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Interleukin-10 -592 promoter polymorphism and primary biliary cirrhosis: No association with disease susceptibility or severity
April 1998 PLC-'/1 and maps to the distal portion of mouse chromosome 2. Reverse transcriptase PCR assay utilizing Cded specific primers demonstrate Cded mRNAs in liver, heart, and brain tissues, throughout mid-embryonic mouse gestation. Analysis of the Cded promoter region shows high GC-content, high ratio of CpG/GpC, multiple GC-boxes, an absent TATA box, as well as AP2, and HNF1 binding sites. These characteristics are also found in several genes important in the regulation of cell growth or DNA synthesis, such as transforming growth factor-131, c-Ha-ras, nerve growth factor and epidermal growth factor receptor. Conclusions: The structural details described here are significant because Cded, by tethering other proteins with its PH domain, could play a vital role in liver development, particularly by involving the signal transduction process in hepatocyte formation. L0707
EFFECT OF DEVELOPMENTAL STAGE OR DIFFERENTIATION MODULATORS ON FUNCTIONAL EXPRESSION OF MAXI°K CHANNELS IN CULTURED CHICK HEPATOCYTES. R.S. Yu, S. James, F.-Y. Han, D.C. P o n & C.E. Hill, GI Diseases Research Unit, Hotel Dieu Hospital and Department of Physiology, Queen's University, Kingston, Ontario, Canada. Large conductance, Ca2÷ and voltage-gated K÷ ('Maxi-K') channels are present in the basolateral plasma membranes of many secretory epithelia and are thought to provide the driving force for water secretion in exocrine tissue. We have identified these channels in short-term cultured embryonic chick hepatocytes and suggest that they may have similar functions in generating some of the driving force for bile formation in the liver. We have shown previously that functional Maxi-K channels in hepatocytes appear over time in culture following trypsin dissociation of ten day embryonic chick livers. We proposed that the functional expression of these channels is correlated with cell-cell contact formation and/or hepatocellular differentiation state. Chick liver passes through a major differentiation point in the day seven embryo. To test our hypothesis we quantified the functional expression of Maxi-K channels isolated from (i) six or ten day embryos, and cultured for 6 to 45 hours in the presence or absence of 1.0 taM dexamethasone, a corticosteroid known to induce cell-cell contact formation and differentiation in the liver, or (ii) ten day embryos, and cultured for 24 hours in serum-free medium (SFM) supplemented with either insulin,, transferrin, epidermal growth factor and albumin (GIBCO 'HepatoZYME-SFM') or 1% fetal calf serum (FCS). Through polymerase chain reaction, we also amplified complementary DNA obtained from messenger RNA isolated from intact embryonic liver tissue using degenerate primers for the mouse brain Maxi-K channel transcript (mSlo). This demonstrated that channel expression in culture was not a consequence of hepatocyte dedifferentiation in culture. The cell-attached configuration of the patch clamp technique was used to monitor channel activity in hepatocytes. Age did not significantly effect either the rate of appearance (calculated as the doubling time) of functional channels, or the time at which 50% of channels were present (tso time). In contrast, dexamethasone significantly decreased the doubling time for appearance of channels in hepatocytes derived from both embryonic ages. The doubling time decreased from 8.5 -+ 2.8 and 6.4 _+0.2 hours respectively in six and ten day controls to 4.5 -+ 0.4 and 2.9 _+0.1 hours respectively in six and ten day cells cultured in the presence of the steroid. Functional channel expression in cells cultured in SFM was not significantly different from those exposed to 7.5% FCS (22 _+8.0% and 13 _+0.03% respectively) but was significantly greater than those maintained in 1% FCS (5.9-+5.7%). Our results demonstrate that Maxi-K channels appear early in liver development and that their functional expression may be linked to cell-cell contact formation, possibly through cytoskeletal reorganization and hepatocellular polarization into a fully functioning bile secreting structure. This study was financially supported by the Natural Sciences and Engineering Research Council (NSERC) of Canada.
AASLD A1371 • L0708 THE PREDICTIVE VALUE OF DIFFUSION CAPACITY (DLCO) IN PATIENTS WITH PRIMARY BILIARY CIRRHOSIS PRIOR TO ORTHOTOPIC LIVER TRANSPLANTATION. DL Zack Z, RM Dy 1, AA Floreani 2, TM McCashland 1. Sections of Gastroenterology 1 and Pulmonology2, University of Nebraska Medical Center, Omaha, NE. Primary biliary cirrhosis (PBC) is a chronic cholestatic liver disease that has been reported to be associated with fibrosing alveolitis and a variety of autoimmune diseases resulting in lung function abnormalities. Decreased DLCO has been reported in up to 30% of patients with FBC. The clinical implications of an abnormal DLCO remain unknown in this population. AIMS: 1) To assess the utility of DLCO to predict pulmonary complications prior to orthotopic liver transplantation (OLT) in PBC patients compared to those with primary sclerosing cholangitis (PSC) and alcoholic liver disease (ALD). 2) To determine if laboratory values, histological stage, and presence of clinical syndromes of autoimmune disease correlated with abnormal DLCO. METHODS: A retrospective chart review of patients undergoing OLT for PBC, PSC and ALD from 1985 through 1996 was conducted. Data collected at time of OLT evaluation included PFI"s, tobacco use, Child's class, extubation time, and pulmonary complications (pneumonia, hypoxemia, reintubation, ARDS, death) for all groups. For those patients with PBC, histological stage of explant and presence of Sj~igrens's, CREST and sicca syndrome according to history and physical exam was included. RESULTS: Study groups included 93 patients (M: 8, F: 85) with PBC, 101 (M: 61, F: 40) with PSC and 79 (M: 65, F: 13) with ALD, Pulmonary complications were common in all groups (PBC 22%, PSC 23%, ALD 30%, p=NS). The median % of predicte d DLCO within each group was similar, regardless of the presence or absence of pulmonary complications (PBC 86 vs. 81, PSC 80 vs. 85, ALD 74 vs. 69, all p=NS). Group comparisons with pulmonary complications did not reveal a significant difference in the % of predicted DLCO. In contrast, the ALD patients with no complications had a lower % of predicted DLCO (ALD 69 vs. PBC 81 vs. PSC 85, p=0.007 and p=0.002). The incidence of abnormal DLCO (<70% of predicted) was higher in ALD (53.6%), but similar in PBC (23.2%) and PSC (18.6%). Extubation time was approximately 1 day among all groups when the % of predicted DLCO<70, p=NS. In contrast, ALD patients with % of predicted DLCO >70 had longer median extubation time (ALD 1.5 d vs. PBC 1 d vs. PSC 1 d, p=0.01 and p=0.0001). A greater number of PBC patients with histological stage IV disease had % of predicted DLCO<70 when compared to stage II and I1 disease (29% vs. 9%, p=0.02). No relationship was identified between SjOgrens's, CREST or sicca syndrome and DLCO in patients with PBC. When the % of predicted DLCO<70, 6.6% of the patients had CREST, 6.6% had SjOgrens's and 28.5% had sicca syndrome, p=NS. CONCLUSION: The incidence of abnormal DLCO was highest in the ALD, however similar in the PBC and PSC groups. The DLCO is unable to accurately predict which PBC patients are at highest risk to develop postoperative complications and long intubation time. Histological stage appears to correlate with decreased DLCO in PBC patients. The presence of autoimmune diseases such as Sj0grens's, CREST and sicca syndrome did not impact the measure of gas exchange efficiency. • L0709 INTERLEUKIN-10 -592 PROMOTER POLYMORPHISM A N D PRIMARY BILIARY CIRRHOSIS: NO ASSOCIATION WITH DISEASE SUSCEPTIBILITY OR SEVERITY. F. Zappala, J. Grove, F.E. Watt, A.K. Daly, C.P. Day, M.F. Bassendine, D.E.J. Jones. Center for Liver Research, University of Newcastle, Newcastle-upon-Tyne, UK. Primary biliary cirrhosis (PBC) is a chronic cholestatic liver disease with an autoimmune aetiology. Family studies, which have shown a significantly increased incidence of PBC in the close relatives of patients, suggest that genetic factors play a significant role in determining disease susceptibility. Several studies have identified loci which appear to play a role in determining both disease susceptibility (HLA DR8, C4A*0) and severity (DR8, TNF-ct -308). Here we have studied another candidate susceptibility/severity locus in PBC, an apparently functional biallelic polymorphism at position -592 in the promoter region of the gene encoding the immuno-modulatory cytokine interleukin-10 (IL-10). IL-10 plays an important role in the functional control in vivo of autoreactive. Th-1 type CD4+ T-cells, with experimental manipulation of 1L-10 leading to significant modulation of disease development in animal models of autoimmunity. IL-10 -592 genotypes were studied by PCR in 171 well characterised, histologically staged, PBC patients and 141 geo-graphically matched North-Eastern controls. 99/171 (58%) of PBC patients were homozygous for the commoner allele (C/C), 68/171 (40%) were heterozygotes (A/C) whilst 4/171 (2%) were homozygous for the rarer allele (A/A). These genotype frequencies were not significantly different to those seen in controls (p=0.49, OR 1.2 [0.8-1.9]). No variation in allele frequency was seen when PBC patients were subdivided according to disease stage (C allele frequency 0.81 (stage I/II) v 0.77 (stage III/IV). Conclusions: These findings suggest that IL-10 -592 is neither a susceptibility nor a severity locus in PBC.
EFFECT OF DEVELOPMENTAL STAGE OR DIFFERENTIATION MODULATORS ON FUNCTIONAL EXPRESSION OF MAXI°K CHANNELS IN CULTURED CHICK HEPATOCYTES. R.S. Yu, S. James, F.-Y. Han, D.C. P o n & C.E. Hill, GI Diseases Research Unit, Hotel Dieu Hospital and Department of Physiology, Queen's University, Kingston, Ontario, Canada. Large conductance, Ca2÷ and voltage-gated K÷ ('Maxi-K') channels are present in the basolateral plasma membranes of many secretory epithelia and are thought to provide the driving force for water secretion in exocrine tissue. We have identified these channels in short-term cultured embryonic chick hepatocytes and suggest that they may have similar functions in generating some of the driving force for bile formation in the liver. We have shown previously that functional Maxi-K channels in hepatocytes appear over time in culture following trypsin dissociation of ten day embryonic chick livers. We proposed that the functional expression of these channels is correlated with cell-cell contact formation and/or hepatocellular differentiation state. Chick liver passes through a major differentiation point in the day seven embryo. To test our hypothesis we quantified the functional expression of Maxi-K channels isolated from (i) six or ten day embryos, and cultured for 6 to 45 hours in the presence or absence of 1.0 taM dexamethasone, a corticosteroid known to induce cell-cell contact formation and differentiation in the liver, or (ii) ten day embryos, and cultured for 24 hours in serum-free medium (SFM) supplemented with either insulin,, transferrin, epidermal growth factor and albumin (GIBCO 'HepatoZYME-SFM') or 1% fetal calf serum (FCS). Through polymerase chain reaction, we also amplified complementary DNA obtained from messenger RNA isolated from intact embryonic liver tissue using degenerate primers for the mouse brain Maxi-K channel transcript (mSlo). This demonstrated that channel expression in culture was not a consequence of hepatocyte dedifferentiation in culture. The cell-attached configuration of the patch clamp technique was used to monitor channel activity in hepatocytes. Age did not significantly effect either the rate of appearance (calculated as the doubling time) of functional channels, or the time at which 50% of channels were present (tso time). In contrast, dexamethasone significantly decreased the doubling time for appearance of channels in hepatocytes derived from both embryonic ages. The doubling time decreased from 8.5 -+ 2.8 and 6.4 _+0.2 hours respectively in six and ten day controls to 4.5 -+ 0.4 and 2.9 _+0.1 hours respectively in six and ten day cells cultured in the presence of the steroid. Functional channel expression in cells cultured in SFM was not significantly different from those exposed to 7.5% FCS (22 _+8.0% and 13 _+0.03% respectively) but was significantly greater than those maintained in 1% FCS (5.9-+5.7%). Our results demonstrate that Maxi-K channels appear early in liver development and that their functional expression may be linked to cell-cell contact formation, possibly through cytoskeletal reorganization and hepatocellular polarization into a fully functioning bile secreting structure. This study was financially supported by the Natural Sciences and Engineering Research Council (NSERC) of Canada.
AASLD A1371 • L0708 THE PREDICTIVE VALUE OF DIFFUSION CAPACITY (DLCO) IN PATIENTS WITH PRIMARY BILIARY CIRRHOSIS PRIOR TO ORTHOTOPIC LIVER TRANSPLANTATION. DL Zack Z, RM Dy 1, AA Floreani 2, TM McCashland 1. Sections of Gastroenterology 1 and Pulmonology2, University of Nebraska Medical Center, Omaha, NE. Primary biliary cirrhosis (PBC) is a chronic cholestatic liver disease that has been reported to be associated with fibrosing alveolitis and a variety of autoimmune diseases resulting in lung function abnormalities. Decreased DLCO has been reported in up to 30% of patients with FBC. The clinical implications of an abnormal DLCO remain unknown in this population. AIMS: 1) To assess the utility of DLCO to predict pulmonary complications prior to orthotopic liver transplantation (OLT) in PBC patients compared to those with primary sclerosing cholangitis (PSC) and alcoholic liver disease (ALD). 2) To determine if laboratory values, histological stage, and presence of clinical syndromes of autoimmune disease correlated with abnormal DLCO. METHODS: A retrospective chart review of patients undergoing OLT for PBC, PSC and ALD from 1985 through 1996 was conducted. Data collected at time of OLT evaluation included PFI"s, tobacco use, Child's class, extubation time, and pulmonary complications (pneumonia, hypoxemia, reintubation, ARDS, death) for all groups. For those patients with PBC, histological stage of explant and presence of Sj~igrens's, CREST and sicca syndrome according to history and physical exam was included. RESULTS: Study groups included 93 patients (M: 8, F: 85) with PBC, 101 (M: 61, F: 40) with PSC and 79 (M: 65, F: 13) with ALD, Pulmonary complications were common in all groups (PBC 22%, PSC 23%, ALD 30%, p=NS). The median % of predicte d DLCO within each group was similar, regardless of the presence or absence of pulmonary complications (PBC 86 vs. 81, PSC 80 vs. 85, ALD 74 vs. 69, all p=NS). Group comparisons with pulmonary complications did not reveal a significant difference in the % of predicted DLCO. In contrast, the ALD patients with no complications had a lower % of predicted DLCO (ALD 69 vs. PBC 81 vs. PSC 85, p=0.007 and p=0.002). The incidence of abnormal DLCO (<70% of predicted) was higher in ALD (53.6%), but similar in PBC (23.2%) and PSC (18.6%). Extubation time was approximately 1 day among all groups when the % of predicted DLCO<70, p=NS. In contrast, ALD patients with % of predicted DLCO >70 had longer median extubation time (ALD 1.5 d vs. PBC 1 d vs. PSC 1 d, p=0.01 and p=0.0001). A greater number of PBC patients with histological stage IV disease had % of predicted DLCO<70 when compared to stage II and I1 disease (29% vs. 9%, p=0.02). No relationship was identified between SjOgrens's, CREST or sicca syndrome and DLCO in patients with PBC. When the % of predicted DLCO<70, 6.6% of the patients had CREST, 6.6% had SjOgrens's and 28.5% had sicca syndrome, p=NS. CONCLUSION: The incidence of abnormal DLCO was highest in the ALD, however similar in the PBC and PSC groups. The DLCO is unable to accurately predict which PBC patients are at highest risk to develop postoperative complications and long intubation time. Histological stage appears to correlate with decreased DLCO in PBC patients. The presence of autoimmune diseases such as Sj0grens's, CREST and sicca syndrome did not impact the measure of gas exchange efficiency. • L0709 INTERLEUKIN-10 -592 PROMOTER POLYMORPHISM A N D PRIMARY BILIARY CIRRHOSIS: NO ASSOCIATION WITH DISEASE SUSCEPTIBILITY OR SEVERITY. F. Zappala, J. Grove, F.E. Watt, A.K. Daly, C.P. Day, M.F. Bassendine, D.E.J. Jones. Center for Liver Research, University of Newcastle, Newcastle-upon-Tyne, UK. Primary biliary cirrhosis (PBC) is a chronic cholestatic liver disease with an autoimmune aetiology. Family studies, which have shown a significantly increased incidence of PBC in the close relatives of patients, suggest that genetic factors play a significant role in determining disease susceptibility. Several studies have identified loci which appear to play a role in determining both disease susceptibility (HLA DR8, C4A*0) and severity (DR8, TNF-ct -308). Here we have studied another candidate susceptibility/severity locus in PBC, an apparently functional biallelic polymorphism at position -592 in the promoter region of the gene encoding the immuno-modulatory cytokine interleukin-10 (IL-10). IL-10 plays an important role in the functional control in vivo of autoreactive. Th-1 type CD4+ T-cells, with experimental manipulation of 1L-10 leading to significant modulation of disease development in animal models of autoimmunity. IL-10 -592 genotypes were studied by PCR in 171 well characterised, histologically staged, PBC patients and 141 geo-graphically matched North-Eastern controls. 99/171 (58%) of PBC patients were homozygous for the commoner allele (C/C), 68/171 (40%) were heterozygotes (A/C) whilst 4/171 (2%) were homozygous for the rarer allele (A/A). These genotype frequencies were not significantly different to those seen in controls (p=0.49, OR 1.2 [0.8-1.9]). No variation in allele frequency was seen when PBC patients were subdivided according to disease stage (C allele frequency 0.81 (stage I/II) v 0.77 (stage III/IV). Conclusions: These findings suggest that IL-10 -592 is neither a susceptibility nor a severity locus in PBC.