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Assessment of response to interferon (α-IFN) in obese and non-obese patients with chronic HCV
April 1995
LOWERING EFFECTOF BRANCHED CHAIN AMONO ACID AND ARGININE ON BLOOD AMMONIA LEVEL IN PATIENTSWITH LIVERCIRRHOSIS. K. Kurosawa, M. Kato, K. Yamamoto, N. HayasN, A. Kaneko, T. Michida, M. Shiomi. N. Park, H. Ishida, and M. Masuzawa Department of Gastroenterology, Osaka National Hospital, Osaka, 540, JAPAN. Hepatic encephalopathy is a multifactorial disorder and ammonia is considered to be one of the candidates contributing to its pathogenesis. Infusion of amino acid solutions composed mainly of branched chain amino acid (BCAA) or of argenine (Arg) is often used for treatment of patients with hyperammonemia due to liver cirrhosis. In this study we measured changes in the level of blood ammonia caused by administration of BCAA or Arg, and analyzed the results in correlation with liver functions. METHQD$: Nineteen pathients with liver cirrhosis or hepatocellular carcinoma were infused with BCAA(n=13) or Arg(n=6) solution. Before and after the infusion, blood samples were taken for determination of ammonia, blood chemistry, and concentration of amino acids. RESULTS: We found that of 13 patients infused with BCAA, levels of blood ammonia increased in 8 patients(Group A), and decreased in 5 patients(Group B). Administration of Arg brought about significant decrease in ammonia levels in all the patients tested(Group C). Blood levels of albumin, choline esterase and indirect bilirubin of each group are as follows; AIb Ch E Indir. Bil Group A 2.57+0.51 62,1,17 1.28,1,0.61" *p
• ASSESSMENT OF RESPONSE TO INTERFERON (u-IFN) IN OBESE AND NON-OBESE PATIENTS WITH CHRONIC HCV. N.P. Lain*, D.L. Pitrak, R.C. Speralakis°, T.E. Wiley, T.J Layden. Depts. of Medicine and Pharmacy Practice*, University of Illinois, Chicago, Illinois We have shown that one of the variables that predicts an unfavorable response to ct-IFN treatment in HCV patients (pts) is obesity (Dig Dis Sci, accepted for publication). The mechanisms for this negative effect is unclear but may relate to 1) obese pts have more advanced liver disease; 2) impaired absorption or distribution of cMFN in obesity; 3) higher pre-treatment HCV RNA levels; 4) and/or impaired cellular response to ct-IFN. To determine whether one or more of these variables is altered in obese pts, we examined 6 obese (O) (weight > 30% of IBW) and 5 non-obese (NO) pts with chronic HCV infection before and after a 10 mlU single-dose t~-IFN injection. This high dose of ct-IFN was chosen in order to obtain measurable serum t~-IFN levels. Variables studied include: Knodell Histologic Acitivity Index (HA1); serum HCV RNA levels by quantitative branched DNA; 2'5'-oligoadenlylate synthetase (2-50AS) levels in peripheral blood mononuclear cells (PBMC) as a biologic response index; and serum ct-IFN levels over 24 h post injection. The mean HAl was similar in O and NO pts. Although baseline HCV RNA levels were higher in O (15.34-15.9 equiv/ml) than NO (7.74-6.0 equiv/ml), values were not statisticallydifferent. Peak a-IFN levels and total ~-IFN levels integrated over 24 h were inversely proportional to body weight (for peak c~IFN r=-0.69, p---0.02). Despite lower integral~ed ct-IFN levels, HCV RNA titers were equally reduced at 24 h in O and NO pts, averaging 1.14-2.0 equiv/ml and 0.4+0.7 equiv/ml respectively. Baseline 2-5 OAS levels were inversely proportional to body weight (p < .004); peak 2-50AS were inversely proportional (r=-0.6, p < 0.03) to peak eMFN levels; and 2-50AS levels were not predictive of viral clearance in O and NO. In summary, 1) neither the HAl nor the level of viremia was significantly different in O vs. NO subjects. 2) PBMC 2-50AS levels appear not to be a good biologic indicator of ct-IFN response or serum viral clearance; 3) serum viral clearance with this dose of c~-IFN is independent of body weight and pretreatment serum HCV RNA levels; 4) serum t~-IFN levels attained after a single, 10 mlU dose of u-IFN were lower in obese patients. Lower serum ct-IFN levels may reflect poor absorption, differences in distribution, and/or metabolism of c¢-IFN in obese patients. (This research was funded in pa(t by the Schering Corp., Kenilworth, NJ.)
AASLD
Al105
• CDI8/ICAM-1-DEPENDENT P R O C E S S F O R K U P F F E R C E L L INDUCED M I T O C H O N D R I A L DYSFUNCTION IN H E P A T O M A C E L L S : I N F L U E N C E S OF C H R O N I C E T H A N O L - F E E D I N G ON N I T R I C OXIDE (NO) SYNTHESIS. I Kurose, H Ebinuma, H Higuchi, H Saito, S Kato, S Miura, and H Ishii. Department ~lnternal Medicine,
Keio University School of Medicine, Tokyo 160. JAPAN. Recent studies have shown that NO released from Kupffer cells (KCs) modulates mitochondrial function in cocultured rat hepatoma cells (AH70) (Cancer Res 53:2676,1993). The present study was designed to investigate 1) the mechanism(s) by which KCs release NO in response to coculture with hepatoma cells, and 2) whether chronic ethanol-feeding alters NO production of KCs and subsequent mitochondriat dysfunction in hepatoma cells. To that effect, KCs isolated from male Wistar rats were cocultured with AH70 cells. The confocaI laser scanning microscopic observations revealed that coculture of non-activated KCs and AH70 cells increased cytosolic Ca ++ level in KCs indicated as the fura-2 fluorescence, and reduced mitochondrial energization in AH70 cells indicated as the rhodamine 123 (Rh t23) fluorescence. The sum of nitrite and nitrate levels increased in the culture medium of KCs with AH70 cells as compared with in that of KCs alone. Either L-NMMA, or aminoguanidine effectively attenuated NO production of KCs and the reduction of Rh 123 fluorescence in AH70 cells, suggesting that NO produced by inducible NO synthase of KCs modulates hepatoma cell mitochondrial function. Furthermore, either nicardipine (a voltage-dependent Ca ++ channel blocker), monoclonal antibody against CDI8 or ICAM-I attenuated the NO production of KCs as well as fluorographic alterations. A flowcytometeric analysis provided the evidence that the coculture increases ICAM-I expression on AH70 cells and CDI8 on KCs. These results suggest that the CD18/ICAM-1dependent close contact stimulates Ca ++ mobilization and NO production in KCs. In another set of experiment, KCs isolated from rats, which were pair-fed with Lieber's ethanol-containing liquid diet, were used in the same investigations. KCs from ethanol-fed rats did not show the Ca ++ mobilization, increase NO production or reduce Rh123 fluorescence in AH70 cells after the coculture, whereas an increased CD 18 expression was still observed. The present study, therefore, supports a scenario that the chronic ethanol-feeding diminishes the NO production of KCs via altering the Ca ++ channel sensitivity, and reduces cytotoxic activities of KCs against hepatoma cells.
EFFECT OF HIGH-DOSE ALPHAqNTERFERON (IFN) ON VIREMIA OF RESISTANT HCV GENOTYPES. N.P. Lain*, D.R. Gretch**, M.J. Chung**, T.J. Layden***. Depts. of Medicine*** and Pharmacy Practice*, University of Illinois, Chicago, IL; and Hepatitis Lab **, University of Washington Medical Center, Seattle, WA, U.S.A. It has been suggested that HCV genotypes correlate with response to IFN, and that patients infected with HCV Type I have a lower response to the standard dose of IFN (3 mlU). The effect of high-dose IFN on improving the response of resistant HCV genotypes to treatment have not been clearly defined. This study examined the effect of a single 10 mlU injection of IFN on viral clearance in ten patients who are infected with HCV (Type la, n=5; Type lb, n=5) over 48 hours. Serial serum samples were collected at time 0 (baseline) and at 6, t0, 24 and 48 hours post-IFN injection. Quantitative HCV RNA levels were then determined using branched DNA assay. All patients had a significant reduction in HCV RNA levels at 24 hour post-lFN, of which 5 had undetectable levels. However, by 48 hour post-IFN, a rise in HCV RNA levels were noted in 7 patients, two of which had undetectable levels at 24 hour. Nevertheless, the HCV RNA levels remained significantly lower than the baseline values. The results suggest that a single, high-dose IFN significantly reduces the viremia of resistant HCV genotypes 24 hours after treatment, and the effect has been transient in these patients. Further studies should evaluate the benefit of incorporating high-dose and/or more frequent dosing of IFN for treating patients infected with resistant HCV genotypes. The effect of 1FN on the HCV viremia in the peripheral blood mononuclear cells (PBMC) and liver cells should also be assessed. Mean (4- s.d.) HCV RNA levels (x l&) in equivalents/ml at different time points post-IFN injection Baseline
6-hr
10-hr
24-hr
48-hr'
13.0+13.0
7.8_6.1
8.14-8.0
0.4+0.5
1.34-1.4
This reseach was funded in part by the Schering Corporation, Kenilworth, NJ.
LOWERING EFFECTOF BRANCHED CHAIN AMONO ACID AND ARGININE ON BLOOD AMMONIA LEVEL IN PATIENTSWITH LIVERCIRRHOSIS. K. Kurosawa, M. Kato, K. Yamamoto, N. HayasN, A. Kaneko, T. Michida, M. Shiomi. N. Park, H. Ishida, and M. Masuzawa Department of Gastroenterology, Osaka National Hospital, Osaka, 540, JAPAN. Hepatic encephalopathy is a multifactorial disorder and ammonia is considered to be one of the candidates contributing to its pathogenesis. Infusion of amino acid solutions composed mainly of branched chain amino acid (BCAA) or of argenine (Arg) is often used for treatment of patients with hyperammonemia due to liver cirrhosis. In this study we measured changes in the level of blood ammonia caused by administration of BCAA or Arg, and analyzed the results in correlation with liver functions. METHQD$: Nineteen pathients with liver cirrhosis or hepatocellular carcinoma were infused with BCAA(n=13) or Arg(n=6) solution. Before and after the infusion, blood samples were taken for determination of ammonia, blood chemistry, and concentration of amino acids. RESULTS: We found that of 13 patients infused with BCAA, levels of blood ammonia increased in 8 patients(Group A), and decreased in 5 patients(Group B). Administration of Arg brought about significant decrease in ammonia levels in all the patients tested(Group C). Blood levels of albumin, choline esterase and indirect bilirubin of each group are as follows; AIb Ch E Indir. Bil Group A 2.57+0.51 62,1,17 1.28,1,0.61" *p
• ASSESSMENT OF RESPONSE TO INTERFERON (u-IFN) IN OBESE AND NON-OBESE PATIENTS WITH CHRONIC HCV. N.P. Lain*, D.L. Pitrak, R.C. Speralakis°, T.E. Wiley, T.J Layden. Depts. of Medicine and Pharmacy Practice*, University of Illinois, Chicago, Illinois We have shown that one of the variables that predicts an unfavorable response to ct-IFN treatment in HCV patients (pts) is obesity (Dig Dis Sci, accepted for publication). The mechanisms for this negative effect is unclear but may relate to 1) obese pts have more advanced liver disease; 2) impaired absorption or distribution of cMFN in obesity; 3) higher pre-treatment HCV RNA levels; 4) and/or impaired cellular response to ct-IFN. To determine whether one or more of these variables is altered in obese pts, we examined 6 obese (O) (weight > 30% of IBW) and 5 non-obese (NO) pts with chronic HCV infection before and after a 10 mlU single-dose t~-IFN injection. This high dose of ct-IFN was chosen in order to obtain measurable serum t~-IFN levels. Variables studied include: Knodell Histologic Acitivity Index (HA1); serum HCV RNA levels by quantitative branched DNA; 2'5'-oligoadenlylate synthetase (2-50AS) levels in peripheral blood mononuclear cells (PBMC) as a biologic response index; and serum ct-IFN levels over 24 h post injection. The mean HAl was similar in O and NO pts. Although baseline HCV RNA levels were higher in O (15.34-15.9 equiv/ml) than NO (7.74-6.0 equiv/ml), values were not statisticallydifferent. Peak a-IFN levels and total ~-IFN levels integrated over 24 h were inversely proportional to body weight (for peak c~IFN r=-0.69, p---0.02). Despite lower integral~ed ct-IFN levels, HCV RNA titers were equally reduced at 24 h in O and NO pts, averaging 1.14-2.0 equiv/ml and 0.4+0.7 equiv/ml respectively. Baseline 2-5 OAS levels were inversely proportional to body weight (p < .004); peak 2-50AS were inversely proportional (r=-0.6, p < 0.03) to peak eMFN levels; and 2-50AS levels were not predictive of viral clearance in O and NO. In summary, 1) neither the HAl nor the level of viremia was significantly different in O vs. NO subjects. 2) PBMC 2-50AS levels appear not to be a good biologic indicator of ct-IFN response or serum viral clearance; 3) serum viral clearance with this dose of c~-IFN is independent of body weight and pretreatment serum HCV RNA levels; 4) serum t~-IFN levels attained after a single, 10 mlU dose of u-IFN were lower in obese patients. Lower serum ct-IFN levels may reflect poor absorption, differences in distribution, and/or metabolism of c¢-IFN in obese patients. (This research was funded in pa(t by the Schering Corp., Kenilworth, NJ.)
AASLD
Al105
• CDI8/ICAM-1-DEPENDENT P R O C E S S F O R K U P F F E R C E L L INDUCED M I T O C H O N D R I A L DYSFUNCTION IN H E P A T O M A C E L L S : I N F L U E N C E S OF C H R O N I C E T H A N O L - F E E D I N G ON N I T R I C OXIDE (NO) SYNTHESIS. I Kurose, H Ebinuma, H Higuchi, H Saito, S Kato, S Miura, and H Ishii. Department ~lnternal Medicine,
Keio University School of Medicine, Tokyo 160. JAPAN. Recent studies have shown that NO released from Kupffer cells (KCs) modulates mitochondrial function in cocultured rat hepatoma cells (AH70) (Cancer Res 53:2676,1993). The present study was designed to investigate 1) the mechanism(s) by which KCs release NO in response to coculture with hepatoma cells, and 2) whether chronic ethanol-feeding alters NO production of KCs and subsequent mitochondriat dysfunction in hepatoma cells. To that effect, KCs isolated from male Wistar rats were cocultured with AH70 cells. The confocaI laser scanning microscopic observations revealed that coculture of non-activated KCs and AH70 cells increased cytosolic Ca ++ level in KCs indicated as the fura-2 fluorescence, and reduced mitochondrial energization in AH70 cells indicated as the rhodamine 123 (Rh t23) fluorescence. The sum of nitrite and nitrate levels increased in the culture medium of KCs with AH70 cells as compared with in that of KCs alone. Either L-NMMA, or aminoguanidine effectively attenuated NO production of KCs and the reduction of Rh 123 fluorescence in AH70 cells, suggesting that NO produced by inducible NO synthase of KCs modulates hepatoma cell mitochondrial function. Furthermore, either nicardipine (a voltage-dependent Ca ++ channel blocker), monoclonal antibody against CDI8 or ICAM-I attenuated the NO production of KCs as well as fluorographic alterations. A flowcytometeric analysis provided the evidence that the coculture increases ICAM-I expression on AH70 cells and CDI8 on KCs. These results suggest that the CD18/ICAM-1dependent close contact stimulates Ca ++ mobilization and NO production in KCs. In another set of experiment, KCs isolated from rats, which were pair-fed with Lieber's ethanol-containing liquid diet, were used in the same investigations. KCs from ethanol-fed rats did not show the Ca ++ mobilization, increase NO production or reduce Rh123 fluorescence in AH70 cells after the coculture, whereas an increased CD 18 expression was still observed. The present study, therefore, supports a scenario that the chronic ethanol-feeding diminishes the NO production of KCs via altering the Ca ++ channel sensitivity, and reduces cytotoxic activities of KCs against hepatoma cells.
EFFECT OF HIGH-DOSE ALPHAqNTERFERON (IFN) ON VIREMIA OF RESISTANT HCV GENOTYPES. N.P. Lain*, D.R. Gretch**, M.J. Chung**, T.J. Layden***. Depts. of Medicine*** and Pharmacy Practice*, University of Illinois, Chicago, IL; and Hepatitis Lab **, University of Washington Medical Center, Seattle, WA, U.S.A. It has been suggested that HCV genotypes correlate with response to IFN, and that patients infected with HCV Type I have a lower response to the standard dose of IFN (3 mlU). The effect of high-dose IFN on improving the response of resistant HCV genotypes to treatment have not been clearly defined. This study examined the effect of a single 10 mlU injection of IFN on viral clearance in ten patients who are infected with HCV (Type la, n=5; Type lb, n=5) over 48 hours. Serial serum samples were collected at time 0 (baseline) and at 6, t0, 24 and 48 hours post-IFN injection. Quantitative HCV RNA levels were then determined using branched DNA assay. All patients had a significant reduction in HCV RNA levels at 24 hour post-lFN, of which 5 had undetectable levels. However, by 48 hour post-IFN, a rise in HCV RNA levels were noted in 7 patients, two of which had undetectable levels at 24 hour. Nevertheless, the HCV RNA levels remained significantly lower than the baseline values. The results suggest that a single, high-dose IFN significantly reduces the viremia of resistant HCV genotypes 24 hours after treatment, and the effect has been transient in these patients. Further studies should evaluate the benefit of incorporating high-dose and/or more frequent dosing of IFN for treating patients infected with resistant HCV genotypes. The effect of 1FN on the HCV viremia in the peripheral blood mononuclear cells (PBMC) and liver cells should also be assessed. Mean (4- s.d.) HCV RNA levels (x l&) in equivalents/ml at different time points post-IFN injection Baseline
6-hr
10-hr
24-hr
48-hr'
13.0+13.0
7.8_6.1
8.14-8.0
0.4+0.5
1.34-1.4
This reseach was funded in part by the Schering Corporation, Kenilworth, NJ.