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Gene transfer to primate liver using second generation recombinant adenovirus
April 1 9 9 5
A R E LARGE P A R A C E N T H E S I S THE B E S T T R E A T M E N T FOR R E F R A C T O R Y A S C I T E S IN P A T I E N T S W I T H C I R R H O S I S OF THE LIVER ? O.NOUEL, D.BOUTROUX, M.LE BRIS, M.DARTOIS. Hepatology Unit. General Hospital-STBRIEUC- FRANCED u r i n g a four y e a r s period, 783 p a t i e n t s with c i r r h o s i s of the liver w e r e ~ a d m i t t e d in the same H e p a t o l o g y Unit. 483 (61%) p a t i e n t s w e r e a s c i t i c and 17 (3,5%) h a d a r e f r a c t o r y ascites. R e f r a c t o r y a s c i t e s w a s d e f i n e d b y a) i m p o s s i b i l i t y to use diuretics (hyponatremia, res±stance, renal insuffisiency), b) necessity of therapeutic paracenthesis d u r i n g a p e r i o d of three m o n t h c) absence of c l i n i c a l or p a r a c l i n i c a l facts for hepatocarcinoma. Moreover these 17 patients, 16 had a l c o h o l i c c i r r h o s i s and 1 H C V + cirrhosis. After the period test of three month, paracenthesis w e r e p e r f o r m e d e v e r y w e e k for out patients with or without volumetric compensation (2 and 15 p a t i e n t s respectively). The p a t i e n t s w e r e p e r i o d i c l y e x a m i n a t e d d u r i n g a minimal p e r i o d of two years. A m o n g the 16 a l c o h o l i c patients, i0 w e r e no abstinent. F o u r p a t i e n t s died, three w i t h s e v e r e b a c t e r i a l i n f e c t i o n and one (HCV +) a f t e r liver transplantation. For 9 patients, we w e r e surprised to observe a total disparition of a s c i t e s d u r i n g t h e p e r i o d of survey. Moreover, one p a t i e n t c u r e d a b r u p t l y after a 5 years of w e e k l y therapeutic paracenthesis, always as out-patient. C o n c l u s i o n : R e f r a c t o r y ascites is a rare (3,5%) condition. It is not usually admitted that r e f r a c t o r y a s c i t e s do c u r e spontaneously. In our e x P e r i e n c e it is m o s t frequent. This fact should influence therapeutic choice. All therapeutic studies with aggressive procedures ( p e r i t o n e o j u g u l a r shunts, TIPS, portal d e r i v a t i o n s or transplantation) should be matched to p a r a c e n t h e s i s in term of s a f e t y and cost,
• GENE TRANSFER TO PRIMATE LIVER USING SECOND GENERATION RECOMBINANT ADENOVlRUS. F.A, Nunesl, 2, S.E. Raper 1, J.M. Wilson 1. 1Institute for Human Gene Therapy and Division of 2Gastroenterology, University of Pennsylvania, Philadelphia, PA Recombinant adenovirus has clinical potential for liver gene therapy since it can efficiently transfer genes to quiescent hepatocytes'and be produced in high titer and purity. First generation recombinant adenovirus, deleted in the E l a and E l b regions of the adenovirus genome, has been shown to produce high but transient expression in mouse liver, Loss of expression is associated with hepatic inflammation and a cytotoxic lymphocyte response against newly synthesized antigens. In contrast, a more crippled second generation adenovirus, containing a temperature sensitive mutation in the E2a gene, leads to longer duration of expression and less inflammation in mouse liver. In this study the safety and duration of expression of this second generation vector was tested in non-human primate liver. Juvenile rhesus monkeys (n=4) weighing 3-5 kg underwent laparotomy, cannulation of the portal vein and an infusion of 1 X 1013 particles/kg of second generation adenovirus carrying the lacZ reporter gene. Hematologic, biochemical and immunologic parameters were assayed at three days then weekly post infusion. Necropsies were performed on days 3, 7 and 28 after adenovirus administration. Transgene expression in the liver, determined by X-Gal histochemical staining, was high on days 3 and 7 but minimal by day 28 post infusion. Transgene expression was also detectable in the spleen and rarely in the vascular endothelium of thoracic and abdominal organs. Biochemical evidence of liver inflammation developed by day 7, was maximal at day 14 and resolved by day 28 post infusion. There was minimal histologic inflammation on days 3, 7 and 28 post adenovirus administration. The monkey necropsied on day 28 developed a neutralizing antibody response to the recombinant adenovirus by day 21 post infusion. Recombinant adenovirus was not recovered in the pharynx, urine or stool of the animals. Primate data suggests that second generation recombinant adenovirus may be a useful gene therapy vector for the short term treatment of metabolic liver diseases.
AASLD
Al135
DENTAL AND PERIODONTAL DISEASE iN PATIENTS WITH LIVER CIRRHOSIS - ROLE OF ALCOHOL ABUSE. GiNovace.._kk, U. NovacekPlachetzky, R. P~tzi, R. Slevicek, S. Lentner, A. Gangl, P. Ferenci. Department of Internal Medicine IV, University of Vienna, Austria. Dental foci occurs frequently in patients with cirrhosis and may be e potential source for infections, especially in transplant candidates. The aim of the study was to assess the rote of cirrhosis and chronic alcoholism for the development of dental or periodontal disease. Dental (total number of teeth, number of carious teeth and index of oral hygiene = plaque free surface of all teeth in %) and periodontal (loss of attachment = distance of the cemento-enamel junction to the bottom of the clinical pocket in mm) examinations were performed in 97 patients with cirrhosis [alcoholic (A.CIRR): 64, nonalcoholic (NA.CIRR): 33], in 68 alcoholics (ALCO) without cirrhosis and in 71 age matched healthy controls. Results: ALCO and A.CIRR had a lower total number of teeth than NA.CIRR and controls (p<0.05). The number of carious teeth was larger in ALCO (% of remaining teeth: 12.7%, p<0.001) and A.CIRR (21.3%, p<0.001) than in NA.CIRR (1.9%) and controls (1.2%). The time since the last der~tal examination was longer in ALCO (median: 12 months; interquartile range: 4-36 months; p<0.001)and in A.CIRR (21; 695 months; p<0.001) than in NA.CIRR (6; 2-7 months) and in controls (5; 2-10 months) The results of oral hygiene and periodontal disease are given in the table. There was no difference between NA.CIRR and controls in the occurrence of dental and periodontal disease. Severity and duration of liver disease had no influence on dental and periodontal disease. IControts NA.CIRR ALCO ..... A.CIRR index of oral hygiene 151+~26#+* 38+-21#+ 22_+17 ,,, 13_+9 loss of attachment I 21-30 years 126-+06+ 3.4+_0.9 31-40 years [3,1:t0.8#÷ 3.5_+1.5# 4.2_+0.8# 5.5_+1.4+ 41-50 years 14.2+_1.3# 4.4+1.3 5.2+1.6 5.9-+1.9 51-60 years ~4.6+1.3# 4.9_+1.5# 6.2+1.3 6.4_+1.6 mean_+SD; # p<0.05 vs A.CIRR, + p<0.05 vs ALCO, * p<0.05 vs NA.CIRR Conclusion: The occurrence of dental and periodontal disease in patients with liver disease is not due to the presence of cirrhosis itself, but appears to be the consequence of bad oral hygiene and poor dental care in alcoholics. I
ROLE OF BILIARY IMMUNOGLOBULINS ON CHOLESTEROL GALLSTONE DISEASE: MARKED INCREASE OF IgG IN GALLBLADDER BILE OF GALLSTONE PATIENTS. Nu~ez L, Amigo L., Miquel J.F., Rigotti A., Puglielli L., Raddatz. A., Pimentel F.,* Strasberg S., *Harvey R., and Nervi F. Departamento de Gastroenterologiay Centre para la Prevenci6n del CAncer Digestive, Pontificia Universidad CatSlica, Santiago, Chile and *Departmentof Surgery, Washington University,St.Louis, Me. Biliary cholesterol crystallization is a key step in cholesterol gallstone formation. Several biliary proteins have in vitro cholesterol crystallization promoting activity and thus, are potentially important in the formation of gallstones. Immunoglobulin G (IgG) has been proposed as one of the most potent pronucleator ( J. BioL Chem :268 . 5193.1993 ). Aim~ To evaluate the role of biliary Igs in the formation of cholesterolgallstones, in a high risk area by: a) comparing the biliary Ig concentrations (IgA, IgG, IgM ) in patients with and without gallstones, and b) correlating the levels of biliary Igs with the nucleation time( NT ) of native biles. Methods: Bile specimens were obtained from Chilean patients;60 gallbladder bile samples were collected by needle aspiration of the gallbladder at the beginning of cholecystectomy; 40 control samples were obtained during abdominal surgery from non lithiasic patients. The Ig concentrations were measured using an ELISA procedure with specific antibodies against each immunoglobulin. Semiquantitative [~ystopathological analysis of the degree of gallbladder inflamation was correlated with biliary IgG concentrations. The NT was determined by daily observation of cholesterol crystals. In all gallbladder bile samples, haemoglobin was measured to discard blood contamination. Results: The Ig concentrations and the nucleation times of gallbladder bile sampleswere: IqG(u~Ji'nl) laA(uCUtT~I) k]M(ue/ml) NT(days) C_,ontrols(n=40) 52_+7 464_+63 118_+15 21_+3
Gallstone(n=60)
678_+123(*) 486_+64
148_+24
4_+1(*)
Values are the mean +_1S.E (*) p< 0.05. An inverse correlation ( r=-0.3 ; p=0.01) was found between the NT of gallbladder biles and their IgG concentrations. No significant correlations were found between Ig concentrationsand the degree of gallbladder inflamation. A striking observation of this study was the marked increase, > 400% of IgG concentration in gallblader bile of Chilean gallstone patients as compared with North Americans ( J. Biol. Chem 268 : 5193,1993). Conclusiops: Only IgG was markedly increased in gallstone patients. Althought, it is not possible 1o ascertain wether IgG increase in gallbladder bile is a primary, or a secondary phenomena to the presence of gallstones, this study supports the hypothesis that !gG may play an trope[rantrole in the pathogenesisof cholesterol gallstone formation, Supported by Ministero deglJ Affari Esteri d'ltalia and Fondecyt 1940620.
A R E LARGE P A R A C E N T H E S I S THE B E S T T R E A T M E N T FOR R E F R A C T O R Y A S C I T E S IN P A T I E N T S W I T H C I R R H O S I S OF THE LIVER ? O.NOUEL, D.BOUTROUX, M.LE BRIS, M.DARTOIS. Hepatology Unit. General Hospital-STBRIEUC- FRANCED u r i n g a four y e a r s period, 783 p a t i e n t s with c i r r h o s i s of the liver w e r e ~ a d m i t t e d in the same H e p a t o l o g y Unit. 483 (61%) p a t i e n t s w e r e a s c i t i c and 17 (3,5%) h a d a r e f r a c t o r y ascites. R e f r a c t o r y a s c i t e s w a s d e f i n e d b y a) i m p o s s i b i l i t y to use diuretics (hyponatremia, res±stance, renal insuffisiency), b) necessity of therapeutic paracenthesis d u r i n g a p e r i o d of three m o n t h c) absence of c l i n i c a l or p a r a c l i n i c a l facts for hepatocarcinoma. Moreover these 17 patients, 16 had a l c o h o l i c c i r r h o s i s and 1 H C V + cirrhosis. After the period test of three month, paracenthesis w e r e p e r f o r m e d e v e r y w e e k for out patients with or without volumetric compensation (2 and 15 p a t i e n t s respectively). The p a t i e n t s w e r e p e r i o d i c l y e x a m i n a t e d d u r i n g a minimal p e r i o d of two years. A m o n g the 16 a l c o h o l i c patients, i0 w e r e no abstinent. F o u r p a t i e n t s died, three w i t h s e v e r e b a c t e r i a l i n f e c t i o n and one (HCV +) a f t e r liver transplantation. For 9 patients, we w e r e surprised to observe a total disparition of a s c i t e s d u r i n g t h e p e r i o d of survey. Moreover, one p a t i e n t c u r e d a b r u p t l y after a 5 years of w e e k l y therapeutic paracenthesis, always as out-patient. C o n c l u s i o n : R e f r a c t o r y ascites is a rare (3,5%) condition. It is not usually admitted that r e f r a c t o r y a s c i t e s do c u r e spontaneously. In our e x P e r i e n c e it is m o s t frequent. This fact should influence therapeutic choice. All therapeutic studies with aggressive procedures ( p e r i t o n e o j u g u l a r shunts, TIPS, portal d e r i v a t i o n s or transplantation) should be matched to p a r a c e n t h e s i s in term of s a f e t y and cost,
• GENE TRANSFER TO PRIMATE LIVER USING SECOND GENERATION RECOMBINANT ADENOVlRUS. F.A, Nunesl, 2, S.E. Raper 1, J.M. Wilson 1. 1Institute for Human Gene Therapy and Division of 2Gastroenterology, University of Pennsylvania, Philadelphia, PA Recombinant adenovirus has clinical potential for liver gene therapy since it can efficiently transfer genes to quiescent hepatocytes'and be produced in high titer and purity. First generation recombinant adenovirus, deleted in the E l a and E l b regions of the adenovirus genome, has been shown to produce high but transient expression in mouse liver, Loss of expression is associated with hepatic inflammation and a cytotoxic lymphocyte response against newly synthesized antigens. In contrast, a more crippled second generation adenovirus, containing a temperature sensitive mutation in the E2a gene, leads to longer duration of expression and less inflammation in mouse liver. In this study the safety and duration of expression of this second generation vector was tested in non-human primate liver. Juvenile rhesus monkeys (n=4) weighing 3-5 kg underwent laparotomy, cannulation of the portal vein and an infusion of 1 X 1013 particles/kg of second generation adenovirus carrying the lacZ reporter gene. Hematologic, biochemical and immunologic parameters were assayed at three days then weekly post infusion. Necropsies were performed on days 3, 7 and 28 after adenovirus administration. Transgene expression in the liver, determined by X-Gal histochemical staining, was high on days 3 and 7 but minimal by day 28 post infusion. Transgene expression was also detectable in the spleen and rarely in the vascular endothelium of thoracic and abdominal organs. Biochemical evidence of liver inflammation developed by day 7, was maximal at day 14 and resolved by day 28 post infusion. There was minimal histologic inflammation on days 3, 7 and 28 post adenovirus administration. The monkey necropsied on day 28 developed a neutralizing antibody response to the recombinant adenovirus by day 21 post infusion. Recombinant adenovirus was not recovered in the pharynx, urine or stool of the animals. Primate data suggests that second generation recombinant adenovirus may be a useful gene therapy vector for the short term treatment of metabolic liver diseases.
AASLD
Al135
DENTAL AND PERIODONTAL DISEASE iN PATIENTS WITH LIVER CIRRHOSIS - ROLE OF ALCOHOL ABUSE. GiNovace.._kk, U. NovacekPlachetzky, R. P~tzi, R. Slevicek, S. Lentner, A. Gangl, P. Ferenci. Department of Internal Medicine IV, University of Vienna, Austria. Dental foci occurs frequently in patients with cirrhosis and may be e potential source for infections, especially in transplant candidates. The aim of the study was to assess the rote of cirrhosis and chronic alcoholism for the development of dental or periodontal disease. Dental (total number of teeth, number of carious teeth and index of oral hygiene = plaque free surface of all teeth in %) and periodontal (loss of attachment = distance of the cemento-enamel junction to the bottom of the clinical pocket in mm) examinations were performed in 97 patients with cirrhosis [alcoholic (A.CIRR): 64, nonalcoholic (NA.CIRR): 33], in 68 alcoholics (ALCO) without cirrhosis and in 71 age matched healthy controls. Results: ALCO and A.CIRR had a lower total number of teeth than NA.CIRR and controls (p<0.05). The number of carious teeth was larger in ALCO (% of remaining teeth: 12.7%, p<0.001) and A.CIRR (21.3%, p<0.001) than in NA.CIRR (1.9%) and controls (1.2%). The time since the last der~tal examination was longer in ALCO (median: 12 months; interquartile range: 4-36 months; p<0.001)and in A.CIRR (21; 695 months; p<0.001) than in NA.CIRR (6; 2-7 months) and in controls (5; 2-10 months) The results of oral hygiene and periodontal disease are given in the table. There was no difference between NA.CIRR and controls in the occurrence of dental and periodontal disease. Severity and duration of liver disease had no influence on dental and periodontal disease. IControts NA.CIRR ALCO ..... A.CIRR index of oral hygiene 151+~26#+* 38+-21#+ 22_+17 ,,, 13_+9 loss of attachment I 21-30 years 126-+06+ 3.4+_0.9 31-40 years [3,1:t0.8#÷ 3.5_+1.5# 4.2_+0.8# 5.5_+1.4+ 41-50 years 14.2+_1.3# 4.4+1.3 5.2+1.6 5.9-+1.9 51-60 years ~4.6+1.3# 4.9_+1.5# 6.2+1.3 6.4_+1.6 mean_+SD; # p<0.05 vs A.CIRR, + p<0.05 vs ALCO, * p<0.05 vs NA.CIRR Conclusion: The occurrence of dental and periodontal disease in patients with liver disease is not due to the presence of cirrhosis itself, but appears to be the consequence of bad oral hygiene and poor dental care in alcoholics. I
ROLE OF BILIARY IMMUNOGLOBULINS ON CHOLESTEROL GALLSTONE DISEASE: MARKED INCREASE OF IgG IN GALLBLADDER BILE OF GALLSTONE PATIENTS. Nu~ez L, Amigo L., Miquel J.F., Rigotti A., Puglielli L., Raddatz. A., Pimentel F.,* Strasberg S., *Harvey R., and Nervi F. Departamento de Gastroenterologiay Centre para la Prevenci6n del CAncer Digestive, Pontificia Universidad CatSlica, Santiago, Chile and *Departmentof Surgery, Washington University,St.Louis, Me. Biliary cholesterol crystallization is a key step in cholesterol gallstone formation. Several biliary proteins have in vitro cholesterol crystallization promoting activity and thus, are potentially important in the formation of gallstones. Immunoglobulin G (IgG) has been proposed as one of the most potent pronucleator ( J. BioL Chem :268 . 5193.1993 ). Aim~ To evaluate the role of biliary Igs in the formation of cholesterolgallstones, in a high risk area by: a) comparing the biliary Ig concentrations (IgA, IgG, IgM ) in patients with and without gallstones, and b) correlating the levels of biliary Igs with the nucleation time( NT ) of native biles. Methods: Bile specimens were obtained from Chilean patients;60 gallbladder bile samples were collected by needle aspiration of the gallbladder at the beginning of cholecystectomy; 40 control samples were obtained during abdominal surgery from non lithiasic patients. The Ig concentrations were measured using an ELISA procedure with specific antibodies against each immunoglobulin. Semiquantitative [~ystopathological analysis of the degree of gallbladder inflamation was correlated with biliary IgG concentrations. The NT was determined by daily observation of cholesterol crystals. In all gallbladder bile samples, haemoglobin was measured to discard blood contamination. Results: The Ig concentrations and the nucleation times of gallbladder bile sampleswere: IqG(u~Ji'nl) laA(uCUtT~I) k]M(ue/ml) NT(days) C_,ontrols(n=40) 52_+7 464_+63 118_+15 21_+3
Gallstone(n=60)
678_+123(*) 486_+64
148_+24
4_+1(*)
Values are the mean +_1S.E (*) p< 0.05. An inverse correlation ( r=-0.3 ; p=0.01) was found between the NT of gallbladder biles and their IgG concentrations. No significant correlations were found between Ig concentrationsand the degree of gallbladder inflamation. A striking observation of this study was the marked increase, > 400% of IgG concentration in gallblader bile of Chilean gallstone patients as compared with North Americans ( J. Biol. Chem 268 : 5193,1993). Conclusiops: Only IgG was markedly increased in gallstone patients. Althought, it is not possible 1o ascertain wether IgG increase in gallbladder bile is a primary, or a secondary phenomena to the presence of gallstones, this study supports the hypothesis that !gG may play an trope[rantrole in the pathogenesisof cholesterol gallstone formation, Supported by Ministero deglJ Affari Esteri d'ltalia and Fondecyt 1940620.