2.1.1 Hepatitis C patients. Stored serum samples were studied from 75 patients with chronic hepatitis C infection who have been treated with interferon alpha and ribavirin combination therapy. All sera were taken before treatment was started. All patients were treated in the outpatient clinic of the Department of Gastroenterology, Hepatology and Endocrinology of Medical School Hannover, Germany. Another 16 sera and paired peripheral blood mononuclear cells (PBMCs) were collected from patients with chronic hepatitis C infection without antiviral therapy for evaluating cellular immune responses. These 91 patients had a mean age of 46 years ranging from 19-73 years (male: 49; female: 42). Characteristics are summarized in Table 4.
2.1.2 Hepatitis B patients. Serum samples were taken from randomly selected 50 patients with chronic hepatitis B infection (mean age: 47 years, range: 15-72, male 33;
female: 17). The presence of hepatitis B surface antigen (HBsAg) for more than 6 months was required for the diagnosis of chronic HBV infection.
2.1.3 Liver tissue samples
Explanted liver tissues were obtained at the time of liver transplantation from 50 patients with end-stage liver damage undergoing orthotopic liver transplantation for various reasons from 1993 to 2000 at Medical School Hanover (mean age: 47 years, range: 8-69). Of these, 19 had HCV related end-stage liver disease, while 31 had non-HCV liver cirrhosis (e.g. primary biliary cirrhosis, primary sclerosing cholangitis, Morbus Wilson and Polycystic liver disease).
Routine liver biopsy samples were obtained from 32 patients (mean age: 46 years, range: 33-75). Of these, 13 had chronic hepatitis C. 19 biopsies were taken from patients with no evidence of HCV infection.
All of these samples were stored at -20°C until used.
All patients were recruited at Medical School Hannover, Germany.
Role of Parvovirus B19 infection in hepatitis C -- Subjects and Methods
2.1.4 Healthy individuals
Sera and paired PBMCs were collected from 30 laboratory and clinic healthy volunteers (mean age: 37.5 years, range: 25-65, male: 16, female: 14). Among them, 19 were B19-IgG antibody positive while 11 were negative.
2.2 Methods
2.2.1 Serological tests.
Anti-B19 IgM and anti-B19 IgG antibodies were tested using the Parvovirus B19 IgM/IgG Enzyme Immunoassay (Biotrin, Ireland) according to the manufacture’s instruction. Anti-HCV antibodies were tested using ARCHITECT Anti-HCV assay (Abbott, USA).
2.2.2 Extraction of nucleic acids from liver tissue
DNAs were extracted from 20-30mg frozen liver tissue using the QIAamp DNA mini kit (Qiagen, Germany) according to the manufacture’s instruction.
Contamination was excluded by re-extraction of these same samples in another laboratory by a different person.
DNAs from liver biopsy samples were extracted by a person who had never contacted with B19 virus in another laboratory.
2.2.3 Polymerase chain reaction (PCR).
Nested PCR (nPCR) and quantitative real time PCR (qPCR) were performed to detect B19 DNA. Primers specific for the VP1/VP2 structural sequence were employed in nPCR as described elsewhere [64]. Briefly, the primer pairs for the first round of PCR were as follows: sense 5’-AGCATGTGGAGTGAGGGGGC-3’ and anti-sense 5’-AAAGCATCAGGAGCTATACTTCC-3’. The primers for second round were sense GCTAACTCTGTAACTTGTAC-3’ and anti-sense 5’-AAATATCTCCATG GGGTTGAG (NCBI GenBank accession No. U38509). The whole reaction consisted of two 35-cycle programs (1 cycle= 94°C for 30 seconds, 50°C for 30 seconds and 72°C for 45 seconds)
Quantitative real-time PCR (qPCR) was performed using the Parvo B19 artus LC-PCR kit (Qiagen, Germany) according to the manufacture’s instruction. A qPCR of genomic C-reactive protein (CRP) DNA was performed with same samples in
Role of Parvovirus B19 infection in hepatitis C -- Subjects and Methods
order to determine the amount of human CRP DNA representing the actual amount of amplifiable cellular DNA in each sample [65, 66]. B19 copy numbers per cell were calculated from the amplification of B19 divided by the amount of human CRP DNA.
Samples were defined as a “serologically recovered” cohort by the presence of anti-B19 IgG with the absence of IgM and DNA.
2.2.4 Isolation of PBMCs and carboxy fluorescein succinimidyl ester (CFSE)-based T cell proliferation assay.
PBMCs were separated from heparinized blood samples by gradient centrifugation on Ficoll-Paque and stored in liquid nitrogen until used.
CFSE-based T cell proliferation assay was performed as described previously [11]. Briefly, frozen PBMCs were thawed and suspended at 107/ml in PBS plus 0.2%
BSA and incubated at 37°C for 7 min with 2.5ųM CFSE (Sigma, USA). An equal volume of FCS was added thereafter and cells were incubated on ice for 5 min to stop reaction, followed by 3 times washing. Then, labelled cells were resuspended in medium (RPMI 1640 supplemented with 10% inactive AB serum and penicillin), plated at 0.3*106 cells per 200ul per well in round-bottom 96-well microtiter plates and cultured with DMSO alone (background), synthetic peptides (VP1/2 7.2 and VP1/2 4.7 [47], ProImmune, UK, Table 2) at a final concentration of 1ųg/ml and 5ųg/ml, tetanus toxoid (TT, 3ųg/ml) and PHA (6ųg/ml) as positive controls at 37°C with 5% CO2 for 7 days. Each condition was duplicated.
PBMCs from patients with chronic HCV infection were stimulated also with recombinant genotype 1a-derived HCV core, NS3 and NS4 protein (Microgen, Germany) at a final concentration of 1ųg/ml.
Flow cytometric analysis: at the time of harvest, CFSE-labelled PBMCs were washed in PBS containing 2% FCS and 0.05% sodium azide, and stained with the following antibodies: anti-human CD4-APC, CD3-PE (Becton Dickson, Germany) at 4°C. Flow cytometric data (100,000 nongated events) were acquired on a BD FACSCalibur 4-color flow cytometer using BD Cellquest software (both from BD Biosciences). For analysis, BD FlowJo 6.1.1 (Treestar, Ashland, OR, USA) was used to gate on CD4+CD3+ T cell populations. The number of cells that had proliferated was determined by gating on the lineage-positive CFSElow and CFSEhigh subset. The background of CD4+ T cells proliferative frequency (%) was calculated as the number of CFSElow CD4+ T cells /(numbers of CFSElow CD4+ T cells + numbers of CFSEhigh
Role of Parvovirus B19 infection in hepatitis C -- Subjects and Methods
CD4+ T cells) *100 in the absence of antigen. The PF was calculated by subtracting the mean background proliferation from the proliferating fraction in response to specific antigen. The SI was calculated by dividing the antigen-induced PF by the background PF. Both a PF of 1.0% or more and an SI of 2.0 or more are considered as a positive response, as previously defined [67, 68] .
Table 2:Synthetic peptide specification
B19 region CD4+ T-cell-restricted peptide sequence peptide no
VP1/2 FLIPYDPEHHYKVFSPAASS 4.7
VP1/2 LASEESAFYVLEHSSFQLLG 7.2
Role of Parvovirus B19 infection in hepatitis C -- Results