- Email: [email protected]
Hepatitis C virus (HCV) screening following intravenous immunoglobulin therapy
A1068
AASLD ABSTRACTS
GASTROENTEROLOGY, VOI. IOS, NO. 4
• NONISOTOPIC DETECTION OF HEPATITIS C VIRUS IN PERIPHERAL BLOOD MONONUCLEAR CELLS BY REVERSE TRANSCRIPTASE POLYMERASE CHAIN REACTION IN SITU HYBRIDIZATION. K, Fukudal), S. Nishiguchi1), T. Kuroki1), H. Morimoto2), M. Sakurai 2), S. Otani3), A. Matsuhisa4) and K. Kobayashi1). Third Dept. ofintemal Medicine1), Second Dept. of Pathology 2) and Second Dept. of Biochemistry 3), Osaka City University Medical School, Osaka, Japan, and Dept. of Biological Science Research 4), Research & Development Center, FUSO Pharmaceutical Industries, LTD., Osaka, Japan. Hepatitis C virus (HCV) is a positive-stranded RNA virus of about i0 kb which is known to replicate through negative-stranded RNA intermediates. It remains Controversial whether HCV exists in peripheral mononuclear cells (PBMC) and replicates there. In order to determine whether HCV has these properties, we performed nested reverse transcriptase polymerase chain reaction (RT-PCR) and RT-PCR in situ hybridization in PBMC. Material and Methods. PBMC were obtained from patients with chronic hepatitis C. Blood specimens from anti-HCV-negative, healthy individuals were included as negative controls. For detection of negative-stranded RNA by nested RTPCR, cDNA synthesis was performed with a sense oligonucleotide primer instead of an antisense primer and terminated by heating at 10O*Cfor 30 rain to exclude resting RT activity. The RT mixture was then treated with 10 ~tg/mL RNase A at 37"C for 30 rain. After phenol/chloroform extraction and ethanol precipitation, the cDNA was dissolved in dH20 and amplified by PCR. For RT-PCR in situ hybridization, PBMC were fixed in 4% PFA. After RT-PCR, PBMC were smeared on a slide and then in situ hybridization was performed with a digoxigenin cRNA probe. Results and Conclusion. We studied PBMC in an attempt to detect positive or negative strands of HCV RNA by nested RT-PCR as described above, and found that both types of strands were detectable in PBMC. We performed in situ hybridization without RT-PCR using the digoxigenin-labeted cRNA probe in an attempt to detect negative-stranded HCV RNA in PBMC but no signal was observed. Using RT-PCR in situ hybridization, we recognized the amplified products of both strands of HCV in PBMC. No signal was observed in PBMC of Control subjects. Using these methods, we detected both positive- and negative- stranded HCV RNA. RT-PCR in situ hybridization appeared to be sufficiently sensitive for visualization of low copy numbers of HCV with nonradioactive probes. These findings clearly indicate that HCV exist and may replicate in PBMC.
• A SURVEY OF THE FREQUENCY OF GASTROINTESTINAL COMPLAINTS IN PATIENTS WITH CHRONIC LIVER DISEASE. JS Galati, HP Monsour, CH Dyer, S Seagren, EMM Quigley. Dept. of Gastroenterology and Hepatology Univ. of Nebraska Medical Center, Omaha,NE, and Univ. of Texas Health Science Center, Houston,TX. Patients with chronic liver disease (CLD) frequently report gastrointestinal(GI) complaints. We have recently demonstrated that gastric emptying and small bowel transit of a solid meal is delayed in patients with CLD and portal hypertension, suggesting foregut dysfunction. However, the true frequency and specificity of these GI symptoms has never been quantified. These symptoms may also impact on the nutritional status of these patients. A 70 item questionnaire, inquiring about GI symptoms and nutritional status, was administered to 121 consecutive patients with CLD (64 female, 57 male, median age 48.7 yrs, range 19-72) and 94 age and sex matched health controls (50 female, 44 male, median age 38 yrs, range 19-65). None of the CLD patients had any other gastrointestinal disorders. The CLE) patients were stratified by disease type, Child-Pugh class, and the presence of complications. Four symptoms suggestive of foregut dysfunction were significantly common in CLD patients (table). Symptom
CLD N (%)
Control N (%)
p value
Abd Pain
97 (79.5)
44 (46.2)
< .001
Nausea > once/wk
38 (31.4)
7 (7.3)
< .001
Bloating
91 (75.8)
24 (25.8)
<.00i
Early satiety 60 (49.1) 12 (12.6) < .001 The presence of ascites or hel~atic enceph~ Iopathy did not sta:istically influence the frequency of reported bloating or early satiety. Patients with CLD report a strikingly increased frequency o f GI complaints suggestive of foregut dysfunction and supports previous findings describing gastric emptying and small bowel dysfunction. Neither CLD type, Child-Pugh class, nor the presence of ascites or hepatic encephalopathy related to the frequency of the symptoms. The early satiety reported may contribute to the overall poor nutritional status found in CLD 13atients.
HEPATITIS C VIRUS (HCV) SCREENING FOLLOWING INTRAVENOUS IMMUNOGLOBULIN THERAPY S.Furs I.Grimm, Division of Digestive Diseases and Nutrition, Dept. of Med c ne, Un versity of North Carolina, Chapel Hill, NC.
Introduction: A case of HCV transmission possibly linked to administration of the intravenous immunoglobulin (IVIG) product, Polygam® (Baxter Healthcare Corporation) led to withdrawal of this product from the market in February 1994. The Centers for Disease Control subsequently recommend HCV screening of all patients treated with Polygam® between April 1993 and February 1994 (MMWR 1994;43:505). )n October 1994, UNC Hospitals sought to identify and screen all Polygam® recipients for evidence of HCV. Methods: UNC Hospitals pharmacy records were reviewed for patients who were administered Polygam® between 4/93 and 2/94. Patients were contacted by mail and advised to undergo screening. Anti-HCV (EIA-2) and ALT levels were obtained more than 6 months after WIG therapy. Results: 129 recipients were identified; ages, newborn to 87; 67 (52%) were pediatric; 56 (43%) were male; 24 died prior to the screenin (none had clinical evidence of hepatitis). 105 patients were notified, and 50 responded (48%). 48 patients underwent screening. 120 charts were available for review. 61 (50%) patients received prior or subsequent blood product transfusions at UNC. 19 (16%) patients received more than 5 doses of IVIG. The most common indications for IVIG therapy (comprising 65%) were immunodeficiency syndromes (12%), idiopathic immune thrombocytopenia, myasthenia gravis, Kawasaki's disease, and preterm birth with infection. 6/48 patients had an elevated ALT (highest level 105 U/L). One patient of 48 was seropositive for anti-HCV. This individual had known prior hepatitis C infection, and an elevated ALT. No patients had acutely elevated liver enzymes after IVIG transfusion. Patients with elevated ALT will be followed for evidence of seroconversion; none of these patients has immune deficiency. Additional efforts will be made to screen all remaining patients. Conclusions: No evidence of clinical hepatitis or HCV seroconversion was observed among 45 patients screened following administration of Po/ygam®at our institution. The risk of HCV following treatment with Po/ygam®appears to be low.
INVOI,VEMENT OF TIlE APO,11FAS RECEPTOR AND LIGANI) IN LIVER DAMAGE. P.R. Galle, WJ. Hofmann*, G. Otto¶, W. Stremmel, L. Runkel#. Depts. of Internal Medicine & Pathology* & Surgery¶, Zentrum fiir Molekulare Biologic#, University of Heidelberg, Bergheimerstr. 58, 69115 Heidelberg, FRG. Apoptosis occurs in the normal liver and in various forms of liver disease. We have recently demonstrated that primary human liver ceils are acutely sensitive to apoptosis initiated by the APO-1/Fas (CD95) receptor (Hepatology 1994; 20(4): 208A). To investigate the clinical relevance of this finding we examined a panel of explanted liver tissues from patients with various forms of liver failure both for expression of the APO-1/Fas receptor and of the APO-1/Fas ligand. Material and Methods: Hepatectomy specimens were obtained during liver transplantation and snap frozen (normal liver, n=5; alcoholic cirrhosis, n=14; hepatitis B virus (HBV) related cirrhosis, n=l 1; acute hepatic failure, n=14). APO-1/Fas receptor expression was assessed by immunohistology using an lgGl nhouse monoclonal antibody. APO-1/Fas ligand mRNA expression was monitored by in situ hybridization using an 35S-labelled antisense RNA corresponding to the sequence coding for amino acids 100 to 246 of the human APO-1/Fas ligand (Intern Immun 1994; 6: 1567) Results: Immunohistology revealed that hepatocytes in normal livers and in alcoholic cirrhosis express low constitutive levels of APO-I/Fas. APO1/Fas receptor expression was highly elevated in hepatocytes in HBV related cirrhosis and in acute liver failure. By in situ hybridization APO-1/Fas ligand mRNA expression was absent in normal liver but detected at high levels in livers with ongoing liver damage. In cases of HBV related cirrhosis and acute hepatic failure ligand expression was found primarily in lymphocytes. In contrast, patients with alcoholic liver damage showed high APOl/Fas ligand mRNA expression in hepatocytes. Conclusion: These findings suggest that the APO-1/Fas receptor/ligand systemis involved in different forms of hepatic cell death. In hepatitis B, activation of this system and subsequent liver damage seems to be mediated primarily by T-lymphocytes. In alcoholic liver damage, death of hepatocytes might occur by fralricide and paracdne or autocrine mechanisms mediated by the hepatocytes themselves.
AASLD ABSTRACTS
GASTROENTEROLOGY, VOI. IOS, NO. 4
• NONISOTOPIC DETECTION OF HEPATITIS C VIRUS IN PERIPHERAL BLOOD MONONUCLEAR CELLS BY REVERSE TRANSCRIPTASE POLYMERASE CHAIN REACTION IN SITU HYBRIDIZATION. K, Fukudal), S. Nishiguchi1), T. Kuroki1), H. Morimoto2), M. Sakurai 2), S. Otani3), A. Matsuhisa4) and K. Kobayashi1). Third Dept. ofintemal Medicine1), Second Dept. of Pathology 2) and Second Dept. of Biochemistry 3), Osaka City University Medical School, Osaka, Japan, and Dept. of Biological Science Research 4), Research & Development Center, FUSO Pharmaceutical Industries, LTD., Osaka, Japan. Hepatitis C virus (HCV) is a positive-stranded RNA virus of about i0 kb which is known to replicate through negative-stranded RNA intermediates. It remains Controversial whether HCV exists in peripheral mononuclear cells (PBMC) and replicates there. In order to determine whether HCV has these properties, we performed nested reverse transcriptase polymerase chain reaction (RT-PCR) and RT-PCR in situ hybridization in PBMC. Material and Methods. PBMC were obtained from patients with chronic hepatitis C. Blood specimens from anti-HCV-negative, healthy individuals were included as negative controls. For detection of negative-stranded RNA by nested RTPCR, cDNA synthesis was performed with a sense oligonucleotide primer instead of an antisense primer and terminated by heating at 10O*Cfor 30 rain to exclude resting RT activity. The RT mixture was then treated with 10 ~tg/mL RNase A at 37"C for 30 rain. After phenol/chloroform extraction and ethanol precipitation, the cDNA was dissolved in dH20 and amplified by PCR. For RT-PCR in situ hybridization, PBMC were fixed in 4% PFA. After RT-PCR, PBMC were smeared on a slide and then in situ hybridization was performed with a digoxigenin cRNA probe. Results and Conclusion. We studied PBMC in an attempt to detect positive or negative strands of HCV RNA by nested RT-PCR as described above, and found that both types of strands were detectable in PBMC. We performed in situ hybridization without RT-PCR using the digoxigenin-labeted cRNA probe in an attempt to detect negative-stranded HCV RNA in PBMC but no signal was observed. Using RT-PCR in situ hybridization, we recognized the amplified products of both strands of HCV in PBMC. No signal was observed in PBMC of Control subjects. Using these methods, we detected both positive- and negative- stranded HCV RNA. RT-PCR in situ hybridization appeared to be sufficiently sensitive for visualization of low copy numbers of HCV with nonradioactive probes. These findings clearly indicate that HCV exist and may replicate in PBMC.
• A SURVEY OF THE FREQUENCY OF GASTROINTESTINAL COMPLAINTS IN PATIENTS WITH CHRONIC LIVER DISEASE. JS Galati, HP Monsour, CH Dyer, S Seagren, EMM Quigley. Dept. of Gastroenterology and Hepatology Univ. of Nebraska Medical Center, Omaha,NE, and Univ. of Texas Health Science Center, Houston,TX. Patients with chronic liver disease (CLD) frequently report gastrointestinal(GI) complaints. We have recently demonstrated that gastric emptying and small bowel transit of a solid meal is delayed in patients with CLD and portal hypertension, suggesting foregut dysfunction. However, the true frequency and specificity of these GI symptoms has never been quantified. These symptoms may also impact on the nutritional status of these patients. A 70 item questionnaire, inquiring about GI symptoms and nutritional status, was administered to 121 consecutive patients with CLD (64 female, 57 male, median age 48.7 yrs, range 19-72) and 94 age and sex matched health controls (50 female, 44 male, median age 38 yrs, range 19-65). None of the CLD patients had any other gastrointestinal disorders. The CLE) patients were stratified by disease type, Child-Pugh class, and the presence of complications. Four symptoms suggestive of foregut dysfunction were significantly common in CLD patients (table). Symptom
CLD N (%)
Control N (%)
p value
Abd Pain
97 (79.5)
44 (46.2)
< .001
Nausea > once/wk
38 (31.4)
7 (7.3)
< .001
Bloating
91 (75.8)
24 (25.8)
<.00i
Early satiety 60 (49.1) 12 (12.6) < .001 The presence of ascites or hel~atic enceph~ Iopathy did not sta:istically influence the frequency of reported bloating or early satiety. Patients with CLD report a strikingly increased frequency o f GI complaints suggestive of foregut dysfunction and supports previous findings describing gastric emptying and small bowel dysfunction. Neither CLD type, Child-Pugh class, nor the presence of ascites or hepatic encephalopathy related to the frequency of the symptoms. The early satiety reported may contribute to the overall poor nutritional status found in CLD 13atients.
HEPATITIS C VIRUS (HCV) SCREENING FOLLOWING INTRAVENOUS IMMUNOGLOBULIN THERAPY S.Furs I.Grimm, Division of Digestive Diseases and Nutrition, Dept. of Med c ne, Un versity of North Carolina, Chapel Hill, NC.
Introduction: A case of HCV transmission possibly linked to administration of the intravenous immunoglobulin (IVIG) product, Polygam® (Baxter Healthcare Corporation) led to withdrawal of this product from the market in February 1994. The Centers for Disease Control subsequently recommend HCV screening of all patients treated with Polygam® between April 1993 and February 1994 (MMWR 1994;43:505). )n October 1994, UNC Hospitals sought to identify and screen all Polygam® recipients for evidence of HCV. Methods: UNC Hospitals pharmacy records were reviewed for patients who were administered Polygam® between 4/93 and 2/94. Patients were contacted by mail and advised to undergo screening. Anti-HCV (EIA-2) and ALT levels were obtained more than 6 months after WIG therapy. Results: 129 recipients were identified; ages, newborn to 87; 67 (52%) were pediatric; 56 (43%) were male; 24 died prior to the screenin (none had clinical evidence of hepatitis). 105 patients were notified, and 50 responded (48%). 48 patients underwent screening. 120 charts were available for review. 61 (50%) patients received prior or subsequent blood product transfusions at UNC. 19 (16%) patients received more than 5 doses of IVIG. The most common indications for IVIG therapy (comprising 65%) were immunodeficiency syndromes (12%), idiopathic immune thrombocytopenia, myasthenia gravis, Kawasaki's disease, and preterm birth with infection. 6/48 patients had an elevated ALT (highest level 105 U/L). One patient of 48 was seropositive for anti-HCV. This individual had known prior hepatitis C infection, and an elevated ALT. No patients had acutely elevated liver enzymes after IVIG transfusion. Patients with elevated ALT will be followed for evidence of seroconversion; none of these patients has immune deficiency. Additional efforts will be made to screen all remaining patients. Conclusions: No evidence of clinical hepatitis or HCV seroconversion was observed among 45 patients screened following administration of Po/ygam®at our institution. The risk of HCV following treatment with Po/ygam®appears to be low.
INVOI,VEMENT OF TIlE APO,11FAS RECEPTOR AND LIGANI) IN LIVER DAMAGE. P.R. Galle, WJ. Hofmann*, G. Otto¶, W. Stremmel, L. Runkel#. Depts. of Internal Medicine & Pathology* & Surgery¶, Zentrum fiir Molekulare Biologic#, University of Heidelberg, Bergheimerstr. 58, 69115 Heidelberg, FRG. Apoptosis occurs in the normal liver and in various forms of liver disease. We have recently demonstrated that primary human liver ceils are acutely sensitive to apoptosis initiated by the APO-1/Fas (CD95) receptor (Hepatology 1994; 20(4): 208A). To investigate the clinical relevance of this finding we examined a panel of explanted liver tissues from patients with various forms of liver failure both for expression of the APO-1/Fas receptor and of the APO-1/Fas ligand. Material and Methods: Hepatectomy specimens were obtained during liver transplantation and snap frozen (normal liver, n=5; alcoholic cirrhosis, n=14; hepatitis B virus (HBV) related cirrhosis, n=l 1; acute hepatic failure, n=14). APO-1/Fas receptor expression was assessed by immunohistology using an lgGl nhouse monoclonal antibody. APO-1/Fas ligand mRNA expression was monitored by in situ hybridization using an 35S-labelled antisense RNA corresponding to the sequence coding for amino acids 100 to 246 of the human APO-1/Fas ligand (Intern Immun 1994; 6: 1567) Results: Immunohistology revealed that hepatocytes in normal livers and in alcoholic cirrhosis express low constitutive levels of APO-I/Fas. APO1/Fas receptor expression was highly elevated in hepatocytes in HBV related cirrhosis and in acute liver failure. By in situ hybridization APO-1/Fas ligand mRNA expression was absent in normal liver but detected at high levels in livers with ongoing liver damage. In cases of HBV related cirrhosis and acute hepatic failure ligand expression was found primarily in lymphocytes. In contrast, patients with alcoholic liver damage showed high APOl/Fas ligand mRNA expression in hepatocytes. Conclusion: These findings suggest that the APO-1/Fas receptor/ligand systemis involved in different forms of hepatic cell death. In hepatitis B, activation of this system and subsequent liver damage seems to be mediated primarily by T-lymphocytes. In alcoholic liver damage, death of hepatocytes might occur by fralricide and paracdne or autocrine mechanisms mediated by the hepatocytes themselves.