- Email: [email protected]
Two overlapping immunodominant B-cell epitopes in HCV core antigen identified by monoclonal antibody mapping
Al124
AASI.D ABSTRACTS
GASTROENTEROLOGY, VOl. 108, No. 4
L A C K O F E F F E C T O F O M E P R A Z O L E THERAPY AND/OR A C U T E E T H A N O L INGESTION ON PITUITARY GONADAL H O R M O N A L AXIS. Anil Minocha. SriPrakash Mokshagundam, Salman Rashid. Departments of Medicine, University of Louisville, Louisville, Kentucky and University of Arkansas, Little Rock, Arkansas. INTRODUCTION: Omeprazole and ethanol are two commonly used drugs that are known to affect the endocrine and hepatic enzyme systems. It is therefore possible that the two could interact when used simultaneously and have significant impact on the pituitary-gonadal hormonal axis in healthy men. M E T H O D S : We determined the effect of acute ethanol ingestion in doses simulating "social drinking" before, and after a two week course of omeprazole. Eight healthy men without any history of ethanol abuse were studied. Blood was drawn for measurement o f L H , FSH, total and free testosterone in the basal state as well as three hours after the ingestion of ethanol (0.5g/Kg). The test was repeated after administration of omeprazole (20rag b.i.d.) for 2 weeks. All hormones were measured by radio-immunoassay. RESULTS: Hormone
Testosterone (ng/dl) Free Testosterone (ng/dl) LH (U/L) FSH (U/L)
P i e Omeprazole Basal PQSt et0h
Basal
527+94
489+69
565+141 504_.+99
132+35
142+43
129+40 142-1-34
4.6+1.0 5.6+__2,6
4.6+0.7 5.1+1.7
4.8-t-0.9 5.5-1-1.2 5.4-1-2.1 5 . 0 + 1 . 6
Post Omeprazole
Post etoh
CONCLUSIONS: Omeprazole and ethanol either alone or in combination do not have a significant impact on the pituitary_gonadal axis in healthy men,
Z O N A L H E T E R O G E N E I T Y O F COLD S T O R A G E - I N D U C E D M I T O C H O N D R I A L D Y S F U N C T I O N IN I S O L A T E D AND P E R F U S E D RAT L I V E R : R O L E S O F CD18 AND ICAM-1. S_ Miura, 1 Kurose, H Higuchi, S Kato, and H lshii. Department of Internal Medicine, School qf Meidcine, Keio University. Tokyo 160, Japan. Previous studies have implicated that neutrophil infiltration is a cause of ischemia-reperfusion-induced tissue damage. However, the role of lymphocytes in the postischemic liver injury has not been fully investigated. The present study was, therefore, designed to investigate the temporal and spatial relationship(s) between leukocyte (lymphocyte as well as neutrophil) accumulation and hepatic energy synthesis after the cold storage followed by reperfusion. To that effect, the liver of male Wistar rats was isolated and perfused with Euro-Colins solution (37°C, pH7.4) from the catheter inserted into the portal vein. The NADH autofluorescence in the surface of liver was continuously monitored (excitation 335nm, emission 380nm) and analyzed by the computer-assisted fluorescence microscopic system (Hepatology 18:380,1993). During the cold storage, the NADH autofluorescence significantly increased, and decreased to the baseline level immediately after the reperfusion. At 60 minutes after the reperfusion, the NADH autofluorescence significantly increased in the liver subjected to 120 minutes cold storage, but not 60 minutes. The increase in NADH was more enhanced in the pericentral area. In another series of experiments, CFSE-labeled lymphocytes or neutrophils ( l x l 0 8 cells) were injected from portal vein at the beginning of reperfusion after 120 minutes cold ischemia, and the same observation was performed. When either IL-2 (1000 units/ml)-treated lymphocytes or neutrophils were administered, the CFSE-associated fluorescence was accumulated in the periportal area~ and the NADH autofluorescence increased in the corresponding sites. The accumulation of the injected leukOcytes and an increase in NADH both observed in the periportal area were significantly attenuated by the simultaneous injection of monoclonal antibody directed against either CDI8 or ICAM-1. Furthermore, a flowcytometeric analysis demonstrated that treatment with IL-2 up-regulates the CDI 8 expression on leukocytes. Thus, the present study suggests that the c01d storage followed by reperfusion causes metabolic alteration in the pericentral area of rat liver, and that the postischemic mitochondrial dysfunction was enhanced by activated lymphocytes as well as neutrophils especially in the periportal area.
• 13C-GALACTOSE BREATH TEST: COMPARISON WITH GALACTOSE ELIMINATION CAPACITY TO MEASURE LIVER FUNCTION. F. Mion, M. Rousseau*, P. Paliard, Y. Minaire. F~d~ration des Sp~cialit~s Digestives, H6pital E. Herriot; * Inbiomed, Lyon, France. i, The galactose elimination capacity (GEC) test is often used as a measure of liver function. However, GEC is a clearance test rather than an exact evaluation of liver metabolic capacity, lacks of sensitivity in non cirrhotic liver diseases, and relies on repeated blood sampling. The goal of this study was to develop a ~aC-galactose breath test (GBT), and to evaluate its results in patients with non cirrhotic and cirrhotic liver diseases, in comparison with the GEC. This study was performed in 5 healthy volunteers, 6 patients with chronic viral hepatitis, and 16 cirrhotic patients (5 Child A, 7 Child B and 4 Child C). The GBT and GEC were performed at the same time. After an overnight fast, 500 mg/kg of BW of galactose (containing 1% of [1~sC]-galactose (99% APE) were given iv. Blood samples were obtained every 5 minutes between 20 and 75 minutes after the end of galactose infusion, to measure galactosemia and calculate GEC. Breath samples were obtained every 10 minutes between 0 and 90 minutes, and every 30 minutes until 180 minutes.~3C isotopic enrichment in breath was measured by GC-IRMS, and the results were then expressed as % of the dose of galactose recovered in breath. The azC enrichement in breath was linear over time between O and 90 min, in patients as well as in controls. Regression analysis of ~sC excretion curves allowed the calculation of the amount of galactose oxidised by the liver per unit of time. The results of the GBT were significantly different between controls, non cirrhotic and cirrhotic patients (galactose oxidised in ~tmol/min = 134.6+_10.3 vs 84.5+_12.8 vs 56.6+_5.6 respectively, p<9.0001). There was a good correlation with GEC (r2=0,743, p<0.001). GEC and GBT were not found to be dependent on age and sex. However, results of GEC were significantly correlated to patients' body weight, which was not the case for GBT. In cirrhotic patients, there was a significant correlation between the severity of liver disease (as assessed by the Child-Pugh classification), and the results of GBT (p<0.05). In conclusion, GBT appears as a non invasive and very sensitive ~tool to measure liver function. Its place in the follow-up of chronic liver ,diseases remains to be defined.
• TWO OVERLAPPING IMMUNODOMINANT B-CELL EPITOPES IN HCT CORE ANTIGEN IDENTIFIED BY MONOCLON2ALANTIBODY MAPPING Mizokami M, Orito E, Ohba If, Hasegawa A 1, Ohb~ Y , Kohara M3 , Lau JYN4 . Nagoya City
University, Nagoya; Tonen Corporation , Saltama; Tokyo Metropolitan Institute of Medical Science , Tokyo, Japan; University of Florida, Gaine~ville,FL. Background HCV has been shown to have a diameter of 55-60 nm with a 30-33 nm inner core. The HCV core region is relatively well conserved and has a good antlgenicity profile, which on a theoretical basis, may allow for the development of assays for direct detection of HCV particles H.vpothesisConserved immnnodominant B-cen epitopes are present within the HCV core antigen. Ai...mmTo map the immunodominant B-ceil epitopes using a combination of monoclonai antibodies and synthetic overlapping peptides. Methods HCV CoreAntigen Production The HCV core region contains a highly hydrophohic 3' terminus with a low antigenicity pro[de. Accordingly,the truncated HCV core [spanning amino acid (aa) 1-152, HCV type Ib] was expressed as a fusion protein in E. coll. Generation of MonoclonalAntibodies BALB/e female mice 7-10 weeks old were immunized with 15-50pg antigen subcutaneously (SC). A boost 100//g SC at week 4 and 200/ag intravenous at week 7 were given to booster the immune response. The spleen cells were fused with P3.X63Ag8.653 myeloma ceils, cloned, and inject intraperitoneally to produce ascitic fluid containing the antibodies to HCV core. Generation of PeDtides/Sereenine for the Epitopes Chemical peptides were synthesized using a solid phase strategy with the Fast Mac system. These peptides covered the 5'terminns (aa 1-73) since this region has the highest antigenicity prof'de. Mapping of the epitopes were achieved by ELISA. Sequence Comparison/Ph~,logenetic TreeAnalysis The reported deduced HCV core aa sequences were aligned to determine the variations of the mapping epitopes (all 6 major types). The phylogenetic tree relationship of the sequences was confirmed by the construction of a phylogenetic tree based on the Neighbor-jolning method. Results A total of 9 monoelonal antibodies were produced. 36 peptides of various length covering the aa sequence 1-73 were also generated to map the epitopes. The monoelonal antibodies can be divided into two groups. 5 (group I) recognized a minimal epitope spanning aa 26-45 and the remaining 4 (group 2) spanned aa 39-50. Despite a significant overlap of the two epitopes, the two groups of antibodies were not mutually interfering in binding by ELISA, indicating that the overlapping was essential for epitope confirmation instead of binding motifs. Comparison of the aa sequences derived from various HCV types showed that this region is well conserved, apart from type 3 HCV (strain HCV-K3a) where there were 4 aa substitutions within this region. Conclusions (I) There are two overlapping immnnodominant B-cell epitopes in the HCV core region (an 26-50). (2) The conserved nature of this region, and the presence of overlapping B-cell epitopes that are non-mutually exclusive, allows the development of assays for the capture of HCV core antigens in circulation.
AASI.D ABSTRACTS
GASTROENTEROLOGY, VOl. 108, No. 4
L A C K O F E F F E C T O F O M E P R A Z O L E THERAPY AND/OR A C U T E E T H A N O L INGESTION ON PITUITARY GONADAL H O R M O N A L AXIS. Anil Minocha. SriPrakash Mokshagundam, Salman Rashid. Departments of Medicine, University of Louisville, Louisville, Kentucky and University of Arkansas, Little Rock, Arkansas. INTRODUCTION: Omeprazole and ethanol are two commonly used drugs that are known to affect the endocrine and hepatic enzyme systems. It is therefore possible that the two could interact when used simultaneously and have significant impact on the pituitary-gonadal hormonal axis in healthy men. M E T H O D S : We determined the effect of acute ethanol ingestion in doses simulating "social drinking" before, and after a two week course of omeprazole. Eight healthy men without any history of ethanol abuse were studied. Blood was drawn for measurement o f L H , FSH, total and free testosterone in the basal state as well as three hours after the ingestion of ethanol (0.5g/Kg). The test was repeated after administration of omeprazole (20rag b.i.d.) for 2 weeks. All hormones were measured by radio-immunoassay. RESULTS: Hormone
Testosterone (ng/dl) Free Testosterone (ng/dl) LH (U/L) FSH (U/L)
P i e Omeprazole Basal PQSt et0h
Basal
527+94
489+69
565+141 504_.+99
132+35
142+43
129+40 142-1-34
4.6+1.0 5.6+__2,6
4.6+0.7 5.1+1.7
4.8-t-0.9 5.5-1-1.2 5.4-1-2.1 5 . 0 + 1 . 6
Post Omeprazole
Post etoh
CONCLUSIONS: Omeprazole and ethanol either alone or in combination do not have a significant impact on the pituitary_gonadal axis in healthy men,
Z O N A L H E T E R O G E N E I T Y O F COLD S T O R A G E - I N D U C E D M I T O C H O N D R I A L D Y S F U N C T I O N IN I S O L A T E D AND P E R F U S E D RAT L I V E R : R O L E S O F CD18 AND ICAM-1. S_ Miura, 1 Kurose, H Higuchi, S Kato, and H lshii. Department of Internal Medicine, School qf Meidcine, Keio University. Tokyo 160, Japan. Previous studies have implicated that neutrophil infiltration is a cause of ischemia-reperfusion-induced tissue damage. However, the role of lymphocytes in the postischemic liver injury has not been fully investigated. The present study was, therefore, designed to investigate the temporal and spatial relationship(s) between leukocyte (lymphocyte as well as neutrophil) accumulation and hepatic energy synthesis after the cold storage followed by reperfusion. To that effect, the liver of male Wistar rats was isolated and perfused with Euro-Colins solution (37°C, pH7.4) from the catheter inserted into the portal vein. The NADH autofluorescence in the surface of liver was continuously monitored (excitation 335nm, emission 380nm) and analyzed by the computer-assisted fluorescence microscopic system (Hepatology 18:380,1993). During the cold storage, the NADH autofluorescence significantly increased, and decreased to the baseline level immediately after the reperfusion. At 60 minutes after the reperfusion, the NADH autofluorescence significantly increased in the liver subjected to 120 minutes cold storage, but not 60 minutes. The increase in NADH was more enhanced in the pericentral area. In another series of experiments, CFSE-labeled lymphocytes or neutrophils ( l x l 0 8 cells) were injected from portal vein at the beginning of reperfusion after 120 minutes cold ischemia, and the same observation was performed. When either IL-2 (1000 units/ml)-treated lymphocytes or neutrophils were administered, the CFSE-associated fluorescence was accumulated in the periportal area~ and the NADH autofluorescence increased in the corresponding sites. The accumulation of the injected leukOcytes and an increase in NADH both observed in the periportal area were significantly attenuated by the simultaneous injection of monoclonal antibody directed against either CDI8 or ICAM-1. Furthermore, a flowcytometeric analysis demonstrated that treatment with IL-2 up-regulates the CDI 8 expression on leukocytes. Thus, the present study suggests that the c01d storage followed by reperfusion causes metabolic alteration in the pericentral area of rat liver, and that the postischemic mitochondrial dysfunction was enhanced by activated lymphocytes as well as neutrophils especially in the periportal area.
• 13C-GALACTOSE BREATH TEST: COMPARISON WITH GALACTOSE ELIMINATION CAPACITY TO MEASURE LIVER FUNCTION. F. Mion, M. Rousseau*, P. Paliard, Y. Minaire. F~d~ration des Sp~cialit~s Digestives, H6pital E. Herriot; * Inbiomed, Lyon, France. i, The galactose elimination capacity (GEC) test is often used as a measure of liver function. However, GEC is a clearance test rather than an exact evaluation of liver metabolic capacity, lacks of sensitivity in non cirrhotic liver diseases, and relies on repeated blood sampling. The goal of this study was to develop a ~aC-galactose breath test (GBT), and to evaluate its results in patients with non cirrhotic and cirrhotic liver diseases, in comparison with the GEC. This study was performed in 5 healthy volunteers, 6 patients with chronic viral hepatitis, and 16 cirrhotic patients (5 Child A, 7 Child B and 4 Child C). The GBT and GEC were performed at the same time. After an overnight fast, 500 mg/kg of BW of galactose (containing 1% of [1~sC]-galactose (99% APE) were given iv. Blood samples were obtained every 5 minutes between 20 and 75 minutes after the end of galactose infusion, to measure galactosemia and calculate GEC. Breath samples were obtained every 10 minutes between 0 and 90 minutes, and every 30 minutes until 180 minutes.~3C isotopic enrichment in breath was measured by GC-IRMS, and the results were then expressed as % of the dose of galactose recovered in breath. The azC enrichement in breath was linear over time between O and 90 min, in patients as well as in controls. Regression analysis of ~sC excretion curves allowed the calculation of the amount of galactose oxidised by the liver per unit of time. The results of the GBT were significantly different between controls, non cirrhotic and cirrhotic patients (galactose oxidised in ~tmol/min = 134.6+_10.3 vs 84.5+_12.8 vs 56.6+_5.6 respectively, p<9.0001). There was a good correlation with GEC (r2=0,743, p<0.001). GEC and GBT were not found to be dependent on age and sex. However, results of GEC were significantly correlated to patients' body weight, which was not the case for GBT. In cirrhotic patients, there was a significant correlation between the severity of liver disease (as assessed by the Child-Pugh classification), and the results of GBT (p<0.05). In conclusion, GBT appears as a non invasive and very sensitive ~tool to measure liver function. Its place in the follow-up of chronic liver ,diseases remains to be defined.
• TWO OVERLAPPING IMMUNODOMINANT B-CELL EPITOPES IN HCT CORE ANTIGEN IDENTIFIED BY MONOCLON2ALANTIBODY MAPPING Mizokami M, Orito E, Ohba If, Hasegawa A 1, Ohb~ Y , Kohara M3 , Lau JYN4 . Nagoya City
University, Nagoya; Tonen Corporation , Saltama; Tokyo Metropolitan Institute of Medical Science , Tokyo, Japan; University of Florida, Gaine~ville,FL. Background HCV has been shown to have a diameter of 55-60 nm with a 30-33 nm inner core. The HCV core region is relatively well conserved and has a good antlgenicity profile, which on a theoretical basis, may allow for the development of assays for direct detection of HCV particles H.vpothesisConserved immnnodominant B-cen epitopes are present within the HCV core antigen. Ai...mmTo map the immunodominant B-ceil epitopes using a combination of monoclonai antibodies and synthetic overlapping peptides. Methods HCV CoreAntigen Production The HCV core region contains a highly hydrophohic 3' terminus with a low antigenicity pro[de. Accordingly,the truncated HCV core [spanning amino acid (aa) 1-152, HCV type Ib] was expressed as a fusion protein in E. coll. Generation of MonoclonalAntibodies BALB/e female mice 7-10 weeks old were immunized with 15-50pg antigen subcutaneously (SC). A boost 100//g SC at week 4 and 200/ag intravenous at week 7 were given to booster the immune response. The spleen cells were fused with P3.X63Ag8.653 myeloma ceils, cloned, and inject intraperitoneally to produce ascitic fluid containing the antibodies to HCV core. Generation of PeDtides/Sereenine for the Epitopes Chemical peptides were synthesized using a solid phase strategy with the Fast Mac system. These peptides covered the 5'terminns (aa 1-73) since this region has the highest antigenicity prof'de. Mapping of the epitopes were achieved by ELISA. Sequence Comparison/Ph~,logenetic TreeAnalysis The reported deduced HCV core aa sequences were aligned to determine the variations of the mapping epitopes (all 6 major types). The phylogenetic tree relationship of the sequences was confirmed by the construction of a phylogenetic tree based on the Neighbor-jolning method. Results A total of 9 monoelonal antibodies were produced. 36 peptides of various length covering the aa sequence 1-73 were also generated to map the epitopes. The monoelonal antibodies can be divided into two groups. 5 (group I) recognized a minimal epitope spanning aa 26-45 and the remaining 4 (group 2) spanned aa 39-50. Despite a significant overlap of the two epitopes, the two groups of antibodies were not mutually interfering in binding by ELISA, indicating that the overlapping was essential for epitope confirmation instead of binding motifs. Comparison of the aa sequences derived from various HCV types showed that this region is well conserved, apart from type 3 HCV (strain HCV-K3a) where there were 4 aa substitutions within this region. Conclusions (I) There are two overlapping immnnodominant B-cell epitopes in the HCV core region (an 26-50). (2) The conserved nature of this region, and the presence of overlapping B-cell epitopes that are non-mutually exclusive, allows the development of assays for the capture of HCV core antigens in circulation.