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Detection of circulating autoantibodies directed against the asialoglycoprotein receptor with a recombinantly expressed subunit Hl
206A
AASLD ABSTRACTS
HEPATOLOGYO c t o b e r 2 0 0 1
129
130
CHOLEDOCHODUODENOSTOMY FOR BILIARY RECONSTRUCTION AFTER LIVER TRANSPLANTATION FOR PSC. Jan M Langrehr, Volker Schmitz, Ulf N e u m a n n , Thomas Steinmueller, Peter Neuhaus, Department of Surgery, Charit4 C a m p u s Virchow Klinikum, H u m b o l d t University Berlin, Berlin G e r m a n y
DETECTION OF CIRCULATING AUTOANTIBODIES DIRECTED AGAINST THE ASIALOGLYCOPROTEIN RECEPTOR W I T H A RECOMBINANTLY EXPRESSED SUBUNIT H1. Thomas Schreiter, Chao Liu, Guido Gerken, Ulrich Treichel, University Hospital Essen, Essen G e r m a n y
Introduction: Biliary reconstruction after liver transplantation for primary sclerosing cholangitis (PSC) today is performed as choledochojejunostomy (CJ) in most transplant centers. An increased risk for cholangitis, a disturbed resorption of immunosuppressive drugs, possible technical difficulties w h e n mobilising intestinal loops due to previous surgical procedures a n d the lack of endoscopic access constitute disadvantages of CJ. In contrast, the choled o c h o d u o d e n o s t o m y (CD) preserves the endoscopic route a n d additionally is a time-saving, simple procedure. Patients a n d methods: Between 8-88 a n d 7-00 we performed 1186 liver transplants (OLT) in 1085 patients. 50 adult patients received a primary graft for PSC. In 6 patients with peripheral bile duct pathology only, we performed a side-to-side c h o l e d o c h o c h o l e d o c h o s t o m y (SS). In 20 patients we performed a CJ a n d in 24 patients a CD was used for biliary reconstruction. Results: Demografic data, s u c h as age, gender, cold ischemic time a n d duration of operation used were similar in all groups. Kaplan-Meier estimates for patient survival after 5 years were 85.7% (SS-group), 84.4% (CJ group) a n d 88.3% (CD-group). Complications are listed in the table. Leakage a n d an unsuccessful endoscopic intervention in the CJ-group led to re-operations ( n = 2), whereas complfcations in the CD-group besides one re-operation for dehiscence could be m a n a g e d endoscopically. Conclusions: O u r results demonstrate that CD is a simple a n d safe alternative for biliary reconstruction after OLT for PSC. Especially the feasibflty of postoperative endoscopic interventions reduces the n u m b e r of re-operations for biliary complications.
Bitiary complications according to biliary reconstruction in liver transplantation for PSC 8ilian/reconstruction Cholangitis Leakage 8tenosis PSCre/ITBL SS(n=6) 1 (14%) none 1 (14%) t (14%) CJ (n=20) 14 (70%) 2 (10%) I (5%) 3 (15%) CD (n=24) 7 (29%} 2 (8.3%) 2 (8.3%) 4 (17%) SS = side-to-sidecholedochocholedochostorny;CJ = choledochojejunoslomy; CD = chotedochoduodenostomy
Introduction: The determination of circulating autoantibodies is a hallmark for the diagnosis of a u t o i m m u n e hepatitis. However, several autoantigens have been described but none of them is diagnostic for the disease. Moreover, indirect immunofluorescence for detecting antinuclear (ANA) and smooth muscle (SMA) antibodies is still the gold standard but difficult to assess. The h u m a n asialoglycoprotein-receptor (ASGPR) has been s h o w n to be a major target antigen in more than 80 precent of patients with a u t o i m m u n e hepatitis. Anti-ASGPR recognize mainly conformatory epitopes of the native receptor. This is an integral m e m b r a n e protein of the liver cell providing the clearance of asialoglycoproteins by receptor-mediated endocytosis. It consists of two subunits H 1 a n d H2 in a ratio of about 5: I. The aim was to prepare a recombinant tool for the detection of anti-ASGPR in patients. Methods a n d results: W e have transfected a h u m a n derived embryo-kidney cell-line with the cDNA of H 1 and ultimatively gained clones with high expression of the protein. By ligandaffinity c h r o m a t o g r a p h y the r e c o m b i n a n t H1 could be prepared by the identical m e t h o d w h i c h was used for receptor purification from h u m a n liver. Using EIA technique with 200ng/~,l of the proteins as coating solution, 178 sera from a s e r u m b a n k with positive anti-ASGPR antibodies were screened for reactivity with r e c o m b i n a n t HI. The sera came from b o t h patients with a u t o i m m u n e hepatitis a n d other forms of chronic inflammatory liver disease. The optical densities of 170 of these sera (95,5%) were in a range -+15% of the values obtained with the complete receptor from n o r m a l h u m a n liver. Only i of 178 (0,6%) was negative on the r e c o m b i n a n t antigen. Sera that became negative for ASGPR from n o r m a l liver due to repeated freezing-thawing cycles or long term storage also showed no reactivity with r e c o m b i n a n t H I . In a smaller g r o u p of 35 sera the titers were determined a n d showed no deviation of more than one dilution step. The reactivity with the ASGPR from normal liver could be blocked b y preineubation of the diluted s e r u m with less than 21ng//~l of rec o m b i n a n t H1. Conclusion: These results show clearly that (i) the antigenic sites of the ASGPR are mainly located in the subunit H 1 a n d (ii) fully processed r e c o m b i n a n t H1 expressed in a m a m m a l i a n cell-line can replace the complete receptor prepared from h u m a n liver tissue. It remains to be determined whether the assay is capable of detecting patients with active a u t o i m m u n e hepatitis w h i c h are positive for both ANA a n d SMA.
I31
132
DETECTION OF ANTI-SLA AUTO-ANTIBODIES TARGETS BY ONEAND TWO-DIMENSIONAL IMMUNOBLOTTING ANALYSIS. Eric Ballot, A r n a u d Bruneel, O a n a Zamfir, Catherine Johanet, HOpital Saint-Antoine, Paris France
USE OF CONTRAST-ENHANCED ECHOCARDIOGRAPHY (BUBBLE ECHOCARDIOGRAM) AS ROUTINE SCREEN FOR ASYMPTOMATIC HEPATOPULMONARY SYNDROME IN PATIENTS U N D E R G O I N G LIVER TRANSPLANT EVALUATION. Dianne L Rudow, Micheal Goldstein, Steven Lobritto, Lori Rosenthal, Mark W Russo, Patricia Harren, Jean Emond, Robert S Brown Jr., Columbia University College of Physicians & Surgeons, New York, NY Hepatopulmonary syndrome (HP8), characterized by a triad of liver dysfunction, intrapulmonary vascular dilatation, and hypoxemia (room air PaO2 <70), is a well-recognizedpulmonary vascular disorder of advanced liver disease. The incidence of HPS in patients meeting criteria for liver transplantation has been reported as high as 50%. Most of these patients present with signs and symptoms of hepatic failure and not from pulmonary decompensation. In fact, many patients with HPS have no pulmonary symptoms. In an era of cost containment, patients being evaluated for liver transplantation are often screened for pulmonary disease with measurement of arterial PaO2 or alveolar-arterialgradient while breathing room air. Pulmonary function tests and bubble echocardiography are only performed in symptomaticpatients or in patients with history of preexisting pulmonary disease. Given the prevalence of HPS, we argue that HPS is under-diagnosed by conventional screening methods in our transplant population. Aim: To analyzewhether contrastenhanced echocardiographyis a useful tool in diagnosing asymptomaticpatients with HPSprior to liver transplantation. Methods: During liver transplant evaluation, we screened 19 consecutive patients for evidence of pulmonary disease with complete pulmonary function testing, contrastenhanced echocardiography or "bubble" echocardingraphy, chest x-ray, and arterial blood gas. Results: Of the 19 patients screened, 17 bad room air PaO2 > 70 (89%), yet 5/17 (29%) exhibited pulmonary symptoms, specificallydyspnea. Symptomaticpatients tended to have a higher mean PaO2 when compared with asymptomatic patients, 89 ± 14 mmHg and 81 +- 15 mmHg respectively, although not statistically significant. Eleven of 19 patients (58%) showed evidence of intrapulmonary shunting on contrast-enhanced echocardiogram. Seven of ll (64%) shunt-positive patients had no pulmonary symptoms.The diffusion capacitiesfor carbon monoxide (DLCO), another indicator of intrapulmonary vascular dilatation, were reduced accordingly in shunt-positive patients compared with patients lacking evidence of shunting on echocardiogram,50% +- 11 of predicted and 66% + 9 of predicted, respectively(p = 0.01). Traditional screening methods for HPS using room air arterial PaO2 failed to identify 10 patients (91%) with significantlyreduced DLCO and intrapulmonary shunting on echocardingram. All but one patient with shunting on echocardiographyhad DLCO < 60% predicted, 1/8 patients without shunting had a DLCO <60% predicted. Conclusion: Screeningfor lIPS with contrast-enhanced echocardiographyis a sensitive test for identification of patients with subclinical hepatopulmonary syndrome. Early identification of HPSis critical for patient outcome. Rapidprogression of disease and development of pulmonary hypertension causing high mortality rates make early intervention vital. Contrast echocardiography should replace standard echocardiographyin routine fiver transplant evaluation.
Auto-antibodies to soluble liver antigen (SLA) were s h o w n to be an u n c o m m o n b u t very specific marker for a u t o - i m m u n e hepatitis type 1 (AIH-1) allowing to reclassify 15% to 20% of hepatitis previously considered as cryptogenic. As anti-SLA antibodies detection needs a complex inhibition ELISA, molecular targets remain controversial despite recent identification of a main anti-SLA auto-antigen as a tRNA-associated 50,000 protein. W e have u n d e r t a k e n to characterize these antigens b y one a n d two dimensional i m m u n o b l o t t i n g analysis. Sera from 44 patients anti-SLA-positive b y ELISA a n d 70 negative controls were analyzed by one- a n d two-dimensional i m m u n o b l o t t i n g using the 100,000 g s u p e m a t a n t of rat liver homogenate. Eighteen percent gave no reaction, 29% (vs 4% of controls, p < 0.001) stained a 58,000 protein focusing at pl between 6.5 - 7.0, 68% (vs 8.5% of controls, p < 0.0001) a 50,000 protein at pI 6.0 - 6.5 a n d 29% (vs 7%, p < 0.001) a 35,000 protein at pI a r o u n d 6.0. The patterns of controls were different from anti-SLA positive sera. In conclusion, anti-SLA targets were heterogeneous a n d not restricted to the main 50,000 protein. Furthermore, some epitopes seemed conformational a n d were n o t recognized by i m m u n o b l o t performed with denaturated proteins. The high percentage of non-reactive sera by i m m u n o b l o t still incites us to use the reference inhibition ELISA as detection test.
PATIENT CHARACTERISTICS POSITIVE BUBBLE ECHO (tl) PaO2(roomair) <70 1 (t1%) DLCO <60% 10 (90%) Dyspnea 4 (37%)
NEGATIVE BUBBLE ECHO (8)_ 1 (12%) 1 (12%) 2 (25%)
AASLD ABSTRACTS
HEPATOLOGYO c t o b e r 2 0 0 1
129
130
CHOLEDOCHODUODENOSTOMY FOR BILIARY RECONSTRUCTION AFTER LIVER TRANSPLANTATION FOR PSC. Jan M Langrehr, Volker Schmitz, Ulf N e u m a n n , Thomas Steinmueller, Peter Neuhaus, Department of Surgery, Charit4 C a m p u s Virchow Klinikum, H u m b o l d t University Berlin, Berlin G e r m a n y
DETECTION OF CIRCULATING AUTOANTIBODIES DIRECTED AGAINST THE ASIALOGLYCOPROTEIN RECEPTOR W I T H A RECOMBINANTLY EXPRESSED SUBUNIT H1. Thomas Schreiter, Chao Liu, Guido Gerken, Ulrich Treichel, University Hospital Essen, Essen G e r m a n y
Introduction: Biliary reconstruction after liver transplantation for primary sclerosing cholangitis (PSC) today is performed as choledochojejunostomy (CJ) in most transplant centers. An increased risk for cholangitis, a disturbed resorption of immunosuppressive drugs, possible technical difficulties w h e n mobilising intestinal loops due to previous surgical procedures a n d the lack of endoscopic access constitute disadvantages of CJ. In contrast, the choled o c h o d u o d e n o s t o m y (CD) preserves the endoscopic route a n d additionally is a time-saving, simple procedure. Patients a n d methods: Between 8-88 a n d 7-00 we performed 1186 liver transplants (OLT) in 1085 patients. 50 adult patients received a primary graft for PSC. In 6 patients with peripheral bile duct pathology only, we performed a side-to-side c h o l e d o c h o c h o l e d o c h o s t o m y (SS). In 20 patients we performed a CJ a n d in 24 patients a CD was used for biliary reconstruction. Results: Demografic data, s u c h as age, gender, cold ischemic time a n d duration of operation used were similar in all groups. Kaplan-Meier estimates for patient survival after 5 years were 85.7% (SS-group), 84.4% (CJ group) a n d 88.3% (CD-group). Complications are listed in the table. Leakage a n d an unsuccessful endoscopic intervention in the CJ-group led to re-operations ( n = 2), whereas complfcations in the CD-group besides one re-operation for dehiscence could be m a n a g e d endoscopically. Conclusions: O u r results demonstrate that CD is a simple a n d safe alternative for biliary reconstruction after OLT for PSC. Especially the feasibflty of postoperative endoscopic interventions reduces the n u m b e r of re-operations for biliary complications.
Bitiary complications according to biliary reconstruction in liver transplantation for PSC 8ilian/reconstruction Cholangitis Leakage 8tenosis PSCre/ITBL SS(n=6) 1 (14%) none 1 (14%) t (14%) CJ (n=20) 14 (70%) 2 (10%) I (5%) 3 (15%) CD (n=24) 7 (29%} 2 (8.3%) 2 (8.3%) 4 (17%) SS = side-to-sidecholedochocholedochostorny;CJ = choledochojejunoslomy; CD = chotedochoduodenostomy
Introduction: The determination of circulating autoantibodies is a hallmark for the diagnosis of a u t o i m m u n e hepatitis. However, several autoantigens have been described but none of them is diagnostic for the disease. Moreover, indirect immunofluorescence for detecting antinuclear (ANA) and smooth muscle (SMA) antibodies is still the gold standard but difficult to assess. The h u m a n asialoglycoprotein-receptor (ASGPR) has been s h o w n to be a major target antigen in more than 80 precent of patients with a u t o i m m u n e hepatitis. Anti-ASGPR recognize mainly conformatory epitopes of the native receptor. This is an integral m e m b r a n e protein of the liver cell providing the clearance of asialoglycoproteins by receptor-mediated endocytosis. It consists of two subunits H 1 a n d H2 in a ratio of about 5: I. The aim was to prepare a recombinant tool for the detection of anti-ASGPR in patients. Methods a n d results: W e have transfected a h u m a n derived embryo-kidney cell-line with the cDNA of H 1 and ultimatively gained clones with high expression of the protein. By ligandaffinity c h r o m a t o g r a p h y the r e c o m b i n a n t H1 could be prepared by the identical m e t h o d w h i c h was used for receptor purification from h u m a n liver. Using EIA technique with 200ng/~,l of the proteins as coating solution, 178 sera from a s e r u m b a n k with positive anti-ASGPR antibodies were screened for reactivity with r e c o m b i n a n t HI. The sera came from b o t h patients with a u t o i m m u n e hepatitis a n d other forms of chronic inflammatory liver disease. The optical densities of 170 of these sera (95,5%) were in a range -+15% of the values obtained with the complete receptor from n o r m a l h u m a n liver. Only i of 178 (0,6%) was negative on the r e c o m b i n a n t antigen. Sera that became negative for ASGPR from n o r m a l liver due to repeated freezing-thawing cycles or long term storage also showed no reactivity with r e c o m b i n a n t H I . In a smaller g r o u p of 35 sera the titers were determined a n d showed no deviation of more than one dilution step. The reactivity with the ASGPR from normal liver could be blocked b y preineubation of the diluted s e r u m with less than 21ng//~l of rec o m b i n a n t H1. Conclusion: These results show clearly that (i) the antigenic sites of the ASGPR are mainly located in the subunit H 1 a n d (ii) fully processed r e c o m b i n a n t H1 expressed in a m a m m a l i a n cell-line can replace the complete receptor prepared from h u m a n liver tissue. It remains to be determined whether the assay is capable of detecting patients with active a u t o i m m u n e hepatitis w h i c h are positive for both ANA a n d SMA.
I31
132
DETECTION OF ANTI-SLA AUTO-ANTIBODIES TARGETS BY ONEAND TWO-DIMENSIONAL IMMUNOBLOTTING ANALYSIS. Eric Ballot, A r n a u d Bruneel, O a n a Zamfir, Catherine Johanet, HOpital Saint-Antoine, Paris France
USE OF CONTRAST-ENHANCED ECHOCARDIOGRAPHY (BUBBLE ECHOCARDIOGRAM) AS ROUTINE SCREEN FOR ASYMPTOMATIC HEPATOPULMONARY SYNDROME IN PATIENTS U N D E R G O I N G LIVER TRANSPLANT EVALUATION. Dianne L Rudow, Micheal Goldstein, Steven Lobritto, Lori Rosenthal, Mark W Russo, Patricia Harren, Jean Emond, Robert S Brown Jr., Columbia University College of Physicians & Surgeons, New York, NY Hepatopulmonary syndrome (HP8), characterized by a triad of liver dysfunction, intrapulmonary vascular dilatation, and hypoxemia (room air PaO2 <70), is a well-recognizedpulmonary vascular disorder of advanced liver disease. The incidence of HPS in patients meeting criteria for liver transplantation has been reported as high as 50%. Most of these patients present with signs and symptoms of hepatic failure and not from pulmonary decompensation. In fact, many patients with HPS have no pulmonary symptoms. In an era of cost containment, patients being evaluated for liver transplantation are often screened for pulmonary disease with measurement of arterial PaO2 or alveolar-arterialgradient while breathing room air. Pulmonary function tests and bubble echocardiography are only performed in symptomaticpatients or in patients with history of preexisting pulmonary disease. Given the prevalence of HPS, we argue that HPS is under-diagnosed by conventional screening methods in our transplant population. Aim: To analyzewhether contrastenhanced echocardiographyis a useful tool in diagnosing asymptomaticpatients with HPSprior to liver transplantation. Methods: During liver transplant evaluation, we screened 19 consecutive patients for evidence of pulmonary disease with complete pulmonary function testing, contrastenhanced echocardiography or "bubble" echocardingraphy, chest x-ray, and arterial blood gas. Results: Of the 19 patients screened, 17 bad room air PaO2 > 70 (89%), yet 5/17 (29%) exhibited pulmonary symptoms, specificallydyspnea. Symptomaticpatients tended to have a higher mean PaO2 when compared with asymptomatic patients, 89 ± 14 mmHg and 81 +- 15 mmHg respectively, although not statistically significant. Eleven of 19 patients (58%) showed evidence of intrapulmonary shunting on contrast-enhanced echocardiogram. Seven of ll (64%) shunt-positive patients had no pulmonary symptoms.The diffusion capacitiesfor carbon monoxide (DLCO), another indicator of intrapulmonary vascular dilatation, were reduced accordingly in shunt-positive patients compared with patients lacking evidence of shunting on echocardiogram,50% +- 11 of predicted and 66% + 9 of predicted, respectively(p = 0.01). Traditional screening methods for HPS using room air arterial PaO2 failed to identify 10 patients (91%) with significantlyreduced DLCO and intrapulmonary shunting on echocardingram. All but one patient with shunting on echocardiographyhad DLCO < 60% predicted, 1/8 patients without shunting had a DLCO <60% predicted. Conclusion: Screeningfor lIPS with contrast-enhanced echocardiographyis a sensitive test for identification of patients with subclinical hepatopulmonary syndrome. Early identification of HPSis critical for patient outcome. Rapidprogression of disease and development of pulmonary hypertension causing high mortality rates make early intervention vital. Contrast echocardiography should replace standard echocardiographyin routine fiver transplant evaluation.
Auto-antibodies to soluble liver antigen (SLA) were s h o w n to be an u n c o m m o n b u t very specific marker for a u t o - i m m u n e hepatitis type 1 (AIH-1) allowing to reclassify 15% to 20% of hepatitis previously considered as cryptogenic. As anti-SLA antibodies detection needs a complex inhibition ELISA, molecular targets remain controversial despite recent identification of a main anti-SLA auto-antigen as a tRNA-associated 50,000 protein. W e have u n d e r t a k e n to characterize these antigens b y one a n d two dimensional i m m u n o b l o t t i n g analysis. Sera from 44 patients anti-SLA-positive b y ELISA a n d 70 negative controls were analyzed by one- a n d two-dimensional i m m u n o b l o t t i n g using the 100,000 g s u p e m a t a n t of rat liver homogenate. Eighteen percent gave no reaction, 29% (vs 4% of controls, p < 0.001) stained a 58,000 protein focusing at pl between 6.5 - 7.0, 68% (vs 8.5% of controls, p < 0.0001) a 50,000 protein at pI 6.0 - 6.5 a n d 29% (vs 7%, p < 0.001) a 35,000 protein at pI a r o u n d 6.0. The patterns of controls were different from anti-SLA positive sera. In conclusion, anti-SLA targets were heterogeneous a n d not restricted to the main 50,000 protein. Furthermore, some epitopes seemed conformational a n d were n o t recognized by i m m u n o b l o t performed with denaturated proteins. The high percentage of non-reactive sera by i m m u n o b l o t still incites us to use the reference inhibition ELISA as detection test.
PATIENT CHARACTERISTICS POSITIVE BUBBLE ECHO (tl) PaO2(roomair) <70 1 (t1%) DLCO <60% 10 (90%) Dyspnea 4 (37%)
NEGATIVE BUBBLE ECHO (8)_ 1 (12%) 1 (12%) 2 (25%)