fibrosis and in individual liver cell fractions following acute CCI 4 intoxication. Total RNA from rat hepatic lipocytes, obtained by pronase/collagenase perfusion of normal rat liver, was subjected to Northern analysis. TIMP-1 expression was found to be upregulated with activation by culture on plastic as defined by procollagen-1 expression. In contrast, IC mRNA was expressed in early culture ( < 4 days) becoming undetectable in more activated cells (7-21 days). Expression of TIMP-1, interstitial collagenase, and procollagen-I mRNAs were studied by ribonuclease protection analysis of total liver RNA from rat models of parenchymal (IP CC14 2 x weekly) and biliary injury (bile duct ligation). TIMP-1 mRNA was demonstrated to rise in acute CC14 liver injury (24-72 hours post first injection), then decrease to control levels in early fibrosis (1 week post first injection), before becoming persistently elevated to at least 4 x control values during progressive fibrosis (4 weeks post first injection). TIMP-1 expression was also enhanced relative to control at 6, 24 hours and 3 days after bile duct ligation. Moreover, expression of T1MP-1 predated procollagen-1 in both models. In contrast, IC mRNA levels remained similar to control values after IP CC14 and bile duct ligation throughout both models of injury. To determine the cellular source(s) of TIMP-I during liver injury in vivo; total cellular RNA was extracted from freshly isolated hepatocytes, lipocytes and Kupffer cells prepared from rat livers 48 hours after IP CC14. By Northern analysis, TIMP-1 transcripts were observed in RNA from lipocytes and Kupffer cells only. Taken together, these data indicate that TIMP-1 expression by hepatic lipocytes may promote progression of liver fibrosis by preventing remodelling of secreted coUagen-1 during liver injury in vivo.