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ulcerative colitis (UC)
GASTROENTEROLOGY Vol. 114, No. 4
A1218 AASLD ABSTRACTS
• L0078 FAMILIAL IDIOPATHIC ADULTHOOD BILE D U C T O P E N I A : A REPORT OF FIVE CASES IN THREE GENERATIONS. K. Burak*. M. Swain*, D. Pearson4', J. Kelly'*, S. Urbanski +, R. Bridges*, Division of Gastroenterology* and Department of Pathology +, University of Calgary, Calgary, AB, and Victoria General Hospital~, Victoria, BC, Canada. Idiopathic adulthood ductopenia (lAD) has been described with most cases being sporadic. We describe a family with adult onset bile duct depletion involving five members of an extended family spanning three generations. All cases were from a extended family of British extraction living in rural Alberta, Canada. There was no consanguinity. The proband, a 46 year old man presented in 1989 with elevated cholestatic and hepatocellular enzymes. Extensive workup, including ERCP, excluded other causes of chronic liver disease. Serial liver biopsies demonstrated progression from normal in 1989, to a striking loss of intralobular bile ducts in 1992. He remains asymptomatic and ursodeoxycholic acid (UDCA) has resulted in normalization of his liver enzymes. The proband's brother underwent orthotopic liver transplantation in 1985, at age 35, for cryptogenic chronic hepatitis. The explanted liver showed cirrhosis, extreme cholestasis and loss of bile ducts in medium and small portal tracts. The proband's sister, age 42, has had intermittent elevation of liver enzymes since 1971. ERCP was normal and liver biopsy revealed portal inflammation and ductular damage. Of the proband's three children, a 21 year old asymptomatic son, has elevated liver enzymes. Liver biopsy is consistent with IAD. The proband and his son have a normal 46 XY karyotype and share HLA A2, B51, DR8, DQ1, and DQ4 loci in common. The proband's father had a liver biopsy at age 70 during investigation of a possible liver mass. This revealed extensive fibrosis and striking bile ductular destruction. The proband's mother died at age 67 of gallbladder adenocarcinoma. The proband's maternal grandmother also died of an unsubstantiated liver tumor. Cases described were antimitochondrial antibody negative with no cause for ductopenia identified. This is the largest series of familial lAD reported and the first with multiple generations described. In this family, disease expression varied from asymptomatic to that requiring transplantation, and showed a propensity for the possible development of hepatobiliary tumors. Medical therapy with UDCA has resulted in improvement of liver enzymes in one case, although the impact on the natural history of this disorder remains unknown. • L0079 MOLECULAR CLONING AND EXPRESSION OF MOUSE ITIH-4: ENCODES A SCAFFOLDING PROTEIN IN LIVER FORMATION. Tao Cai 1, Ping Yu 1, Satdarshan P.S. Monga 1, Bibhuti Mishra 2, Lopa Mishra 1 1Laboratory of Developmental Molecular Biology, VA Medical Center, Washington, DC; and Fels Institute for Cancer Research and Molecular Biology, Temple University, Philadelphia; 2National Human Genome Research Institute, Clinical Gene Therapy Branch, National Institutes of Health, Bethesda, MD. Introduction: Inter a trypsin inhibitors (ITI) belong to a family of serine plasma protease inhibitors, which are involved in the stabilization and scaffolding of extracellular matrix proteins, crucial features in liver formation. Utilizing a specific screening strategy to identify genes important in three dimensional structure of the liver, we have successfully isolated mouse itih-4. Methods and Results: Mouse itih-4 (GenBank accession number, AF023919) was obtained from subtraction hybridization of embryonic liver cDNA libraries. The itih-4 ORF is 3030 basepalrs, and encodes a 942 amino acid protein (H4P). H4P comprises a hyaluronic acid (HA) binding site, and a putative amino terminus collagen-binding region homologous to the von Willebrand factor (vWF) domain. In addition, two EF-hand motifs (aa 278 to 289 and aa 303 to 314) are present in the vWF domain, suggesting a potential calcium-binding function. Northern blot analysis revealed a 3.1 kb transcript with prominent expression in liver, and to a lesser extent in lung and heart tissue. Reverse transcriptase PCR assay utilizing itih-4 specific primers demonstrated itih-4 mRNA in embryonic liver, heart and brain throughout mid-embryonic mouse gestation. Quantitative scanning of the resulting southern blot indicated that itih-4 mRNA levels were significantly increased in embryonic liver from post-coital day 10.5 to 14.5; itih-4 mRNAs in heart and brain tissue at these stages were lower but detectable with RT-PCR. Conclusions: These results suggest that regulation of itih-4 gene expression plays an important role during early embryonic development and growth, particularly during normal liver development, morphogenesis and remodelling. • L0080
CHARACTERIZATION OF GASTRIC MUCOSAL DAMAGE INDUCED BY ASPIRIN IN PORTAL HYPERTENSIVE RATS. S. Calatavud. M.C. Ramfrez, M.J. Sanz, J.M. Piqu6, J. Bosch, J.V. Esplugues. Dept. of Pharmacology, University of Valencia, Spain. B A C K G R O U N D : Portal hypertension is related to an increased formation of
the vasoactive and gastroprotective mediator nitric oxide (NO) (Hepatology 1993;18:628-634). AIM: The present study characterizes the response of portal hypertensive rats to the gastrolesive action of aspirin. METHODS: Portal hypertension was induced in Sprague-Dawley rats by partial portal vein ligation (PPVL). Control rats underwent a sham operation (SO) and the
experiments were carried out 14 days after surgery. Animals received a gastrolesive dose of aspirin (200 mg/kg in HCI 0.2N, p.o.) and were killed three hours later. Gastric emptying in these conditions was evaluated by adding the non absorbable marker phenol red to the aspirin suspension. Some animals were treated with the NO synthesis inhibitor L-NAME (3mg/kg, i.p.) 15 min before aspirin administration. Injury was evaluated macroscopically as % of gastric mucosal area damaged. In some experiments, gastric bleeding was measured by the use of Cr51-1abelled erythrocytes. RESULTS: Portal hypertensive rats exhibited a 74 + 5% diminution (n=7, p<0.05) in the level of gastric damage induced by aspirin in control animals (5.8 ± 1.4, n=8). This effect was not related to an alteration in the gastric emptying of this NSAID (79 ± 3 % and 72 ± 3 % in SO and PPVL rats respectively, n=4 both). Pretreatment with L-NAME restored the damaging effect of aspirin in PPVL rats (5.0 ± 1.1, n=10, p<0.05) without modifying the lesion index of control animals (5.4 ± 2.1, n=7). Gastric bleeding was similar in PPVL and SO rats (4.4 ± 0.6 and 4.1 ± 0.5 ~tl red blood cells, respectively), and this means that there is an increased blood leakage by damaged area unit in PPVL rats (p<0.05). CONCLUSION: The gastric mucosa of portal hypertensive rats shows an enhanced resistance to exogenous injury and this effect involves an increased synthesis of NO. However, gastric lesions in these animals show an augmented bleeding per unit of injury. • L0081
CHARACTERIZATION OF THE ANTIGEN FOR THE ANTINEUTROPHIL CYTOPLASMIC ANTIBODY SPECIFIC FOR PRIMARY SCLEROSING CHOLANGITIS (PSC)/ULCERATIVE COLITIS (UC). BJ Cameron 1, DS Bansi 2, R Ali, 1 RW Chapman3 and KA Fleming ~ University of Oxford, Nuffield Dept. of Pathology ~ and Dept. of Gastroenterology ~, Oxford Radcliffe Hospital Trust, Dept. of Gastroenterology 2, The Middlesex Hospital, London, U.K. Perinuclear antineutrophil cytoplasmic antibodies (ANCA) are found in up to 87% of patients with primary sclerosing cholangitis with/without concomitant ulcerative colitis ( ± UC), 65% of UC alone and up to 50% with autoimmune hepatitis (AIH). Identification of the neutrophil antigen(s) against which ANCA is directed may provide insight into the role of these antibodies in pathogenesis and diagnosis. Aim: Characterisation and identification of the antigen recognised by the anti-neutrophil cytoplasmic antibody specific for primary sclerosing cholangitis/ulcerative colitis. Methods: A variety of lysis procedures were analysed. Dot blots of lysed neutrophils were performed with subsequent detection by patients' sera. The effect of enzymes and heat treatment of the antigen was examined. Affinity chromatography using ANCA +ve and ANCA -ve agarose beads was attempted in order to partially purify the antigen by elution. SDS-PAGE and Western transfer was then employed to analyse the eluates. Results: The antigen co-segregated with nuclei. It is destroyed by incubation with pepsin or trypsin, is heat labile (30 minutes at 37°C) and it is sensitive to DNase digestion (10mg/ml, 10 minutes, 20°C) but not to RNase. Affinity chromatography with ANCA +ve and ANCA -VE agarose beads followed by SDS-PAGE and protein transfer indicated that the antigen is approximately 65-70 kDa. However, these latter results are variable and difficult to reproduce. Conclusions: The antigen co-segregates with the nuclei of lysed neutrophils, is protein in nature and is DNA-associated. Attempts to analyse the antigen have been hampered as it is extremely labile. Long term work will be aimed at partial amino acid sequencing, identifying the gene for the antigen, determining its structure and function and its possible relationship to the pathogenesis Of PSC and UC. • L0082
THE EFFECT OF ALCOHOL WITHDRAWAL ON LIVER TRANSAMINASES AND TISSUE INHIBITOR OF METALLOPROTEINASE-1 (TIMP-1). S Campbell 1,3, PM Timms2, PR Maxwell 2, BJ DaneshL Department of Gastroenterology l, Biochemistry 2, Stobhill Hospital, Human Nutrition 3, Glasgow Royal Infirmary, Glasgow UK. Background During acute alcohol withdrawal, portal blood flow may decrease whilst hepatic oxygen demand remains elevated. This may precipitate hepatic ischaemia. TIMP-1 is an inhibitor of interstitial collagenase. Hepatic and plasma TIMP-1 levels are increased in fibrotic alcoholic liver disease (ALD), suggesting that the net increase in fibrous tissue is due to a decrease in collagen degradation. Aims To determine if there is evidence for a worsening in hepatic biochemical tests, or in plasma TIMP-1 levels, following abrupt alcohol withdrawal. Methods Twenty-four alcoholic patients (all consumed > 150g alcohol per day) attending for inpatient alcohol withdrawal were recruited. The high rate of complications in severe ALD may confound biochemical observations, and therefore a population with clinically mild disease was chosen. No subjects had evidence of hepatic decompensation (ascites, encephalopathy). Blood samples were taken within 24 hours of last alcohol consumed, and daily for the next 7 days, and at day 14 and 28 for routine biochemical liver function tests (LFF's) and TIMP-1 (ELISA- Amersham, UK). Blood samples were also obtained from 22 control subjects with no history of physical disease or alcoholism. Our arbitrary criteria for worsening of LFT's were: a 50% rise from baseline value with the elevated value above the laboratory normal reference range.
A1218 AASLD ABSTRACTS
• L0078 FAMILIAL IDIOPATHIC ADULTHOOD BILE D U C T O P E N I A : A REPORT OF FIVE CASES IN THREE GENERATIONS. K. Burak*. M. Swain*, D. Pearson4', J. Kelly'*, S. Urbanski +, R. Bridges*, Division of Gastroenterology* and Department of Pathology +, University of Calgary, Calgary, AB, and Victoria General Hospital~, Victoria, BC, Canada. Idiopathic adulthood ductopenia (lAD) has been described with most cases being sporadic. We describe a family with adult onset bile duct depletion involving five members of an extended family spanning three generations. All cases were from a extended family of British extraction living in rural Alberta, Canada. There was no consanguinity. The proband, a 46 year old man presented in 1989 with elevated cholestatic and hepatocellular enzymes. Extensive workup, including ERCP, excluded other causes of chronic liver disease. Serial liver biopsies demonstrated progression from normal in 1989, to a striking loss of intralobular bile ducts in 1992. He remains asymptomatic and ursodeoxycholic acid (UDCA) has resulted in normalization of his liver enzymes. The proband's brother underwent orthotopic liver transplantation in 1985, at age 35, for cryptogenic chronic hepatitis. The explanted liver showed cirrhosis, extreme cholestasis and loss of bile ducts in medium and small portal tracts. The proband's sister, age 42, has had intermittent elevation of liver enzymes since 1971. ERCP was normal and liver biopsy revealed portal inflammation and ductular damage. Of the proband's three children, a 21 year old asymptomatic son, has elevated liver enzymes. Liver biopsy is consistent with IAD. The proband and his son have a normal 46 XY karyotype and share HLA A2, B51, DR8, DQ1, and DQ4 loci in common. The proband's father had a liver biopsy at age 70 during investigation of a possible liver mass. This revealed extensive fibrosis and striking bile ductular destruction. The proband's mother died at age 67 of gallbladder adenocarcinoma. The proband's maternal grandmother also died of an unsubstantiated liver tumor. Cases described were antimitochondrial antibody negative with no cause for ductopenia identified. This is the largest series of familial lAD reported and the first with multiple generations described. In this family, disease expression varied from asymptomatic to that requiring transplantation, and showed a propensity for the possible development of hepatobiliary tumors. Medical therapy with UDCA has resulted in improvement of liver enzymes in one case, although the impact on the natural history of this disorder remains unknown. • L0079 MOLECULAR CLONING AND EXPRESSION OF MOUSE ITIH-4: ENCODES A SCAFFOLDING PROTEIN IN LIVER FORMATION. Tao Cai 1, Ping Yu 1, Satdarshan P.S. Monga 1, Bibhuti Mishra 2, Lopa Mishra 1 1Laboratory of Developmental Molecular Biology, VA Medical Center, Washington, DC; and Fels Institute for Cancer Research and Molecular Biology, Temple University, Philadelphia; 2National Human Genome Research Institute, Clinical Gene Therapy Branch, National Institutes of Health, Bethesda, MD. Introduction: Inter a trypsin inhibitors (ITI) belong to a family of serine plasma protease inhibitors, which are involved in the stabilization and scaffolding of extracellular matrix proteins, crucial features in liver formation. Utilizing a specific screening strategy to identify genes important in three dimensional structure of the liver, we have successfully isolated mouse itih-4. Methods and Results: Mouse itih-4 (GenBank accession number, AF023919) was obtained from subtraction hybridization of embryonic liver cDNA libraries. The itih-4 ORF is 3030 basepalrs, and encodes a 942 amino acid protein (H4P). H4P comprises a hyaluronic acid (HA) binding site, and a putative amino terminus collagen-binding region homologous to the von Willebrand factor (vWF) domain. In addition, two EF-hand motifs (aa 278 to 289 and aa 303 to 314) are present in the vWF domain, suggesting a potential calcium-binding function. Northern blot analysis revealed a 3.1 kb transcript with prominent expression in liver, and to a lesser extent in lung and heart tissue. Reverse transcriptase PCR assay utilizing itih-4 specific primers demonstrated itih-4 mRNA in embryonic liver, heart and brain throughout mid-embryonic mouse gestation. Quantitative scanning of the resulting southern blot indicated that itih-4 mRNA levels were significantly increased in embryonic liver from post-coital day 10.5 to 14.5; itih-4 mRNAs in heart and brain tissue at these stages were lower but detectable with RT-PCR. Conclusions: These results suggest that regulation of itih-4 gene expression plays an important role during early embryonic development and growth, particularly during normal liver development, morphogenesis and remodelling. • L0080
CHARACTERIZATION OF GASTRIC MUCOSAL DAMAGE INDUCED BY ASPIRIN IN PORTAL HYPERTENSIVE RATS. S. Calatavud. M.C. Ramfrez, M.J. Sanz, J.M. Piqu6, J. Bosch, J.V. Esplugues. Dept. of Pharmacology, University of Valencia, Spain. B A C K G R O U N D : Portal hypertension is related to an increased formation of
the vasoactive and gastroprotective mediator nitric oxide (NO) (Hepatology 1993;18:628-634). AIM: The present study characterizes the response of portal hypertensive rats to the gastrolesive action of aspirin. METHODS: Portal hypertension was induced in Sprague-Dawley rats by partial portal vein ligation (PPVL). Control rats underwent a sham operation (SO) and the
experiments were carried out 14 days after surgery. Animals received a gastrolesive dose of aspirin (200 mg/kg in HCI 0.2N, p.o.) and were killed three hours later. Gastric emptying in these conditions was evaluated by adding the non absorbable marker phenol red to the aspirin suspension. Some animals were treated with the NO synthesis inhibitor L-NAME (3mg/kg, i.p.) 15 min before aspirin administration. Injury was evaluated macroscopically as % of gastric mucosal area damaged. In some experiments, gastric bleeding was measured by the use of Cr51-1abelled erythrocytes. RESULTS: Portal hypertensive rats exhibited a 74 + 5% diminution (n=7, p<0.05) in the level of gastric damage induced by aspirin in control animals (5.8 ± 1.4, n=8). This effect was not related to an alteration in the gastric emptying of this NSAID (79 ± 3 % and 72 ± 3 % in SO and PPVL rats respectively, n=4 both). Pretreatment with L-NAME restored the damaging effect of aspirin in PPVL rats (5.0 ± 1.1, n=10, p<0.05) without modifying the lesion index of control animals (5.4 ± 2.1, n=7). Gastric bleeding was similar in PPVL and SO rats (4.4 ± 0.6 and 4.1 ± 0.5 ~tl red blood cells, respectively), and this means that there is an increased blood leakage by damaged area unit in PPVL rats (p<0.05). CONCLUSION: The gastric mucosa of portal hypertensive rats shows an enhanced resistance to exogenous injury and this effect involves an increased synthesis of NO. However, gastric lesions in these animals show an augmented bleeding per unit of injury. • L0081
CHARACTERIZATION OF THE ANTIGEN FOR THE ANTINEUTROPHIL CYTOPLASMIC ANTIBODY SPECIFIC FOR PRIMARY SCLEROSING CHOLANGITIS (PSC)/ULCERATIVE COLITIS (UC). BJ Cameron 1, DS Bansi 2, R Ali, 1 RW Chapman3 and KA Fleming ~ University of Oxford, Nuffield Dept. of Pathology ~ and Dept. of Gastroenterology ~, Oxford Radcliffe Hospital Trust, Dept. of Gastroenterology 2, The Middlesex Hospital, London, U.K. Perinuclear antineutrophil cytoplasmic antibodies (ANCA) are found in up to 87% of patients with primary sclerosing cholangitis with/without concomitant ulcerative colitis ( ± UC), 65% of UC alone and up to 50% with autoimmune hepatitis (AIH). Identification of the neutrophil antigen(s) against which ANCA is directed may provide insight into the role of these antibodies in pathogenesis and diagnosis. Aim: Characterisation and identification of the antigen recognised by the anti-neutrophil cytoplasmic antibody specific for primary sclerosing cholangitis/ulcerative colitis. Methods: A variety of lysis procedures were analysed. Dot blots of lysed neutrophils were performed with subsequent detection by patients' sera. The effect of enzymes and heat treatment of the antigen was examined. Affinity chromatography using ANCA +ve and ANCA -ve agarose beads was attempted in order to partially purify the antigen by elution. SDS-PAGE and Western transfer was then employed to analyse the eluates. Results: The antigen co-segregated with nuclei. It is destroyed by incubation with pepsin or trypsin, is heat labile (30 minutes at 37°C) and it is sensitive to DNase digestion (10mg/ml, 10 minutes, 20°C) but not to RNase. Affinity chromatography with ANCA +ve and ANCA -VE agarose beads followed by SDS-PAGE and protein transfer indicated that the antigen is approximately 65-70 kDa. However, these latter results are variable and difficult to reproduce. Conclusions: The antigen co-segregates with the nuclei of lysed neutrophils, is protein in nature and is DNA-associated. Attempts to analyse the antigen have been hampered as it is extremely labile. Long term work will be aimed at partial amino acid sequencing, identifying the gene for the antigen, determining its structure and function and its possible relationship to the pathogenesis Of PSC and UC. • L0082
THE EFFECT OF ALCOHOL WITHDRAWAL ON LIVER TRANSAMINASES AND TISSUE INHIBITOR OF METALLOPROTEINASE-1 (TIMP-1). S Campbell 1,3, PM Timms2, PR Maxwell 2, BJ DaneshL Department of Gastroenterology l, Biochemistry 2, Stobhill Hospital, Human Nutrition 3, Glasgow Royal Infirmary, Glasgow UK. Background During acute alcohol withdrawal, portal blood flow may decrease whilst hepatic oxygen demand remains elevated. This may precipitate hepatic ischaemia. TIMP-1 is an inhibitor of interstitial collagenase. Hepatic and plasma TIMP-1 levels are increased in fibrotic alcoholic liver disease (ALD), suggesting that the net increase in fibrous tissue is due to a decrease in collagen degradation. Aims To determine if there is evidence for a worsening in hepatic biochemical tests, or in plasma TIMP-1 levels, following abrupt alcohol withdrawal. Methods Twenty-four alcoholic patients (all consumed > 150g alcohol per day) attending for inpatient alcohol withdrawal were recruited. The high rate of complications in severe ALD may confound biochemical observations, and therefore a population with clinically mild disease was chosen. No subjects had evidence of hepatic decompensation (ascites, encephalopathy). Blood samples were taken within 24 hours of last alcohol consumed, and daily for the next 7 days, and at day 14 and 28 for routine biochemical liver function tests (LFF's) and TIMP-1 (ELISA- Amersham, UK). Blood samples were also obtained from 22 control subjects with no history of physical disease or alcoholism. Our arbitrary criteria for worsening of LFT's were: a 50% rise from baseline value with the elevated value above the laboratory normal reference range.