- Email: [email protected]
Levels of tumor necrosis factor-α in portal vein ligated rats
HEPATOLOGY Vol. 22, No. 4, P t . 2, 1995
AASLD
1401 LEVELS OF~ TUMOR NECROSIS FACTOR-a IN PORTAL VEIN
ABSTRACTS
1402 REFERRAL PATTERN AND MANAGEMENT OF POST
LIGATED RATS. Ipp Michielsen. 1.2AM Ramon. IPA Pelckmans, ~Division of Gastroenterology, University of Antwerp (UIA), Belgium; 2International Institute of Immunopathology,Cologne, FRG.
LAPAROSCOPIC CHOLECYSTECTOMY BILE DUCT INJURIES. DF Mirza, T IsmaU, BH Ferraz-Neto~ N Narsimhan, AD Mayer, P McMaster, JAC Buckels. The Liver Unit, Queen Elizabeth Hospital, Birmingham, UK.
We recently demonstrated increased nitric oxide production in the wall of thoracic aortic rings from portal vein ligated (PVL) rats (Eur J Pharmacol 1995;273:167). Tumor necrosis factor-a (TNF-a) has been suggested to be involved in the pathogenesis of the hyperdynamic circulation in PVL rats (HEPATOLOGY 1993;18:140A). In order to study the role of circulating TNF-c~, plasma levels were determined in male Wistar rats, that underwent either PVL, n=16, or sham operation (SO), n=13. At 3 weeks postoperatively, cardiac blood was sampled percutaneously under ether anesthesia and using sterile technique by means of an endotoxin-free vacutainer sampling tube (EndoTube ET, Chromogenix AB, Mtilndal, Sweden). Plasma was separated within 2 hours and stored at -25°C before assay. The samples were evaluated with Factor-Test-XT M mouse TNF-a ELISA kit (Genzyme, Cambridge, MA), which has also been used successfully to quantitate rat TNF-a. Plasma' samples and standards are incubated on a microtiter plate precoated with monoclonal anti-mouse TNF-c~. After washing again, a peroxidase-conjugated polyclonal anti-mouse TNF-c= which binds to captured TNF-a is added. After washing, a substrate solution is added which initiates a peroxidase catalyzed color change that is stopped by acidification. The absorbance measured at 450 nm is proportionate to the concentration of TNF-a present in the standards or samples. A linear coefficient of correlation between the individual mean delta absorbance of the standards versus their respective TNF-a concentration of 0.99 was obtained for measurements between 35 and 560 pg/ml. The detection limit was 15 pg/ml. The TNF~ concentrations in the experimental samples are determined using the standard curve. Differences between the 2 independent groups of rats were analyzed by nonparametric Mann-Whitney test corrected for ties. Results: In the SO rats, TNF-a levels were 17.5+1.2 pg/ml, in the PVL rats 19.4+1.4 pg/ml, not significantly different. Conclusion: Our findings do not support a role for circulating TNF-a in the pathogenesisof the hyperdynamiccirculation in PVL rats.
1403
CONDITIONS FOR QUANTITATION OF HCV RNA IN CLINICAL PRACTICE. E Miskovsky~ A Carella*, K Gutekunsh T Quinn, D Thomas. The Johns Hopkins School of Medicine, Baltimore, Md and Roche Molecular Systems, Branchburg, NJ. Measurements of the quantity of hepatitis C virus (HCV) RNA are clinically useful for predicting the likelihood of response to and the effect of therapy for chronic hepatitis C. However, concern with the instability of HCV RNA has limited the utilization of these assays. In order to assess the optimal clinical conditions for collection, transportation, and storage of blood specimens obtained for HCV RNA quantitation, we collected blood from 10 HCV-infected patients attending the Johns Hopkins Hepatitis Clinic. From each patient 20 3mi tubes of blood were drawn, 10 EDTA (purple), 5 serum (red), and 5 ACD (yellow). Within 10 minutes of collection, 2 EDTA tubes, 1 serum tube, and 1 ACD tube were centrifuged and aliquots were stored at -70°C. Four EDTA robes were placed at 4°C and the remaining tubes were placed in a storage rack at room temperature. At time points 2, 6, 24, and 48 hours, 1 of each tube type was centrifuged and aliquots promptly stored at -70°C. The quantity of HCV RNA was then measured on stored specimens in 2 independent laboratories using a quantitative PCR assay, the AMPLICOR HCV MONITORTM TEST (Roche Molecular Systems, Branchburg NJ). In both laboratories the following results were obtained: 1) the quantity of HCV RNA was similar (within 1 log) in all storage conditions and tube types; 2) measurements of the quantity of HCV RNA were similar at all time points and between laboratories; and 3) 1-4 log decreases in HCV RNA were detected when specimens were diluted 1:10, 100, 1000, and 10,000, respectively. Using the HCV MONITOR test, clinicians may reliably measure the quantity of HCV RNA within 48 hours of collection in blood stored in EDTA, serum, or ACD tubes. The study was partially funded by Roche Molecular Systems.
457A
Laparoscopic cholecystectomy (LC) is the treatment of choice for cholelithiasis. However this is associated with an increased incidence of bile duct injury(BDI). Methods: From 1991-1994, 20 patients(12 females, median age 49 years, range 25-67, all elective cholecystectomies) were referred to this tertiary unit (14 regional cases, 6 extra. regional) with BDI. At LC, surgery was described as difficult in 10 cases, and 4 BDIs were recognised intraoperatively. Patients were transferred at a median of 26(0-990) days post-LC, although initial symptoms were recorded at a median 3(0-700) days post-LC. Eleven patients underwent additional surgical procedures before referral. Results: BDI were classified as leak from subvesical duct(l), right hepatic duct transection(2), lateral injury common hepatic duct(CHD)(1), CHD division > 2cm from hilum(t), CHD division < 2cm from hilum(8), CHD division at confluence (4), injury involving secondary bile ducts (3); two patients had associated duodenal fistulae. Management included hepatico-jejunostomy(l 1), right hepaticojejunostomy(2), right+left hepatico-jejunostomies(l), laparotomyldrainage (1), ERC+stent(2), pigtail drainage(2) and PTC with dilatation/stent(3). Further complications included: bile leak(2), stricture(2), cholangitis(4), portal hernia(l), intraabdominal abscess(I), secondary biliary cirrhosis(1 ), multiorgan failure and death(I). The median postreferral inpatient stay was 21(7-78) days, the median follow-up was 20(4-42) months. Conclusion: BDI following LC is a complex management problem resulting in significant postoperative morbidity, and is best managed by specialist units.
1404
HEPATIC EXPRESSION OF HEPATOCYTE GROWTH FACTORL I ~ A C R O P H A G E - S T I M U L A T I N G PROTEIN mRNA IN LIVER CIRRHOSIS. Y Miwa. PM Harrison. RD Hu~hes. SJF De,an*, Roeer Williams. Institute of Liver Studies, King's College School of Medicine and Dentistry, London, UK, and *Division of Basic Science Research, Children's Hospital Research Foundation, Cincinnati, Ohio Hepatocytegrowth factor-like/macrophagestimulatingprotein(HGFL/MSP) is a serum protein produced by hepatocytes which stimulates macrophage phagocytosis and induces chemotactic response to C5a. Patients with liver cirrhosis are susceptible to infection partly due impared Kupffer cell phagocytic activity. We have therefore investigated whether reduced hepatic synthesis of HGFL/MSP could account for the impaired Kupffer cell function in these patients. Materials and Methods Liver tissue was obtained from 14 patients with liver cirrhosis (8 viral hepatitis, 4 alcoholic hepatitis, 2 primary biliary cirrhosis) and control tissue was obtained from 5 donor liver grafts. Total RNA was isolated by the guanidinium-acid/phenol/chloroformmethod and fractionated by gel elcctrophoresis. Northern blot analysis was performed using r~p_ labelled eDNA probes for HGFL/MSP, prothrombin and glyceraldchydc-3phosphate dehydroganase (GAPDH). Results The levels of HGFL/MSP mRNA, measured by densitometry and normalized to the expression of GAPDH were reduced in liver cirrhosis compared with normal liver. Prothrombin mRNA levels were also reduced in the patients, but not to the extent of HGFL/MSP. Conclusion Reduced HGFL/MSP production may contribute to the susceptibility to infection in liver cirrhosis. The mechanism for the downregulation of HGFL/MSP mRNA expression in cirrhosis is not known, but the lower HGFL/MSP mRNA levels relative to prothrombin mRNA suggests that it is not simply due to hepatocyte loss.
AASLD
1401 LEVELS OF~ TUMOR NECROSIS FACTOR-a IN PORTAL VEIN
ABSTRACTS
1402 REFERRAL PATTERN AND MANAGEMENT OF POST
LIGATED RATS. Ipp Michielsen. 1.2AM Ramon. IPA Pelckmans, ~Division of Gastroenterology, University of Antwerp (UIA), Belgium; 2International Institute of Immunopathology,Cologne, FRG.
LAPAROSCOPIC CHOLECYSTECTOMY BILE DUCT INJURIES. DF Mirza, T IsmaU, BH Ferraz-Neto~ N Narsimhan, AD Mayer, P McMaster, JAC Buckels. The Liver Unit, Queen Elizabeth Hospital, Birmingham, UK.
We recently demonstrated increased nitric oxide production in the wall of thoracic aortic rings from portal vein ligated (PVL) rats (Eur J Pharmacol 1995;273:167). Tumor necrosis factor-a (TNF-a) has been suggested to be involved in the pathogenesis of the hyperdynamic circulation in PVL rats (HEPATOLOGY 1993;18:140A). In order to study the role of circulating TNF-c~, plasma levels were determined in male Wistar rats, that underwent either PVL, n=16, or sham operation (SO), n=13. At 3 weeks postoperatively, cardiac blood was sampled percutaneously under ether anesthesia and using sterile technique by means of an endotoxin-free vacutainer sampling tube (EndoTube ET, Chromogenix AB, Mtilndal, Sweden). Plasma was separated within 2 hours and stored at -25°C before assay. The samples were evaluated with Factor-Test-XT M mouse TNF-a ELISA kit (Genzyme, Cambridge, MA), which has also been used successfully to quantitate rat TNF-a. Plasma' samples and standards are incubated on a microtiter plate precoated with monoclonal anti-mouse TNF-c~. After washing again, a peroxidase-conjugated polyclonal anti-mouse TNF-c= which binds to captured TNF-a is added. After washing, a substrate solution is added which initiates a peroxidase catalyzed color change that is stopped by acidification. The absorbance measured at 450 nm is proportionate to the concentration of TNF-a present in the standards or samples. A linear coefficient of correlation between the individual mean delta absorbance of the standards versus their respective TNF-a concentration of 0.99 was obtained for measurements between 35 and 560 pg/ml. The detection limit was 15 pg/ml. The TNF~ concentrations in the experimental samples are determined using the standard curve. Differences between the 2 independent groups of rats were analyzed by nonparametric Mann-Whitney test corrected for ties. Results: In the SO rats, TNF-a levels were 17.5+1.2 pg/ml, in the PVL rats 19.4+1.4 pg/ml, not significantly different. Conclusion: Our findings do not support a role for circulating TNF-a in the pathogenesisof the hyperdynamiccirculation in PVL rats.
1403
CONDITIONS FOR QUANTITATION OF HCV RNA IN CLINICAL PRACTICE. E Miskovsky~ A Carella*, K Gutekunsh T Quinn, D Thomas. The Johns Hopkins School of Medicine, Baltimore, Md and Roche Molecular Systems, Branchburg, NJ. Measurements of the quantity of hepatitis C virus (HCV) RNA are clinically useful for predicting the likelihood of response to and the effect of therapy for chronic hepatitis C. However, concern with the instability of HCV RNA has limited the utilization of these assays. In order to assess the optimal clinical conditions for collection, transportation, and storage of blood specimens obtained for HCV RNA quantitation, we collected blood from 10 HCV-infected patients attending the Johns Hopkins Hepatitis Clinic. From each patient 20 3mi tubes of blood were drawn, 10 EDTA (purple), 5 serum (red), and 5 ACD (yellow). Within 10 minutes of collection, 2 EDTA tubes, 1 serum tube, and 1 ACD tube were centrifuged and aliquots were stored at -70°C. Four EDTA robes were placed at 4°C and the remaining tubes were placed in a storage rack at room temperature. At time points 2, 6, 24, and 48 hours, 1 of each tube type was centrifuged and aliquots promptly stored at -70°C. The quantity of HCV RNA was then measured on stored specimens in 2 independent laboratories using a quantitative PCR assay, the AMPLICOR HCV MONITORTM TEST (Roche Molecular Systems, Branchburg NJ). In both laboratories the following results were obtained: 1) the quantity of HCV RNA was similar (within 1 log) in all storage conditions and tube types; 2) measurements of the quantity of HCV RNA were similar at all time points and between laboratories; and 3) 1-4 log decreases in HCV RNA were detected when specimens were diluted 1:10, 100, 1000, and 10,000, respectively. Using the HCV MONITOR test, clinicians may reliably measure the quantity of HCV RNA within 48 hours of collection in blood stored in EDTA, serum, or ACD tubes. The study was partially funded by Roche Molecular Systems.
457A
Laparoscopic cholecystectomy (LC) is the treatment of choice for cholelithiasis. However this is associated with an increased incidence of bile duct injury(BDI). Methods: From 1991-1994, 20 patients(12 females, median age 49 years, range 25-67, all elective cholecystectomies) were referred to this tertiary unit (14 regional cases, 6 extra. regional) with BDI. At LC, surgery was described as difficult in 10 cases, and 4 BDIs were recognised intraoperatively. Patients were transferred at a median of 26(0-990) days post-LC, although initial symptoms were recorded at a median 3(0-700) days post-LC. Eleven patients underwent additional surgical procedures before referral. Results: BDI were classified as leak from subvesical duct(l), right hepatic duct transection(2), lateral injury common hepatic duct(CHD)(1), CHD division > 2cm from hilum(t), CHD division < 2cm from hilum(8), CHD division at confluence (4), injury involving secondary bile ducts (3); two patients had associated duodenal fistulae. Management included hepatico-jejunostomy(l 1), right hepaticojejunostomy(2), right+left hepatico-jejunostomies(l), laparotomyldrainage (1), ERC+stent(2), pigtail drainage(2) and PTC with dilatation/stent(3). Further complications included: bile leak(2), stricture(2), cholangitis(4), portal hernia(l), intraabdominal abscess(I), secondary biliary cirrhosis(1 ), multiorgan failure and death(I). The median postreferral inpatient stay was 21(7-78) days, the median follow-up was 20(4-42) months. Conclusion: BDI following LC is a complex management problem resulting in significant postoperative morbidity, and is best managed by specialist units.
1404
HEPATIC EXPRESSION OF HEPATOCYTE GROWTH FACTORL I ~ A C R O P H A G E - S T I M U L A T I N G PROTEIN mRNA IN LIVER CIRRHOSIS. Y Miwa. PM Harrison. RD Hu~hes. SJF De,an*, Roeer Williams. Institute of Liver Studies, King's College School of Medicine and Dentistry, London, UK, and *Division of Basic Science Research, Children's Hospital Research Foundation, Cincinnati, Ohio Hepatocytegrowth factor-like/macrophagestimulatingprotein(HGFL/MSP) is a serum protein produced by hepatocytes which stimulates macrophage phagocytosis and induces chemotactic response to C5a. Patients with liver cirrhosis are susceptible to infection partly due impared Kupffer cell phagocytic activity. We have therefore investigated whether reduced hepatic synthesis of HGFL/MSP could account for the impaired Kupffer cell function in these patients. Materials and Methods Liver tissue was obtained from 14 patients with liver cirrhosis (8 viral hepatitis, 4 alcoholic hepatitis, 2 primary biliary cirrhosis) and control tissue was obtained from 5 donor liver grafts. Total RNA was isolated by the guanidinium-acid/phenol/chloroformmethod and fractionated by gel elcctrophoresis. Northern blot analysis was performed using r~p_ labelled eDNA probes for HGFL/MSP, prothrombin and glyceraldchydc-3phosphate dehydroganase (GAPDH). Results The levels of HGFL/MSP mRNA, measured by densitometry and normalized to the expression of GAPDH were reduced in liver cirrhosis compared with normal liver. Prothrombin mRNA levels were also reduced in the patients, but not to the extent of HGFL/MSP. Conclusion Reduced HGFL/MSP production may contribute to the susceptibility to infection in liver cirrhosis. The mechanism for the downregulation of HGFL/MSP mRNA expression in cirrhosis is not known, but the lower HGFL/MSP mRNA levels relative to prothrombin mRNA suggests that it is not simply due to hepatocyte loss.