Hepatitis B Virus DNA In Peripheral Blood Mononuclear Cells Research Articles

Detection and Quantification of Hepatitis B Virus Genomes in Peripheral Blood Mononuclear Cells of Chronic Hepatitis B Virus Infection, Cirrhosis, and Hepatocellular Carcinoma Patients

Background: Several studies have revealed that the hepatitis B virus (HBV) exists in peripheral blood mononuclear cells (PBMCs). It remains poorly understood whether HBV DNA and covalently closed circular DNA (cccDNA) can emerge in PBMCs of patients with different stages of HBV infection. Objectives: This study aimed to compare the detection of HBV DNA and quantification and presence of cccDNA within PBMC from patients with chronic hepatitis B (CHB), cirrhosis, and hepatocellular carcinoma (HCC). Methods: The present study was conducted on 120 participants (30 CHB patients, 30 cirrhosis patients, 30 HCC patients, and 30 healthy controls) from Tehran, Iran. HBV serological markers were tested by enzyme-linked immunosorbent assay (ELISA). PBMCs of all individuals were assayed for HBV DNA detection, quantification, and the presence of cccDNA. Results: Of 90 HBV patients, 58 (64.4%) were positive for HBV DNA in PBMCs. HBV DNA was detected in PBMCs isolated from 13/30 CHB, 20/30 cirrhosis, and 25/30 HCC patients. In addition, 6 (20%) CHB, 13 (43.3%) cirrhosis, and 16 (15.3%) HCC patients were cccDNA positive. The HBV viral loads in serums were statistically higher than the HBV viral loads of PBMCs (P < 0.001). A positive correlation was found between HBV DNA loads in serums and PBMCs of patients. Moreover, HBV DNA quantity of serums and PBMCs showed a significant association in terms of hepatitis B e antigen (HBeAg) status. Conclusions: HBV quantity in PBMCs correlated with serum HBV viral loads. HBV genomes in PBMCs may be a risk factor for HBV disease progression.

Low prevalence of occult hepatitis B virus infection in chronic haemodialysis and kidney transplant patients

Occult hepatitis B virus infection (OBI) is defined as the presence of hepatitis B virus (HBV) DNA in serum and/or liver in HBsAg-negative patients. We investigated the prevalence of OBI in large chronic haemodialysis (CHD) and kidney transplant recipients (KTxR) cohorts, including determination of HBV DNA in peripheral blood mononuclear cells (PBMCs). HBV DNA was determined in both serum and PBMCs in 417 CHD patients, 417 KTxR, 20 HBsAg-positive non-CHD non-KTx patients (positive controls) and 40 HBsAg-negative healthy subjects (negative controls). Chronic haemodialysis group: two of 376 patients were HBsAg-positive. The 374 HBsAg-negative patients tested negative for HBV DNA in both serum and PBMCs. KTxR group: 14 of 417 patients were HBsAg-positive. One of 403 HBsAg-negative patients tested positive for HBV DNA in serum but not in PBMCs. Positive controls: six of 20 patients were under antiviral therapy and had negative HBV DNA in both serum and PBMCs. In 11 of 14 remaining patients, HBV DNA was detected in serum and in both serum and PBMCs in 3 patients. Negative controls: All 34 patients were anti-HBc-negative and HBV DNA-negative in both serum and PBMCs. In the long term, the only case of anti-HBc-negative OBI lost anti-HBs 5years after inclusion in the study and showed HBV reactivation with HBsAg re-seroconversion. We found nil prevalence of OBI in CHD patients and a very low prevalence (<1%) in KTxR suggesting that routine screening for HBV DNA is not required in CHD population in our region. However, in KTxR, pretransplant screening with HBV DNA should be considered. Testing for HBV DNA in PBMCs does not seem to be of additional value.

Real-time PCR quantitation of hepatitis B virus total DNA and covalently closed circular DNA in peripheral blood mononuclear cells from hepatitis B virus-infected patients

It remains unclear whether hepatitis B virus (HBV) replicates in extrahepatic tissues, and particularly in peripheral blood mononuclear cells (PBMCs), which may serve as a reservoir for the maintenance of infection. A real-time PCR assay for the quantitation of total and covalently closed circular (ccc) HBV DNA in serum and in PBMCs was developed. This assay was highly sensitive (detection limit: 27 IU/mL), linear over a wide range (9 log 10), and was displayed high inter- and intra-assay reproducibility for the quantitation of total DNA. Genotypes A to E were detected and the results were consistent with those obtained with the COBAS Amplicor HBV Monitor Test. The specificity of the methodology was increased by prior treatment with an enzyme that digests relaxed circular DNA, and the elimination of background signals from virus adsorbed to the surface of PBMCs. HBV DNA was detected in the serum and PBMCs of 12 HBsAg-positive patients, with less than 1% in the cccDNA form. In conclusion, the quantitation of total and ccc HBV DNA in PBMCs is potentially useful as a non-invasive marker, and may help to increase our knowledge of the natural history of hepatitis B.

Dynamic changes of HBV DNA in serum and peripheral blood mononuclear cells of chronic hepatitis patients after lamivudine treatment

To study the dynamic changes of hepatitis B virus (HBV) DNA in serum and peripheral blood mononuclear cells (PBMCs) of patients after lamivudine therapy. A total of 72 patients with chronic HBV infection were included in this study. All patients were confirmed to have the following conditions: above 16 years of age, elevated serum alanine aminotransferase (ALT), positive hepatitis B e antigen (HBeAg), positive HBV DNA in serum and PBMCs, negative antibodies against HAV, HCV, HDV, HEV. Other possible causes of chronic liver damages, such as drugs, alcohol and autoimmune diseases were excluded. Seventy-two cases were randomly divided into lamivudine treatment group (n = 42) and control group (n = 30). HBV DNA was detected both in serum and in PBMCs by fluorescence quantitative polymerase chain reaction (PCR), during and after lamivudine treatment. In the treatment group, HBV DNA became negative both in serum and in PBMC, of 38 and 25 out of 42 cases respectively during the 48 wk of lamivudine treatment, the negative rate was 90.5% and 59.5% respectively. In the control group, the negative rate was 23.3% and 16.7% respectively. It was statistically significant at 12, 24 and 48 wk as compared with the control group (P<0.005). The average conversion period of HBV DNA was 6 wk (2-8 wk) in serum and 16 wk (8-24 wk) in PBMC. Lamivudine has remarkable inhibitory effects on HBV replication both in serum and in PBMCs. The inhibitory effect on HBV DNA in PBMCs is weaker than that in serum.

Configuration and replication competence of hepatitis B virus DNA in peripheral blood mononuclear cells from chronic hepatitis B patients and patients who have recovered from acute self-limited hepatitis

Several studies have reported the presence of hepatitis B virus (HBV) DNA in peripheral blood mononuclear cells (PBMCs). However, the mode of existence, infectivity and replication competence of HBV DNA in PBMCs remain unclear. To clarify these questions, we assayed for covalently closed circular DNA (cccDNA) and integrated HBV DNA in PBMCs from ten patients who had recovered from acute self-limited hepatitis B (AHB) and 14 chronic hepatitis B (CHB) patients. HBV DNA was detected in all six CHB patients who were positive for hepatitis B e antigen (eAg), and cccDNA was detected in five. HBV DNA was detected in seven of the eight CHB patients who were negative for eAg, and cccDNA was detected in three. HBV DNA and cccDNA was also detected in five and two of the ten AHB patients, respectively. Integrated HBV was not detected in PBMCs of any patients. HBV DNA presence and replication competence in PBMCs are associated with eAg-positive phenotype. Sequence analysis revealed that the source of HBV DNA differed between PBMCs and serum. Even in patients who have recovered from AHB, replicable HBV may persist in PBMCs.

Selective unresponsiveness to HBsAg vaccine in newborns related with an in utero passage of HBV DNA

Thirty-four out of 158 (22%) newborns to mothers chronically infected by the hepatitis B virus (HBV) did not produce antibodies (Ab) to HBsAg 1 month after the last injection of the HBV vaccine supplemented with HBV specific immunoglobulins. At birth the HBV genome was detected by polymerase chain reaction in the peripheral blood mononuclear cells (PBMC) of a large majority (28 out of 34) of these non-responder newborns but never in the other newborns who responded to the HBsAg vaccine. HBV genome was detected in serum, only in some cases (nine out of 34) and never in absence of HBV DNA in PBMC. For nine out of 14 followed newborns, the absence of response was transitory since anti-HBs Abs appeared after 15 months, without booster, while the HBV had disappeared. Unresponsiveness was specific of the HBV envelope protein since all late responders and 15 months non-responders to the HBsAg vaccine produced normal levels of Abs to the three poliovirus serotypes, to tetanus toxoid and to the pneumococcus polysaccharides. An in utero induced immune tolerance to low doses of HBsAg appears as the most plausible hypothesis to explain this unresponsiveness to HBV vaccine.

Selective unresponsiveness to HBsAg vaccine in newborns related with an in utero passage of hepatitis B virus DNA

Thirty four out of 158 (22%) newborns to mothers chronically infected by the hepatitis B virus (HBV) did not produce antibodies (Ab) to HBsAg 1 month after the last injection of the HBV vaccine supplemented with HBV specific immunoglobulins. At birth, HBV genome was detected by polymerase chain reaction (PCR) in the peripheral blood mononuclear cells (PBMC) of a large majority (28 out of 34) of these non-responder newborns but never in the other newborns who responded to the HBsAg vaccine. HBV genome was detected in serum, only in some cases (nine out of 34) and never in the absence of HBV DNA in PBMC. For nine out of 14 followed newborns, the absence of response was transitory since anti-HBs Abs appeared after 15 months, without booster, while the HBV genome had disappeared. Unresponsiveness was specific to the HBV envelope protein since all late responders and 15-months-non-responders to the HBsAg vaccine produced normal levels of Abs to the three poliovirus serotypes, to tetanus toxoid and to the pneumococcus polysaccharides. An in utero induced immune tolerance to low doses of HBsAg appears as the most plausible hypothesis to explain this unresponsiveness to HBV vaccine.

Heroin addicts infected by HBV and HIV have a low prevalence of HBV DNA in peripheral blood mononuclear cells.

Several reports show that the prevalence of HBV (hepatitis B virus) carriers in HIV (human immunodeficiency virus) infected populations is significantly higher than in HIV seronegative individuals, independent of the risk group for HIV, that is, homosexuals or drug abusers. In this context, evaluation of the simultaneous presence of HBV and HIV in PBMCs (peripheral blood mononuclear cells) is of particular interest for at least 2 reasons: 1) the possible reciprocal influence of the 2 viruses when they infect the same cell; 2) the possibility that HIV-induced hematological disorders could indirectly influence the settling of HBV in blood cell populations. We report data on the frequency of PCR positivity for HBV DNA in PBMCs from 62 HIV infected patients, rigorously selected by risk group, that is, intravenous use of heroin for at least 3 years and syringe promiscuity. Sixty-seven HIV negative individuals who never used any drug formed the control group. The analysis of the cases positive for HBV DNA in PBMCs showed that: 1) the overall prevalence of PCR positivity found in HIV infected patients was significantly lower than that registered in the control group; 2) PCR positivity among the subjects who were HBsAg negative and anti-HBV positive was extremely low in the HIV infected patients (3.7%) but quite frequent in the HIV negative subjects (37.0%). The results support the hypothesis that, unlike the HIV negative individuals, our HIV infected patients exhibited HBV DNA in PBMCS almost exclusively when they presented with active HBV replication.

Interleukin-2, interleukin-2 receptor and γ-interferon synthesis by peripheral blood mononuclear cells in chronic hepatitis delta virus infection

The behaviour of the immune system during liver damage caused by chronic hepatitis delta virus (HDV) infection was evaluated by assessing, in 16 patients with HBsAg+ chronic liver disease and HDV superinfection (15 HbeAg−, 1 HBeAg+), phytohaemagglutinin (PHA)-induced interleukin-2 synthesis and interleukin-2 receptor expression, PHA and staphylococcal enterotoxin B (SEB)-induced γ-interfeton synthesis and, in some cases, the presence of hepatitis B virus DNA (HBV-DNA) within peripheral blood mononuclear cells (PBMC). The results were compared to those obtained in 13 patients without HBV replication (i.e., serum HBV-DNA and liver HBcAg-negative), in 15 with HBV replication (i.e., serum HBV-DNA and/or liver HBcAg-positive) with chronic liver disease without HDV superinfection, and in 15 HBsAg-negative healthy control subjects. The lymphokine pattern in HDV infection was comparable to that of healthy subjects and of HBV non-replicating patients without HDV superinfection. Interleukin-2 receptor expression and γ-interferon synthesis were however significantly decreased in HDV-negative patients with active HBV replication. HBV-DNA was detected in PBMC from 8 of 23 patients, without any correlation with the lymphokine pattern. Our results suggest that in HDV-related chronic liver disease, immune system alterations are unlikely. HDV superinfection does not affect the occurrence of HBV-DNA sequences within the leukocytes. HBV-DNA in PBMC does not interfere with the interleukin-2 system nor with the γ-interferon response in HBV- and HDV-related chronic liver disease.

State of hepatitis B virus DNA in peripheral blood mononuclear cells from persistently infected individuals: correlation with e antigen and viral DNA in the serum as well as with the activity of liver disease.

Peripheral blood mononuclear cells (PBMC) were harvested from 76 asymptomatic carriers of hepatitis B virus (HBV) and 100 patients with type B chronic liver disease. DNA was extracted from cells and tested for the binding with radiolabeled HBV DNA probe by Southern blot hybridization technique. Among 34 asymptomatic carriers who had hepatitis B e antigen (HBeAg) and HBV DNA in the serum, HBV DNA was detected in 29 (85%) PBMC. In remarkable contrast, of the remaining 42 carriers seronegative for HBeAg, only 1 (2%) had HBV DNA in the serum, and none exhibited HBV DNA in PBMC. Among 32 patients seropositive for HBeAg, HBV DNA was detected in 28 (88%) sera, and in 24 (75%) PBMC. Of 68 patients seronegative for HBeAg, HBV DNA was found in 10 (15%) sera, and in 7 (10%) PBMC. Among 62 cases with HBV DNA in PBMC, only 1 had it integrated into the host's DNA. The remaining 61 had free HBV DNA in PBMC. Only 8 of them possessed replicative intermediate forms of HBV DNA, with molecular sizes less than 2.0 kilobases as observed in liver infected with HBV, and they all were patients with chronic active hepatitis. HBV DNA was not detectable in PBMC from 172 controls comprising 44 healthy individuals and 128 patients with non-A, non-B hepatitis. Based on these results, the state of HBV DNA in PBMC reflects the phase of HBV infection with HBeAg in the serum, and a high activity of hepatitis accompanied by active HBV replication.