Christian Brechot

Infection of peripheral mononuclear blood cells by hepatitis C virus

We investigated the infection of peripheral blood mononuclear cells (PBMNC) by hepatitis C virus (HCV) in 5 patients with HCV-related chronic hepatitis. The presence of HCV-RNA-positive and -negative strands was tested with the polymerase chain reaction (PCR) method. In all subjects, HCV-RNA was shown in PBMNC. In 3 cases, HCV-RNA was shown in the T- and B-cell populations, with viral RNA also present in the monocyte-macrophage fraction of two of these. HCV-RNA-negative stranded molecules, indicative of the viral multiplication, were significantly increased in cells maintained in cultures with PHA/PMA stimulation. The results indicate that HCV infect blood mononuclear cells, thus suggesting that this cellular tropism may play a role in HCV infection.

Passive immunoprophylaxis after liver transplantation in HBsAg-positive patients

110 HBsAg-positive patients underwent orthotopic liver transplantation and received long-term anti-hepatitis B virus (HBV) passive immunoprophylaxis with anti-HBs immunoglobulin. During a mean follow-up period of 20 months, all patients became HBsAg negative after transplantation but circulating HBsAg reappeared in 25 (22.7%). Overall 1-year survival was 83.6% and overall 2 year actuarial recurrence of HBsAg was 29% (59% after posthepatitis B cirrhosis, 13% after posthepatitis B-delta cirrhosis, and 0% after fulminant hepatitis B). Patients with HBV cirrhosis who were HBV-DNA positive had a much greater risk of HBsAg recurrence than patients who were HBV-DNA negative (96% vs 29% at 2 years). Reappearance of HBsAg was associated with evidence of HBV replication and abnormal histological findings in the graft. Long-term passive anti-HBV immunoprophylaxis significantly reduced HBV reinfection and improved survival in patients without evidence of active HBV replication before orthotopic liver transplantation.

Polymerase Chain Reaction to Detect Hepatitis B Virus DNA and RNA Sequences in Primary Liver Cancers from Patients Negative for Hepatitis B Surface Antigen

BACKGROUND AND METHODS The role of hepatitis B virus (HBV) in the course of patients with primary liver cancer who are negative for hepatitis B surface antigen has been debated. We used the polymerase chain reaction to evaluate 28 such patients for the presence of DNA and RNA sequences of the virus; 22 of these patients had associated cirrhosis. The patients were from areas with different prevalences of HBV infection (South Africa, Italy, France, and Japan). RESULTS Antibodies to the surface and core antigens of HBV were detected in 10 of the 23 patients tested. HBV DNA sequences were detected in 17 of the 28 patients, including 8 of the 10 with HBV antibodies and 6 of 13 without HBV serologic markers. HBV RNA molecules were found in four of five tumors tested. CONCLUSIONS Our investigation indicates that transcriptionally active HBV genomes are present in various geographic areas among patients with liver cancer who are negative for hepatitis B surface antigen. This observation is consistent with an etiologic role for the virus in the development of these tumors.

DETECTION OF HTV1 DNA IN INFANTS AND CHILDREN BY MEANS OF THE POLYMERASE CHAIN REACTION

The polymerase chain reaction (PCR) assay was used to investigate the possibility of HIV1 DNA detection in uncultured peripheral blood mononuclear cells from newborn infants and children of HIV-infected mothers. HIV1 DNA sequences were detected in mononuclear cells of six of fourteen symptom-free newborn infants of seropositive mothers. Only one of these infants had detectable HIV antigenaemia. In addition, HIV1 DNA was identified in the mononuclear cells of five of ten children (2-5 years old) of infected mothers who had become seronegative 12-15 months after birth; among these, four children had only mild clinical features related to HIV infection, while the other had none. HIV1 DNA was shown in all of eight seropositive children with HIV infection and none of fifteen normal seronegative controls. The PCR assay thus provides an early and direct identification of HIV infection in newborn infants and seronegative children born to infected mothers.

Hepatitis C virus RNA and hepatitis B virus DNA in serum and liver of patients with fulminant hepatitis

BACKGROUND The role of hepatitis C virus (HCV) in the development of fulminant hepatitis is poorly understood. METHODS The polymerase chain reaction was used to detect HCV RNA and hepatitis B virus (HBV) DNA in serum and liver of 40 patients with fulminant or subfulminant hepatitis. RESULTS HCV RNA was detected in none of the 23 subjects with hepatitis B surface antigen (HBsAg)-negative hepatitis of unknown etiology. HBV DNA was found in 1 of these 23 subjects. In contrast, 8 of the 17 patients with HBsAg-positive hepatitis were HCV RNA positive. Among the latter, 5 had immunoglobulin (Ig) M antibody to hepatitis B core antigen (anti-HBc) and thus were acutely coinfected by HBV and HCV, whereas 3 others without IgM anti-HBc had superinfection by HCV. The active replication of HCV in the liver was shown by detection of high titers of HCV RNA and of negative-stranded HCV RNA. CONCLUSION This investigation therefore shows no evidence of a role for HCV infection in sporadic cases of fulminant hepatitis without defined etiology. However, it suggests that HCV might be implicated in a significant number of patients with HBV-related fulminant hepatitis.

Soluble tumor necrosis factor receptors in chronic hepatitis C: a correlation with histological fibrosis and activity.

BACKGROUND/AIMS Tumor necrosis factor-alpha (TNF) is a mediator of inflammation and cellular immune response. Soluble TNF receptors (sTNFR) sTNF-R55 and sTNF-R75, which compete with cellular receptors for the binding of TNF, have been detected at high levels in infectious diseases including human immunodeficiency virus and HBV infection. In order to investigate the activation of the TNF system in HCV infection, we have analyzed the balance between TNF and sTNF-R in 60 HCV-infected subjects according to their clinical, biological, virological and histological characteristics. METHODS Serum TNF, sTNF-R55 and sTNF-R75 levels were determined by ELISA before any therapy and were compared to a control group of 60 healthy subjects and a group of 34 HBV-infected patients. RESULTS Mean TNF levels were 50.5+/-4.5 pg/ml in HCV patients, and undetectable (<5 pg/ml) in the control subjects. sTNF-R55 and sTNF-R75 levels were significantly higher in HCV-infected patients than in the controls: 2.88+/-0.14 ng/ml vs. 1.30+/-0.05, (p = 0.0001), and 9.54+/-0.58 ng/ml vs. 4.19+/-016, (p = 0.0001), respectively. sTNF-R55 and TNF-alpha levels in HCV patients were not significantly different from levels in HBV patients. sTNF-R75 levels were slightly lower than in HBV patients (9.54+/-0.58 vs. 11.4+/-0.79 ng/ml, p = 0.03). In contrast to other infectious diseases, there was no correlation between levels of sTNF-R and TNF. sTNF-R75 but not TNF levels were correlated with aminotransferases levels (p = 0.0001 and p = 0.0015 for aspartate and alanine aminotransferase, respectively), while sTNF-R55 levels were significantly correlated only with aspartate aminotransferase levels (p = 0.003). sTNF-R75 levels were significantly correlated with the Metavir activity index (p = 0.01), and sTNF-R55 and sTNF-R75 levels were significantly higher in patients with vs. without cirrhosis (3.22+/-0.21 vs. 2.54+/-0.17 ng/ml (p<0.02) and 11.6+/-0.86 vs. 7.5+/-0.53 ng/ml (p<0.001), respectively). sTNF-R55, sTNF-R75 and TNF levels were not correlated with viral load, genotype or response to interferon therapy. CONCLUSIONS Levels of soluble TNF receptors, and particularly sTNF-R75, are significantly correlated with the severity of the disease but not with virological parameters such as quantitative viremia and genotype. High TNF-R production could thus suggest that HCV-related liver disease involves immunological mechanisms, including activation of the TNF system.

Induction or expansion of T-cell responses by a hepatitis B DNA vaccine administered to chronic HBV carriers

Despite the availability of effective hepatitis B vaccines for many years, over 370 million people remain persistently infected with hepatitis B virus (HBV). Viral persistence is thought to be related to poor HBV‐specific T‐cell responses. A phase I clinical trial was performed in chronic HBV carriers to investigate whether HBV DNA vaccination could restore T‐cell responsiveness. Ten patients with chronic active hepatitis B nonresponder to approved treatments for HBV infection were given 4 intramuscular injections of 1 mg of a DNA vaccine encoding HBV envelope proteins. HBV‐specific T‐cell responses were assessed by proliferation, ELISpot assays, and tetramer staining. Secondary end points included safety and the monitoring of HBV viraemia and serological markers. Proliferative responses to hepatitis B surface antigen were detected in two patients after DNA injections. Few HBV‐specific interferon γ–secreting T cells were detectable before immunization, but the frequency of such responses was significantly increased by 3 DNA injections. Immunization was well tolerated. Serum HBV DNA levels decreased in 5 patients after 3 vaccine injections, and complete clearance was observed in 1 patient. In conclusion, this study provides evidence that HBV DNA vaccination is safe and immunologically effective. We demonstrate that DNA vaccination can specifically but transiently activate T‐cell responses in some chronic HBV carriers who do not respond to current antiviral therapies. Supplementary material for this article can be found on the HEPATOLOGYwebsite (http://interscience.wiley.com/jpages/0270‐9139/suppmat/index.html). (HEPATOLOGY 2004;40:874–882.)

Persistent Hepatitis B Virus Infection Of Mononuclear Blood Cells Without Concomitant Liver Infection: The Liver Transplantation Model

We have investigated the recurrence of Hepatitis B virus (HBV) in 30 patients treated by orthotopic liver transplantation and given high doses of anti-HBs immunoglobulin. The polymerase chain reaction (PCR) assay showed no evidence of HBV DNA sequences in the liver of 23/24 patients who remained serum HBsAg-negative during a mean follow-up of 13 months (2-24 months) after OLT. However, the liver scored positive in all the 6 individuals in whom HBsAG reappeared. The PCR assay identified HBV DNA sequences in the peripheral blood mononuclear cells of 7 of 11 subjects who were serum HBsAG-negative and liver HBV DNA-negative by PCR. Therefore, this application of the sensitive PCR assay demonstrates persistent infection of PBMC in the absence of liver HBV--thus OLT provides a model for studying the interaction between HBV, PBMC, and the liver.

Impact of cytomorphological detection of circulating tumor cells in patients with liver cancer.

The clinical impact of circulating tumor cell (CTC) detection is controversial, mainly due to drawbacks of molecular approaches applied to this field. We sought to determine if the specific identification and counting of circulating tumor cells by cytomorphologic analysis has clinical usefulness. Peripheral blood (6 mL), treated using isolation by size of epithelial tumor cells, was obtained from 44 patients with primary liver cancer (PLC) and without metastases, 30 patients with chronic active hepatitis, 39 with liver cirrhosis, and 38 healthy individuals, and followed up for a mean period of 1 year. We searched for β‐catenin mutations in 60 single microdissected CTCs. One patient with liver cancer developed extrahepatic metastases during follow‐up. CTCs and microemboli were found in 23 of the 44 patients with liver cancer and in none of the patients with chronic active hepatitis, patients with cirrhosis, or healthy subjects. Their presence was significantly associated with tumor diffusion (P = .0001) and portal tumor thrombosis (P = .006). Both the presence (P = .01) and number (P = .02) of CTCs and microemboli were significantly associated with a shorter survival. β‐Catenin mutations were found in 3 of 60 CTCs, arguing against their impact on the initial step of tumor cell invasion. In conclusion, the highly sensitive and specific detection of CTCs and microemboli may have clinical implications for cancer staging and outcome prediction. We also show the feasibility of molecular studies of individual circulating tumor cells, aimed at identifying gene mutations involved in tumor invasion. (HEPATOLOGY 2004;39:792–797.)

Specific Vaccine Therapy in Chronic Hepatitis B: Induction of T Cell Proliferative Responses Specific for Envelope Antigens

In a pilot study, it was established that specific therapy by standard anti-hepatitis B virus (HBV) vaccination may be effective in reducing HBV replication and canceling the immune tolerance to hepatitis B surface antigen (HBsAg) particles in about 50% of persons with chronic active HBV replication. In the present study, the vaccine-induced immune responses were analyzed during an ongoing controlled multicenter vaccine trial. Vaccination elicited peripheral blood mononuclear cell proliferative responses specific for envelope antigen in 7 of 27 subjects given HBsAg. The responses induced by the vaccines were mediated by CD4+ T lymphocytes, and at least three different epitopes were recognized. HBV-specific CD4+ T lymphocytes produced high levels of interferon-gamma [corrected] and belonged to a T helper 1 subset. Reduction of serum HBV DNA in some of these persons suggests that induction of CD4+ T cell responses could be important in controlling viremia during vaccine therapy of chronic HBV carriers.