A 2010 update on occult hepatitis B infection.

Occult hepatitis B virus infection is a challenging issue whose virological and clinical relevance has been a source of long-lasting debate. By definition, OBI is characterized by the persistence of HBV-DNA in the liver tissue (and in some cases also in the serum) in absence of HBsAg. According to the HBV serological profile, OBI may be antibody (anti-HBc alone or together with anti-HBs) positive (seropositive OBI) or antibody negative (seronegative OBI). OBI is a complex biological entity with possible relevant clinical implications, mainly related to the intrahepatic persistence of viral cccDNA and to a strong suppression of viral replication and gene expression. Clinical observations suggest that OBI carriers may be a source of HBV transmission through blood transfusion or orthotopic liver transplantation (OLT). The state of suppression of viral replication and gene expression may be discontinued when an immunosuppressive status occurs, leading to typical hepatitis B with severe - and some times - fulminant course. The long-lasting persistence of the virus in the liver may provoke a very mild but continuing necro-inflammation that (if other causes of liver damage cohexist) may contribute over time to the progression of the chronic liver damage towards cirrhosis. In addition, OBI is supposed to be an important risk factor to HCC development since it maintains the pro-oncogenic properties typical of the overt infection.

Is there a downgrading in the alert about the hepatitis B virus infection in Italy?

BACKGROUND AND AIM: There is suspicion of a decrease in warning regarding the hepatitis B virus as a health problem both by the infected individuals and their doctors. The aim of this study was to investigate whether the clinical/virology investigation of chronic hepatitis B virus infected individuals is at present accurate. METHODS: The chronic hepatitis B virus surface antigen carriers consecutively attending 13 different hospital divisions in Calabria from July to December 2005 were evaluated to investigate the available information on the grade of their liver disease, their virologic profile and the hepatitis B virus status of their family members. RESULTS: Four-hundred-thirty hepatitis B virus surface antigen positive individuals were enrolled, 417 of whom were Calabrians. Most of them had a diagnosis of chronic liver disease, but a liver biopsy had been performed only in 13.5% of the cases, whereas more than 1/3 of them had not been tested for hepatitis Delta virus co-infection. The majority of these individuals were unaware of the hepatitis B virus status of their family members. Moreover, anti-hepatitis B virus vaccination procedures were not performed in most of the hepatitis B virus surface antigen carrier families. CONCLUSIONS: This study revealed that fundamental clinical, virological, and epidemiological aspects of chronic hepatitis B virus infection are not investigated in many hepatitis B virus surface antigen carriers, suggesting that the general knowledge as regards hepatitis B virus is mostly inadequate.

TT virus has a ubiquitous diffusion in human body tissues: analyses of paired serum and tissue samples.

The tissue tropism and possible correlation with liver disease of the TT virus (TTV) as well as its prevalence and genotype distribution remain undefined. TTV-DNA was investigated in paired sera and tissue samples from 144 patients, and sera and cerebrospinal fluids (CSF) from additional six subjects. Of the 144 tissue samples, 128 were liver biopsy specimens from subjects with hepatic disease while 16 were surgically obtained nonliver specimens from patients with extrahepatic disease. TTV cloning, sequencing and genotype analyses were performed on isolates from sera, tissue specimens and peripheral blood mononuclear cells of two patients with hepatic and four patients with extrahepatic pathologies, as well as from sera and CSFs of two subjects. TTV was found in 100% of the examined tissues and in 60.1 and 50% of sera from patients with hepatic and extrahepatic pathologies, respectively. Moreover, TTV was detected in four of the six CSFs analysed but only in two correspondent sera. Genotyping revealed the coexistence of multiple TTV genotypes and genetic variants in each infected individual, and the analysis of TTV mRNA showed the presence of transcripts in all the six different tissues studied. These results indicate that the entire adult population in our area is more likely infected by TTV, although several subjects are not viraemic and that TTV infects many different human tissues and is able to invade the central nervous system.

Analysis of haemochromatosis gene mutations in a population from the Mediterranean Basin.

BACKGROUND/AIMS: The C282Y mutation in the haemochromatosis gene (HFE) located on chromosome 6 has been identified as the main genetic basis of hereditary haemochromatosis (HH). Two more mutations of that gene, H63D and S65C, appear to be associated with milder forms of HH. A high allele frequency for C282Y and H63D mutations was reported in populations from North Europe, while incomplete information is available for individuals from the Mediterranean Basin where C282Y homozygotes comprise a smaller percentage of HH cases. In this study we investigated the allele frequency of HFE mutations and the association between HFE mutations and cases of HH in a population from the South of Italy (Sicily and Calabria). In addition, we evaluated a possible association between HFE mutations and either chronic liver disease or type II diabetes. PATIENTS AND METHODS: Three hundred and twenty-seven individuals (654 chromosomes) were tested for C282Y, H63D and S65C mutations of the HFE gene by restriction fragment length polymorphism. Four had HH, 23 had hepatocellular carcinoma, 100 had chronic liver disease, 100 had type II diabetes, and 100 were healthy controls. RESULTS: Both C282Y and S65C mutations were each detected in one of the 654 chromosomes analysed (allele frequency=0.15%), while H63D change was found in 122 chromosomes (allele frequency=18.6%) and was equally distributed in all the categories examined. One healthy individual had compound heterozygosity for C282Y and H63D mutations. The frequency of C282Y in this Southern Italian sample was the lowest yet reported for a population of European origin. None of the four HH patients was either homozygous or heterozygous for C282Y. CONCLUSIONS: In Mediterranean populations from Southern Italy the C282Y mutation occurs sporadically and HFE polymorphisms seem to have little diagnostic relevance.

Occult hepatitis B virus infection.

Many studies have shown that hepatitis B virus infection may also occur in hepatitis B surface antigen-negative patients. This occult infection has been identified both in patients with cryptogenic liver disease and in patients with hepatitis C virus-related chronic hepatitis, and much evidence suggests that it may be a risk factor of hepatocellular carcinoma development. However several aspects of this occult infection remain unclear such as its prevalence and the factor(s) involved in the lack of circulating hepatitis B surface antigen. Moreover, it is uncertain whether the occult hepatitis B virus infection may contribute to chronic liver damage, considering that it is usually associated with a suppressed viral replication. Evidence and hypotheses concerning this fascinating field of bio-medical research are reviewed.

Pro-apoptotic effect of the hepatitis B virus X gene.

The hepatitis B virus (HBV) is a common human pathogen that causes acute and chronic liver disease. Persistent HBV infection is strongly associated with the development of hepatocellular carcinoma. The contribution of the viral regulatory protein HBx in liver oncogenesis has been supported by our recent studies in a transgenic mouse model, showing that HBx cooperates with c-myc by accelerating the onset of primary liver tumors. Here we show that liver expression of HBx is associated with increased rates of spontaneous apoptosis in liver cells from two different transgenic lines. In transient transfection assays, overexpression of HBx in the established hepatocyte cell line MMHD3 and in human hepatoma cells HepG2 was found to induce apoptosis in a dose-dependent manner. These data suggest that HBx might trigger an apoptotic process in HBV-infected hepatocytes, in turn possibly favoring liver regeneration and accumulation of genetic alterations, ultimately leading to liver cell transformation in chronically infected patients.

p53-independent apoptotic effects of the hepatitis B virus HBx protein in vivo and in vitro.

The hepatitis B virus protein HBx is a promiscuous transactivator implicated in both cell growth and death and in the development of hepatocellular carcinoma. We recently reported that HBx can potentiate c-myc-induced liver oncogenesis in a transgenic model where low level expression of HBx induces no pathology. To assess if HBx could affect the hepatocyte turnover, we investigated the HBx-elicited apoptotic responses in transgenic livers and in primary hepatocyte cultures. Here we show that transgenic expression of HBx is associated with a twofold increase of spontaneous cell death in the mouse liver. The finding that apoptosis was enhanced to similar extents in HBx mice carrying homozygous p53 null mutations implied that functionally intact p53 was not required to transduce the death signal. A direct, dose-dependent apoptotic function of HBx was demonstrated in transient transfections of liver-derived cell lines. We further show that stable expression of HBx at low, presumably physiological levels in primary hepatocytes, induced cellular susceptibility to diverse apoptotic insults, including growth factor deprivation, treatment with anti-Fas antibodies or doxorubicine and oxidative stress. HBx expression, but not p53 status profoundly affected the commitment of cells to die upon apoptotic stimuli. These data strengthen the notion that HBX may contribute to HBV pathogenesis by enhancing apoptotic death in the chronically infected liver.

Pre-S2 defective hepatitis B virus infection in patients with fulminant hepatitis.

Controversial data were recently published concerning the association of hepatitis B virus (HBV) variants with fulminant hepatitis (FH). In this study, we first analyzed the complete nucleotide sequences of HBV genomes isolated from serum samples from a surgeon and his mother, who was accidentally infected by the son; both died of FH. The infecting viruses were genetically almost identical in both patients; all the clones examined carried a double nucleotide mutation in the start codon of the pre-S2 region that prevented the synthesis of the corresponding protein. Analyses of different serum samples from the son revealed only wild-type precore sequences in a high viremic serum, whereas hepatitis B e antigen (HBeAg)-defective strains were prevalent when the viremia had decreased. Subsequently, we extended the analysis to the viral genomes isolated from 18 additional patients with acute HBV infection and different clinical behaviors: 3 of 5 patients with FH and without previous liver disease had pre-S2 start codon mutations preventing pre-S2 protein synthesis, whereas none of the 13 control cases had similar genomic rearrangements. Analysis of the precore region showed that viral populations normally producing HBeAg were the only or the prevalent viral strains in all of these cases. In summary, our results support the hypothesis that the pre-S2 protein is not essential for HBV infectivity. They also show that infection by pre-S2-defective virus is frequently associated with FH, indicating that this variant might play a pathogenetic role in cases of acute liver failure. Finally, they suggest that the emergence of HBeAg-defective viruses might be a late event in the course of FH, occurring when HBeAg-producing viruses have been mostly cleared.

Outcome of liver disease and response to interferon treatment are not influenced by hepatitis B virus core gene variability in children with chronic type B hepatitis.

BACKGROUND/AIMS: Hepatitis B virus (HBV) core gene heterogeneity may influence the outcome of liver disease and the response to interferon (IFN) therapy in adult HBV carriers. The aim of this study was to evaluate the possible association between HBV core gene variability and evolution of chronic hepatitis in children. METHODS: We examined serum samples from 25 children with HBV chronic hepatitis and HBe antigen (HBeAg) positivity who were followed-up for a mean of 7.4 years. Seven cases spontaneously seroconverted to anti-HBe, becoming HBV healthy carriers; nine cases were successfully treated with IFN; nine cases were non-responders to IFN therapy. HBV-DNA was extracted from one serum sample ("I") collected during the HBeAg positive phase, and from a second sample ("II") collected after the anti-HBe seroconversion or, in non-responders, after stopping therapy. The entire core gene of the HBV isolates was amplified and sequenced. RESULTS: Each isolate showed single or no missense mutation independently of the clinical behavior of the patients. HBeAg-defective viruses were detected in one case in both samples and in two cases only in sample "II". CONCLUSIONS: Core gene variability does not seem to be involved either in the outcome of infection or in the response to IFN treatment in children with HBV chronic hepatitis. Considering that most of the HBV carriers in our area acquire the infection in childhood, our data suggest that core gene heterogeneity is not a major cause of progression to chronicity.

Evaluation of hepatitis B and C virus infections in patients with non-Hodgkin's lymphoma and without liver disease.

Hepatitis B virus (HBV) and hepatitis C virus (HCV) are hepatotropic and lymphotropic viruses endemic to Sicily. To evaluate whether these viruses may chronically infect patients with non-Hodgkin's lymphoma (NHL) and without liver disease, we examined serum samples from 24 such patients. Five cases (20.8%) revealed HCV infection, as shown by the detection of viral RNA through the polymerase chain reaction technique, while HBV-DNA was not found in any of them by the same method. These results provide one more epidemiological element supporting the hypothesis that the association between HCV infection and lymphoproliferative diseases is not a casual event, and show that HCV may chronically infect patients with NHL without producing liver damage.

PreS and core gene heterogeneity in hepatitis B virus (HBV) genomes isolated from patients with long-lasting HBV chronic infection.

Several reports have been recently published concerning the identification of HBV variants due to rearrangements of the preS1/preS2 or core regions of the viral genome. To evaluate the frequency of the natural occurrence of such variants and whether the heterogeneity of these genomic regions correlates with the severity of the liver disease, we have examined the preS1/preS2 region and the entire core gene sequences of HBV DNA isolated from sera of 30 chronic HBV carriers, 7 with chronic persistent hepatitis, 10 with chronic active hepatitis, 7 with cirrhosis, and 6 with hepatocellular carcinoma. We found no significant rearrangement in any of the preS1 regions examined, while genomic modifications precluding the preS2 protein production were detected in 4 cases, 2 with cirrhosis and 2 with hepatocellular carcinoma. The analysis of the core gene showed the presence of various numbers of missense mutations in the core region of most cases, independent of the grade of liver disease. Moreover, in contrast with previous reports, neither mutation cluster region nor deletion was observed. On the contrary, HBV strains with the precore mutation at nucleotide position 1896, effecting the rise of HBeAg-defective viruses, were found in 26 of the 30 cases examined. In conclusion, our data show that the precore mutant is the only HBV genomic variant commonly selected during a chronic infection. Other HBV variants, due to genomic rearrangements outside the precore region, may exist and influence the outcome of the infection and the course of the liver disease, but the emergence of each of these variants seems to be an unusual and probably casual event.

Hepatitis B E antigen detection in formalin-fixed liver biopsy specimens. A tool to investigate wild-type and E-minus variant HBV infection.

The aim of this study is to investigate whether hepatitis B e antigen (HBeAg) reactivity can be detected on formalin-fixed, paraffin-embedded liver tissue, and whether immunohistochemical detection of intrahepatic HBeAg may help to distinguish between "wild-type" and "eminus" hepatitis B virus (HBV) infection. Liver biopsy specimens were analyzed from 27 patients with chronic type B hepatitis: 12 patients had serum HBeAg (group A), and 15 patients were anti-HBe positive (group B). Part of each biopsy fragment was processed for histologic and immunohistochemical studies, and a part was used for HBV-DNA analysis. Dewaxed sections from each specimen were tested with a specific monoclonal anti-HBe antibody; then a Biotin-Streptavidin kit was used as detection system. HBeAg was revealed in 10 of 12 cases of group A and in 6 of the 15 cases of group B. Pre-core region of HBV genomes, isolated from each biopsy specimen, was analyzed by direct sequencing: 10 cases of group A were found to be infected by wild-type HBV alone and 2 cases by both wild and e-minus HBV types. In group B, all the 6 cases with intrahepatic HBeAg reactivity were found to be infected by mixed viral population, whereas the 9 cases negative for such reactivity were found to be infected by e-minus HBV alone. These results show that HBeAg can be detected in formalin-fixed, paraffin-embedded liver specimens, and the method is sensitive and specific. Because the presence of HBeAg in the liver indicates a wild-type HBV infection, and the lack of detection of such antigen in the hepatocytes of anti-HBe positive subjects correlates with unmixed e-minus HBV infection, the authors conclude that this technique is a useful tool for recognizing the viral strains that infect patients with chronic type B hepatitis.

Divergent effects of interleukin-10 on cytokine production by mononuclear phagocytes and endothelial cells.

Interleukin-10 (IL-10), a product of T helper type 2 (TH2) cells and monocytes, inhibits cytokine production in mononuclear phagocytes. Given the similarities and interrelationship between cells of the monocyte-macrophage lineage and endothelial cells, we examined the effects of IL-10 on vascular endothelium. Murine IL-10 induced low levels of IL-6 production and amplified induction of IL-6 by lipopolysaccharide (LPS) or IL-1 in the murine tEND.1 endothelioma line, used for these studies because it retains properties of normal endothelium. The effect was more evident after prolonged (48-72 h) exposure to IL-10. IL-10 had similar activity on other endothelioma lines, whereas it inhibited IL-6 production by peritoneal macrophages. Induction and amplification of cytokine production by IL-10 was associated with higher levels of mRNA, which were maintained longer (up to 48 h) than in controls. In addition to IL-6, murine IL-10 induced or amplified expression of the chemoattractant cytokines monocyte chemotactic protein-1 (MCP-1) and KC. Human IL-10 inhibited IL-6 release by LPS-stimulated human peripheral blood mononuclear cells, whereas it did not interfere with cytokine production by LPS- or IL-1-stimulated human umbilical vein endothelial cells. The selective inhibitory action of IL-10 on mononuclear phagocytes versus endothelial cells may play a role in the pathophysiology of TH2-directed responses.

Regulated expression of vascular cell adhesion molecule-1 in human malignant melanoma.

Expression of the endothelial adhesion molecule VCAM-1 was studied in human malignant melanoma lines by flow cytometry. Clones 2/4 and 2/14 (derived from the same lesion) had appreciable levels of VCAM-1 expression, whereas clone 2/21 and the lines A2058, Mel24, and A375 were negative. Clone 2/14 was selected for further analysis. Exposure to tumor necrosis factor (TNF) markedly augmented VCAM-1 on melanoma cells. Surface VCAM-1 was associated with expression of specific transcripts that were augmented by TNF. Analysis by reverse transcriptase and polymerase chain reaction using appropriate primers revealed that TNF-stimulated melanoma cells expressed both 7 and 6 immunoglobulin domain transcripts with predominance of the longer species. Tumor necrosis factor--stimulated melanoma cells bound more VLA-4-expressing cells (melanoma and monocytes) than resting tumor cells and anti-VCAM-1 monoclonal antibodies significantly inhibited binding, thus suggesting that surface VCAM-1 on melanoma is functional. Analysis of melanoma tissue sections demonstrated that VCAM-1 is not a marker of transformation of melanocytes because it can be detected in benign nevi. Although, unlike ICAM-1, VCAM-1 is not correlated with tumor progression, its expression in a fraction of primary melanomas indicates that it may play a role in regulating host immune response and homotypic interactions in some malignant melanomas.