Squamous cell carcinoma antigen-1 (SERPINB3) polymorphism in chronic liver disease.

BACKGROUND: The serpin squamous cell carcinoma antigen (SCCA, SERPINB3) has been found over-expressed in primary liver cancer and at lower extent in cirrhosis and chronic hepatitis. A novel SCCA-1 variant (SCCA-PD), presenting a single mutation in the reactive centre (Gly351Ala), has been recently identified (rs3180227). AIM: To explore SCCA-1 polymorphism in patients with HCV infection as single etiologic factor and different extent of liver disease. METHODS: One hundred and fourty-eight patients with chronic HCV infection (45 chronic hepatitis, 53 cirrhosis, 50 HCC) and 50 controls were evaluated. SCCA-1 polymorphism was studied by restriction fragment length polymorphism and confirmed randomly by direct sequencing. Circulating SCCA-IgM complex was determined by ELISA. RESULTS: SCCA-PD was detected with higher frequency in cirrhotic patients (45.3%, odds ratio=2.62; 95%CI 1.13-6.10, p=0.038) than in patients with chronic hepatitis or in controls (24.4% and 24%, respectively). Intermediate figures were found in hepatocarcinoma (36.0%). SCCA-IgM in serum was lower in patients carrying SCCA-PD than in wild type patients and the difference was statistically significant in cirrhotic patients (mean+/-S.D.=117.45+/-54.45 U/ml vs. 268.52+/-341.27 U/ml, p=0.026). CONCLUSIONS: The newly identified SCCA-PD variant was more frequently found in liver cirrhosis, suggesting that patients carrying this polymorphism are more prone to develop progressive liver fibrosis.

Surface expression of squamous cell carcinoma antigen (SCCA) can be increased by the preS1(21-47) sequence of hepatitis B virus.

A variant of the serpin squamous cell carcinoma antigen (SCCA) has been identified as a hepatitis B virus binding protein and high expression of SCCA has recently been found in hepatocarcinoma. Since HBV is involved in liver carcinogenesis, experiments were carried out to examine the effect of HBV preS1 envelope protein on SCCA expression. Surface and intracellular staining for SCCA was assessed by FACS analysis. Preincubation of HepG2 cells and primary human hepatocytes with preS1 protein or with preS1(21-47) tetrameric peptide significantly increased the surface expression of SCCA, without modification of its overall cellular burden, suggesting a surface redistribution of the serpin. An increase in HBV binding and internalization was observed after pre-incubation of the cells with preS1 preparations, compared to cells preincubated with medium alone. Pretreatment of cells with DMSO, while not influencing SCCA basal expression, was responsible for an increase in the efficiency of HBV internalization and this effect was additive to that obtained after incubation with preS1 preparations. In conclusion, the HBV preS1(21-47) sequence is able to induce overexpression of SCCA at the cell surface facilitating virus internalization, while the increased efficiency of HBV entry following DMSO addition is not mediated by SCCA.

Overexpression of squamous cell carcinoma antigen variants in hepatocellular carcinoma.

Pathogenetic mechanisms of hepatocellular carcinoma (HCC) are still unclear and new tools for diagnostic and therapeutic purposes are ongoing. We have assessed whether squamous cell carcinoma antigen (SCCA), a serpin overexpressed in neoplastic cells of epithelial origin, is also expressed in liver cancer. Squamous cell carcinoma antigen was evaluated by immunohistochemistry in 65 HCCs of different aetiology and in 20 normal livers. Proliferative activity was assessed using MIB-1 antibody. In 18 surgical samples, tumour and nontumour liver tissue was available for SCCA cDNA amplification and sequencing. Squamous cell carcinoma antigen was detected in 55 out of 65 (85%) tumour specimens, but in none of the 20 controls. In the majority of the cases, the positive signal was found in the cytoplasm of more than 50% of the hepatocytes. Low or undetectable SCCA (scoreor=2 (mean+/-s.d.: 2%+/-2.4 vs 7.5%+/-10.3, P<0.05). Squamous cell carcinoma antigen mRNA could be directly sequenced in 14 out of 18 liver tumours but in none of the corresponding nontumour samples. From sequence alignment, a novel SCCA1 variant (G(351) to A) was identified in five cases, while SCCA1 was revealed in six cases and SCCA2 in three cases. In conclusion, SCCA variants are overexpressed in HCC, independently of tumour aetiology. A novel SCCA1 variant has been identified in one third of liver tumours.

Cloning and expression of a novel hepatitis B virus-binding protein from HepG2 cells.

A direct involvement of the hepatitis B virus (HBV) preS1-(21-47) sequence in virus attachment to cell membrane receptor(s) and the presence on the plasma membranes of HepG2 cells of protein(s) with receptor activity for HBV have been suggested by many previous experiments. In this study, by using a tetravalent derivative of the preS1-(21-47) sequence, we have isolated by affinity chromatography from detergent-solubilized HepG2 plasma membranes a 44-kDa protein (HBV-binding protein; HBV-BP), which was found to closely correspond to the human squamous cell carcinoma antigen 1 (SCCA1), a member of the ovalbumin family of serine protease inhibitors. Comparison of SCCA1 sequence with the sequence of the corresponding HBV-BP cDNA, cloned by polymerase chain reaction starting from RNA poly(A)(+) fractions extracted from HepG2 cells, indicated the presence of only four nucleotide substitutions in the coding region, leading to three amino acid changes. Intact recombinant HBV-BP lacked inhibitory activity for serine proteases such as alpha-chymotrypsin and trypsin but inhibited with high potency cysteine proteases such as papain and cathepsin L. Direct binding experiments confirmed the interaction of recombinant HBV-BP with the HBV preS1 domain. HepG2 cells overexpressing HBV-BP after transfection of corresponding cDNA showed a virus binding capacity increased by 2 orders of magnitude compared with untransfected cells, while Chinese hamster ovary cells, which normally do not bind to HBV, acquired susceptibility to HBV binding after transfection. Native HBV particle entry was enhanced in transfected cells. Both recombinant HBV-BP and antibodies to recombinant HBV-BP blocked virus binding and internalization in transfected cells as well as in primary human hepatocytes in a dose-dependent manner. Our findings suggest that this protein plays a major role in HBV infection.

Hepatitis C virus RNA profiles in chronically infected individuals: do they relate to disease activity?

Fluctuations of hepatitis C virus (HCV)-RNA serum levels were monitored in a multicenter study in 76 chronic HCV carriers who had been followed longitudinally without receiving antiviral therapy to assess their relation with the course of liver disease activity. Forty-four patients had normal transaminases over more than 2 years, while 32 additional patients had fluctuating levels. Viral load was measured in serial serum samples prospectively collected for 10 to 12 months in 54 patients and in sera stored yearly up to 8 years in an additional 22 patients. In patients tested monthly, a lesser extent of fluctuations was detected in cases with constantly normal transaminases as compared with those with fluctuating transaminases. In the former group, the mean difference between maximum and minimum values observed in each individual patient was 0.7 Log, while in the latter group, it was 1.3 Log (P =.0004). Most of these patients experienced, on average, three peaks of viremia over 1 year. The range of variation observed upon yearly testing was between 0.2 and 2.2 Log and did not reach statistical significance between the two groups. In conclusion, a careful viral replication profile can be achieved only by monthly testing, because longer time intervals could miss viremia fluctuations. HCV-RNA levels are more stable in asymptomatic HCV carriers than in patients with biochemical activity of liver disease.

Characteristics of hepatitis C virus before and after interferon treatment in patients with ongoing viraemia but sustained biochemical response.

In hepatitis C virus (HCV) infection, persistent viraemia can occur after successful biochemical response to interferon treatment. To assess whether this unusual profile might be due to trivial amounts of remaining virus or to the emergence of less pathogenic HCV strains, pre- and posttreatment sera from 27 patients who remained with HCV-RNA, despite sustained transaminase normalisation after interferon therapy, were investigated. All but one had infection by genotype 2 (P < 0.0001), and levels of HCV-RNA were not decreased after therapy. Sequence comparison of the 5' untranslated region revealed mixed viral populations and "not compensatory" nucleotide transitions localised at the stem level of the secondary structure of this region in samples taken before and after treatment. Neither quantitative nor qualitative viral changes, at least for the 5' untranslated region, are responsible for interferon-induced biochemical remission in these patients typically infected by genotype 2.

Hepatitis C genotypes in patients with dual hepatitis B and C virus infection.

In patients with chronic hepatitis B and C virus (HBV, HCV) infection, an inverse relationship in the replicative activity of the two viruses has been reported. In the present study the genotype of HCV was evaluated in 34 consecutive cases found with hepatitis B surface antigen (HBsAg) and anti-HCV in the serum, in order to identify its possible influence in determining the pattern of HBV/HCV interaction. Nineteen patients were HCV-RNA positive and could be genotyped: 8 were infected by HCV-1 (3 by HCV-1a and 5 by HCV-1b), 10 by HCV-2, and only 1 by HCV-3. Among these, 3 were HBV-DNA positive, compared to 10 of 15 HCV-RNA-negative patients (P = 0.003), and all 3 were coinfected with HCV-2. Mean alanine aminotransferase (ALT) levels were similar between patients infected with HCV-1 and HCV-2. Among 7 patients with cirrhosis 5 were infected by HCV-2, while 6 of 12 of those without cirrhosis had HCV-1 infection. In conclusion, HBV replication was inhibited more efficiently by HCV-1 than by HCV-2. Cirrhosis was frequently found in patients with dual HBV and HCV-2 infection.

Hepatitis C virus genotypes and severity of chronic liver disease in haemophiliacs.

We studied the activity and stage of chronic liver disease in 45 HCV-seropositive/HIV-seronegative patients with severe haemophilia followed for at least 10 years. HCV-RNA was detected in serum in 36 patients (80%) Viraemic cases were further analysed for HCV genotypes: 10 (28%) were infected by type 1a, 10 (28%) by type 1b, seven (19%) by type 2, four (11%) by type 3, four (11%) had mixed infections (one by 1a + 1b, one by 1a + 2, one by type 2 + 3, and one by 1a + 2 + 3). ALT levels were within the normal range in 55% of the HCV-RNA negative patients but in only 11% of the viraemic cases. Results show a trend for higher levels of ALT in HCV-RNA-positive patients compared with those without viraemia (98 +/- 56 v 60 +/- 61), and particularly with patients with type 3 HCV infection (148 +/- 44). We suggest that a slow progression of chronic liver disease occurs in haemophilic HCV-positive/HIV-negative patients and conclude that presence of HCV-RNA in serum correlates well with cytolitic damage but, in the time-scale of our follow-up period, commonly used clinical-laboratory parameters cannot predict the chronic evolution of liver infection or identify differences in disease progression in relation to specific HCV subtypes.

Distribution of three major hepatitis C virus genotypes in Italy. A multicentre study of 495 patients with chronic hepatitis C.

Different genotypes of hepatitis C virus (HCV) have been shown to have distinct geographical distribution and to associate with variable clinical features. To evaluate their role in chronic hepatitis in Italian patients, we studied 495 consecutive cases with chronic hepatitis C seen in nine sentinel centres homogeneously distributed over Italy. HCV genotyping was carried out using a dot-blot hybridization assay with genotype-specific probes. Four hundred and eleven patients were viraemic and could be evaluated: 57% were found to be infected with HCV-1, 31% with HCV-2, 8% with HCV-3, 1% showed mixed infection and 3% were ascribed to HCV-2b or HCV-4 by direct sequencing. Geographical distribution showed discrete territorial variations. A history of drug addiction was commoner in patients infected with HCV-3. There were no significant differences in activity of liver disease among different HCV genotypes but the response to interferon therapy was reduced in patients infected with HCV-1 compared to HCV-2 or HCV-3.

Clinical and virological profiles in patients with multiple hepatitis virus infections.

BACKGROUND: The "in vivo" interplay between hepatitis B virus (HBV) and hepatitis C virus (HCV) both in terms of replication activity and cytopathic effect on liver cells is poorly understood. The aim of the study was to investigate their reciprocal influence in patients with HBV and HCV coinfection. METHODS: HBV and HCV genomic sequences in the serum and liver of 55 patients with chronic liver disease who were positive for anti-HCV and for markers of HBV were studied. RESULTS: Twenty-five hepatitis B surface antigen-positive patients, without markers of hepatitis D virus (HDV) infection, showed an inverse relation between seropositivity for HCV RNA and for HBV DNA (P < 0.001). HCV genomic sequences were detected in the liver of all patients positive for HBV DNA but negative for HCV RNA in serum. The biochemical activity and the histological severity of liver disease were lower in HCV RNA-positive/HBV DNA-negative patients, compared with HCV RNA (serum)-negative/HBV DNA-positive cases (P < 0.005). Nine of 10 patients with concurrent HDV infection were negative for serum and liver HCV RNA. None of 20 hepatitis B surface antigen-negative/HCV RNA-positive patients with antibodies to HBV had HBV DNA detectable in serum or liver. CONCLUSIONS: Our findings indicate a reciprocal inverse relation between HBV and HCV replication. Patients positive for antibody to HCV with antibodies to HBV usually have no evidence of HBV DNA persistence in the liver.

The preS1 domain of hepatitis B virus and IgA cross-react in their binding to the hepatocyte surface.

Using a solid-phase assay we have demonstrated specific competition between the preS1 sequence of hepatitis B virus and human IgA in their binding to isolated normal human liver plasma membranes, suggesting molecular mimicry. Monoclonal and polyclonal antibodies raised against virus and IgA epitopes were used to detect and map immunological cross-reactivity to the virus sequence involved in liver cell binding. These findings suggest the existence of a common receptor or of closely related receptors for the attachment of HBV and IgA to human liver cells.

Latent hepatitis B virus infection in childhood hepatocellular carcinoma. Analysis by polymerase chain reaction.

The presence of hepatitis B virus (HBV) has been evaluated in liver specimens from 11 children with primary liver tumors and negative results of serologic testing for HBV markers. HBV-DNA sequences were detected by the polymerase chain reaction procedure, using different sets of oligonucleotide primers from highly conserved regions of HBV genome. Two of three children with histologic diagnosis of hepatocellular carcinoma were positive for HBV-DNA in the liver, whereas the remaining children, including six patients with hepatoblastoma, one patient with hemangioma, and one patient with hamartoma, were negative. These findings support the hypothesis of a primary role of HBV in the development of hepatocellular carcinoma in children from nonendemic areas and without overt HBV infection.

Hepatitis B virus binds to peripheral blood mononuclear cells via the pre S1 protein.

The hepatitis B virus has been documented in hepatic and extrahepatic compartments, including bone marrow and peripheral blood cells. The viral protein involved in the attachment to human hepatocytes has been identified within the N-terminus of the pre S1 envelope protein. Using recombinant particles containing the pre S1, pre S2 and S encoded sequences, we studied which virus envelope protein is involved in binding to peripheral blood cells. Mononuclear cells of 20 healthy subjects bound 125I-labelled particles, with a S/N ratio higher than 2.5 (range 2.69-7.77). Binding was abolished by trypsinization. B lymphocytes and monocytes were found to bind viral particles much more efficiently compared to T cells and granulocytes. A monoclonal antibody (MA 18/7), recognizing the (27-49) pre S1 sequence, completely inhibited viral particle attachment to PBMC, while anti-pre S2 (Q 19/10) and anti-S (20/2) monoclonal antibodies had no effect. We conclude that the pre S1 sequence is involved in HBV attachment to PBMC and to hepatocytes. The nature of the cellular attachment site is unknown, but it might be a receptor for physiologic ligands, as occurs with other viruses.

Does hepatitis B virus play a role in primary liver cancer in children of Western countries?

Hepatitis B virus (HBV) infection was evaluated in serum and liver specimens of eight Italian children with primary liver cancer. All children were negative for HBV markers in serum but four of them showed HBV-DNA sequences and/or HBs antigen expression in the liver. In one case, viral DNA was present in both neoplastic and non-neoplastic tissue, while in one case HBV-DNA was detectable only in nontumoral tissue and in one case only in the tumor area. In these cases, scattered HBsAg was expressed in the cytoplasm of cells in normal part of the liver and in another case neoplastic cells secreted HBsAg in culture. In two cases, the histologic diagnosis was HCC; one case had mixed HPB and one had macrotrabecular HPB. All children were more than 1 year of age. The remaining four children were histologically diagnosed as HPB and were less than 1 year of age. These findings suggest that HBV may be a cofactor for the development of liver cancer also in children of Western countries and that the risk of infection progressively increases with age.

Identification of an attachment site for human liver plasma membranes on hepatitis B virus particles.

The surface antigen of hepatitis B virus (HBsAg) exposes three protein domains: preS1, preS2, and S. In a previous study we have shown that preS1 sequences expressed in transfected yeast cells bind specifically to plasma membranes of human liver. In this study we show that purified virus particles from a virus carrier bind also specifically to such membranes. Subviral HBsAg filaments which are rich in preS1 bind well too, while HBsAg 20-nm particles which contain small amounts of preS1 bind to a much lesser degree. The binding can be inhibited by a monoclonal antibody which recognizes a sequential epitope between amino acids 27 and 49 of the preS1 domain.

Serum HBV-DNA in anti-HBe positive patients detected by filter and liquid phase hybridization assays.

Serum HBV-DNA is considered the best parameter for monitoring HBV replication in the liver. The filter hybridization assay (spot test) with 32P-labelled HBV-DNA has been the technique more frequently used to date. A simple solution hybridization assay, in which 125I HBV-DNA is used as labelled probe, has been recently standardized. We have compared the performances of these two assays for the detection of HBV-DNA. The results were similar with the two methods: an agreement was found in 39/44 (89%) samples. Three sera were positive only by the spot assay-and two only by the liquid phase assay. However, in these cases, HBV-DNA levels were near the sensitivity limits of the assay. Therefore, the filter and the liquid phase assays can be considered to be suitable methods to monitor HBV replication, a fundamental index for the clinical assessment and prognosis of patients with HBsAg positive chronic hepatitis.

Hepatitis B virus DNA forms in the liver of chronically infected individuals. Relation to replication profile.

To analyze the profile of HBV-DNA forms in the liver in relation to different levels of virus replication, liver biopsies from 52 chronic HBsAg carriers were studied by Southern blot analysis. Quantitative evaluation of the major HBV-DNA molecules was carried out by densitometry in 27 patients with ongoing hepatitis B virus (HBV) replication in the liver. Significant variations in the intensity of the different bands were noted in individual cases, but statistical correlation between the amount of single stranded forms and levels of serum HBV-DNA was not observed. In contrast, the amount of linear 3.2 kb HBV-DNA appeared to have an inverse correlation with levels of circulating virions. Only the 3.2 kb form was detected in three patients negative for serum HBV-DNA. In these cases with 'inactive' state of episomic HBV genome seroconversion to anti-HBe occurred from 12 months before to 4 months after the time of liver biopsy testing. This 3.2 kb form can therefore be interpreted as a pattern of transition from the replicative to the non-replicative state of the virus.