B. Kallinowski

Peripheral blood leukocytes serve as a possible extrahepatic site for hepatitis C virus replication

To study possible extrahepatic sites for the replication of hepatitis C virus (HCV), we examined fresh and cultured peripheral blood mononuclear leukocytes (PBML), as well as different subpopulations of PBML of HCV-infected patients, for the presence of viral genomic and antigenomic RNA. Sense and antisense oligonucleotide primers derived from HCV sequences were used for reverse transcription (RT) followed by an amplification with the polymerase chain reaction assay (PCR). Using antisense primers for RT, genomic viral RNA could be detected in serum, liver, total PBML and B lymphocytes of chronically infected patients. However, only liver tissue and PBML specimens were positive when a sense primer was used. To demonstrate further the specificity of these findings, total PBML were stimulated using pokeweed mitogen and synthesis of HCV RNA was determined by incorporation of [3H]uridine into nascent viral RNA molecules using a hybrid release assay. Additionally, total PBML from an uninfected person could be infected in vitro using an HCV RNA-positive serum. The PCR products obtained from serum, liver and PBML specimens of an HCV-positive individual were found to have nearly identical sequences. Our findings suggest that PBML could be a site for viral replication of HCV during the natural course of infection and may represent a reservoir for hepatitis C virions.

Early monotherapy with pegylated interferon alpha-2b for acute hepatitis C infection: the HEP-NET acute-HCV-II study

Early treatment of acute hepatitis C with interferon alpha‐2b for 24 weeks prevents chronic infection in almost all patients. Because pegylated interferons have replaced conventional interferon in the therapy of chronic hepatitis C, the aim of this study was to analyze the efficacy of an early treatment of acute hepatitis C with peginterferon alfa‐ 2b. Between February 2001 and February 2004, 89 individuals with acute HCV infection were recruited at 53 different centers in Germany. Patients received 1.5 μg/kg peginterferon alfa‐2b for 24 weeks; treatment was initiated after a median of 76 days after infection (range 14‐150). End‐of‐treatment response and sustained virological response were defined as undetectable HCV RNA at the end of therapy and after 24 weeks of follow‐up, respectively. In the total study population, virological response was 82% at the end of treatment and 71% at the end of follow‐up. Of 89 individuals, 65 (73%) were adherent to therapy, receiving 80% of the interferon dosage within 80% of the scheduled treatment duration. End‐of‐treatment and sustained virological response rates in this subpopulation were 94% and 89%, respectively. A maximum alanine aminotransferase level of more than 500 U/L prior to therapy was the only factor associated with successful treatment. In conclusion, in acute HCV infection, early treatment with peginterferon α2b leads to high virological response rates in individuals who are adherent to treatment. The high number of dropouts underlines the importance of thorough patient selection and close monitoring during therapy. Thus, future studies should identify factors predicting spontaneous viral clearance to avoid unnecessary therapy. (HEPATOLOGY 2006;43:250–256.)

Hemochromatosis and transferrin receptor gene polymorphisms in chronic hepatitis C: impact on iron status, liver injury and HCV genotype.

Mild iron overload in chronic hepatitis C is associated with liver fibrosis, hepatitis C virus (HCV) genotype 1b infection, and an impaired response to interferon therapy. In this study we evaluated whether polymorphisms in the hemochromatosis gene HFE and the transferrin receptor gene TFR1 are associated with these typical findings. The study considered 246 HCV-infected patients and 200 blood donors as controls, in which C282Y, H63D, and S65C mutations (HFE) and the S142G polymorphism (TFR1) were detected. HCV genotype, serum ferritin levels, stainable intrahepatic iron, and grade of fibrosis according to the METAVIR score (F0–F4) were determined. In HCV-infected patients, heterozygosity for the C282Y mutation in HFE was significantly associated with elevated serum ferritin levels, stainable liver iron, and advanced fibrosis or cirrhosis (F2–F4). By multivariate logistic regression analysis the odds ratio for the development of advanced fibrosis or cirrhosis (F2–F4) was 2.5 for HCV-infected patients carrying a heterozygous C282Y mutation and 4.8 for HCV-infected patients with C282Y/H63D and C282Y/S65C compound heterozygosity. Heterozygosity for the C282Y mutation in HFE contributes to iron accumulation and fibrosis progression in chronic hepatitis C.

Epidemiologie der chronischen Hepatitis C in Deutschland - Eine Analyse von 10 326 Hepatitis-C-Virus-Infizierten aus Schwerpunktpraxen und -ambulanzen

Little is known about the epidemiology of chronic hepatitis C (CHC) in Germany and especially about the importance of transmission, duration of infection, genotypes, symptoms and quality of life of the patients. The current study prospectively evaluates epidemiological and clinical data of patients infected with the hepatitis C virus (HCV). Using online data entry, various characteristics of 10,326 untreated patients with CHC were documented from March 2003 until May 2006 in 352 centres all over Germany. Mean age of patients was 43.4 years. Patients infected by i.v. drug abuse were considerably younger (36.5 years) than the remaining patients (49.2 years). As indicated by their native language, 64.4% of the patients came from Germany and 19.2% from Russia. 61.7% were infected with genotype 1 and 34.9% with genotype 2 or 3. 45.5% of the patients had been infected by i.v. drug abuse. In at least 5.4% of the patients liver cirrhosis had been proved by biopsy. 63.5% of the patients felt an impairment of quality of life caused by CHC. In many patients infected with hepatitis C socio-economic issues are existent. This is reflected, i.e., in very high rates of unemployment in special subpopulations. Coinfections with hepatitis B and HIV occurred in 1.5% and 4.7%, respectively. Nearly 80% of patients were managed near their homes. The data of the 10 326 patients represent about 2% of all German patients with CHC. This database is up to now the largest of its kind and gives a representative insight into the epidemiological situation of CHC in Germany.

Accelerated schedule of hepatitis B vaccination in liver transplant candidates.

Liver transplantation has become the treatment of choice worldwide for many patients with end-stage liver disease. In terms of survival and quality of life, the results of the procedure in many centers are very good. However, the long-term function of the grafts may be affected by vascular, immunologic, or infection problems, the latter being a major cause of morbidity and mortality after orthotopic liver transplantation (OLT). Therefore, prophylactic vaccination against hepatitis B virus (HBV) infection is recommended in patients awaiting liver transplantation. Individuals with chronic advanced liver disease are known to be less responsive to HBV vaccination. On the other hand, the widely recommended standard schedule (months 0, 1, and 6) for immunization against hepatitis B takes 6 months, a regimen which may not be completed in time prior to OLT or which may not be completed due to noncompliance, possible reasons for the lower rates of seroprotection in OLT candidates. Studies show that, in principle, complete immunization with an accelerated hepatitis B vaccination protocol (0, 7, 21 days) induces early seroprotection with excellent seroprotection rates and anti-HBs titers in immunocompetent individuals. We therefore performed a prospective clinical trial to assess immunogenicity and reactogenicity of this accelerated vaccination regimen in OLT candidates compared to healthy controls.

Hepatotropism of GB virus C (GBV-C): GBV-C replication in human hepatocytes and cells of human hepatoma cell lines

BACKGROUND/AIMS Recently, GB virus C (GBV-C) has been identified as another virus potentially causing viral hepatitis. However, its hepatotropism and pattern of infection in humans is still unknown. To elucidate the presence and replication of GBV-C in the human liver, we investigated tissue samples of six explanted livers from five GBV-C mono- or GBV-C/HCV co-infected patients for GBV-C RNA plus- and minus-strand RNA. METHODS These tissues were examined using nested RT-PCR followed by Southern blot hybridization as well as fluorescence in situ hybridization on liver cryosections. To further substantiate susceptibility of liver cells for GBV-C, in vitro infection of human hepatoma cells (HuH7, HepG2) with GBV-C mono-infected serum was performed. RESULTS By reverse transcription followed by nested PCR (RT-PCR), 5 of 6 liver specimens (4/5 patients) were positive for GBV-C plus-strand RNA, and viral minus-strand RNA could be detected in 4 of 6 liver specimens (4/5 patients). One liver sample was negative for GBV-C RNA. In two specimens we could identify GBV-C infection by in situ hybridization. Virus infection appeared to be restricted to hepatocytes and detection of minus-strand RNA showed viral replication in a few highly infected liver cells. In vitro infection of HepG2 or HuH7 cells confirmed these findings by a release of virions into supernatant. CONCLUSION In conclusion, our results establish GBV-C as a hepatotropic virus infecting human cells of hepatic origin in vivo and in vitro.

B-lymphocytes are predominantely involved in viral propagation of hepatitis C virus (HCV)

Recent reports have shown that HCV infection is not only restricted to hepatocytes. Like hepatitis B virus (HBV), which also was thought to be strictly hepatotropic in early molecular and cellular investigations, infection of lymphoid cells by HCV in vivo has been demonstrated. We showed that total peripheral blood leukocytes of chronically HCV-infected patients are infected by detection of plus- and minus-stranded HCV RNA using strand-specific oligonucleotide primers in the RT-PCR. These cells also represent extrahepatic sites for the viral replication, as demonstrated by incorporation of [3H]-uridine into nascent RNA after stimulation of the cells with a mitogen. Furthermore, total PBML from an uninfected person could be infected in vitro using an HCV-positive serum. It could be shown that replication of HCV RNA takes place in these cells. Examination of different subsets of PBML showed predominant infection of B-lymphocytes during HCV disease. Additionally, infection of T-lymphocytes was detected in about 50% of all chronically HCV-infected patients.

Immunogenicity of two accelerated hepatitis B vaccination protocols in liver transplant candidates

Objective It is common practice to immunize patients against hepatitis B virus infection prior to orthotopic liver transplantation (OLT). We compared the seroprotection rates of two accelerated schedules with a recombinant hepatitis B vaccine in patients awaiting OLT. Design and methods Patients were prospectively recruited and vaccinated with either 20 μg (group 1, n = 14) or 40 μg (group 2, n = 20) hepatitis B surface antigen per dosage. Thirty-nine healthy volunteers served as a historical control group. Patients in all groups were vaccinated with an accelerated schedule (0, 7 and 21 days). All patients underwent clinical and laboratory examinations (HBs antibodies, CD4/CD8 ratio, transaminases). Results The accelerated hepatitis B vaccination schedules were well tolerated. Eight weeks after the third injection, no significant differences in seroprotection rates were observed between group 1 (31%) and group 2 (26%). There was no correlation with respect to seroconversion rates and gender, smoking habits or CD4/CD8 ratio. Conclusion These data suggest that accelerated vaccination schedules with a recombinant hepatitis B vaccine are safe and well-tolerated, but only achieve poor seroconversion rates in OLT candidates. Increasing the vaccine dose to 40 μg hepatitis B surface antigen per injection did not result in a higher response rate. Because of the low risk of acquiring de novo hepatitis B infection after transplantation, it should be questioned whether routine hepatitis B vaccination with standard recombinant vaccines prior to liver transplantation should be recommended any longer.

ESTABLISHMENT OF A HIGHLY SPECIFIC DETECTION SYSTEM FOR GB VIRUS C (GBV-C)MINUS-STRAND RNA

Although the clinical relevance of GB virus C (GBV-C) is still elusive, this virus has been found with high prevalence in several groups of patients with liver disease. As was shown for hepatitis C virus (HCV), minus-strand RNA is supposed to function as a replicative intermediate. We have established a reliable and sensitive detection system for GBV-C minus-strand RNA based on nested RT-PCR (reverse transcription-polymerase chain reaction) with a tagged primer system. Sensitivity and specificity was extensively tested using in-vitro transcribed GBV-C sequences and genomic viral RNA. Specificity of the amplified fragments was proven by Southern blot hybridization. Using this detection system, we found the presence of GBV-C minus-strand RNA in 6/41 (14.6%) sera of GBV-C infected or GBV-C/HCV coinfected patients. No correlation with virological parameters such as amount of GBV-C plus-strand RNA, genotype or titer of HCV could be detected.

Hepatitic C virus RNA in different blood lymphocyte subsets

Objective Lymphocytes play an important role in the pathogenesis of chronic viral hepatitis. Apart from intrahepatic replication of hepatitis C virus (HCV), there is also strong evidence for an extrahepatic site in mononuclear blood cells. To further characterize the site of HCV replication, we tested resting peripheral blood lymphocytes (PBL) and stimulated PBL for the presence of positive- and negative-stranded HCV RNA. Method PBL from 10 patients with histologically proven chronic active hepatitis C were sorted into CD4+ (T helper) and CD8+ (T suppressor) T cells, CD20+ B cells and CD16+ natural killer (NK) cells by a fluorescence-activated cell sorter. Results HCV RNA was detected by a nested polymerase chain reaction. Positive-standed HCV RNA was present in all 10 sera. Resting B and NK cells were positive in eight out of 10, CD4+ T cells in six out of 10 and in CD8+ T cells in five out of 10 patients for positive-stranded, but negative for negative-stranded HCV RNA. The supernatants were all negative for HCV RNA. In addition, we looked for positive- and negative-stranded HCV RNA in serum, resting PBL, Epstein-Barr virus transformed B cell lines and phytohaemagglutinin-stimulated T cells from two patients with chronic HCV infection. Positive-standed HCV RNA was present in both sera but only in one resting lymphocyte group. Negative-stranded HCV RNA could not be detected in sera nor in the resting lymphocyte group. After stimulation, however, negative-stranded HCV RNA was present in both Epstein-Barr virus transformed B cell lines as well as in phytohaemagglutinin-stimulated T cells. Conclusions We conclude that PBL from HCV-infected patients are also infected with this virus. The predominant sites of HCV are B and NK cells. These cells may, therefore, represent a reservoir for hepatitis C virions. According to our results viral replication occurs in B and probably in T cells.