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Post-resective liver failure is a frequent complication of liver surgery and it is due to portal hyperperfusion of the remnant liver and to arterial vasoconstriction, as buffer response of the hepatic artery. In this context, splenectomy allows a reduction of portal flow and increases the survival chance in preclinical models. SerpinB3 is over-expressed in the liver in oxidative stress conditions, as a mechanism of cell defense to provide survival by apoptosis inhibition and cell proliferation. In this study, the expression of SerpinB3 was assessed as predictor of liver damage in in vivo models of major hepatic resection with or without splenectomy. Wistar male rats were divided into 4 groups: group A received 30% hepatic resection, group B > 60% resection, group C > 60% resection with splenectomy and group D sham-operated. Before and after surgery liver function tests, echo Doppler ultrasound and gene expression were assessed. Transaminase values and ammonium were significantly higher in groups that underwent major hepatic resection. Echo Doppler ultrasound showed the highest portal flow and resistance of the hepatic artery in the group with > 60% hepatectomy without splenectomy, while the association of splenectomy determined no increase in portal flow and hepatic artery resistance. Only the group of rats without splenectomy showed higher shear-stress conditions, reflected by higher levels of HO-1, Nox1 and of Serpinb3, the latter associated with an increase of IL-6. In conclusion, splenectomy controls inflammation and oxidative damage, preventing the expression of Serpinb3.Therefore, SerpinB3 can be considered as a marker of post-resective shear stress.

The posttranslational modification of proteins critically influences many biological processes and is a key mechanism that regulates the function of the RNA-binding protein Hu antigen R (HuR), a hub in liver cancer. Here, we show that HuR is SUMOylated in the tumor sections of patients with hepatocellular carcinoma in contrast to the surrounding tissue, as well as in human cell line and mouse models of the disease. SUMOylation of HuR promotes major cancer hallmarks, namely proliferation and invasion, whereas the absence of HuR SUMOylation results in a senescent phenotype with dysfunctional mitochondria and endoplasmic reticulum. Mechanistically, SUMOylation induces a structural rearrangement of the RNA recognition motifs that modulates HuR binding affinity to its target RNAs, further modifying the transcriptomic profile toward hepatic tumor progression. Overall, SUMOylation constitutes a mechanism of HuR regulation that could be potentially exploited as a therapeutic strategy for liver cancer.

SerpinB3 is a cysteine protease inhibitor involved in several biological activities. It is progressively expressed in chronic liver disease, but not in normal liver. The role in vascular reactivity of this serpin, belonging to the same family of Angiotensin II, is still unknown. Our aim was to evaluate the in vivoand in vitroeffects of SerpinB3 on systemic and splanchnic hemodynamics.

Objective: To define long-term outcome in patients with chronic hepatitis C who remain viremic after sustained biochemical response to interferon-a therapy. Design: Prospective evaluation of an outpatient cohort. Setting: University hospital. Patients: 107 patients with chronic hepatitis C who maintained normal aminotransferase levels as long as 12 months after interferon-[alpha]therapy. Patients were followed prospectively for an additional 6 to 36 months. Measurements: Aminotransferase levels were monitored at 3-month intervals. Serum hepatitis C virus (HCV) RNA was tested by polymerase chain reaction before therapy, at the end of therapy, and 12 months after therapy. The HCV genotype was defined by spot hybridization using serum specimens obtained before treatment. Results: Hepatitis C virus RNA was detected in 27 (25%) patients with sustained biochemical response; 80 (75%) patients were negative for HCV RNA. Patients positive for HCV RNA were older (P < 0.001), had received a smaller interferon-[alpha] dose (P= 0.02), and were more frequently infected with HCV genotype 2 (P < 0.01). Liver histologic findings were active in 57% of patients positive for HCV RNA, despite normal alanine aminotransferase levels, compared with only 12% of patients who were negative for HCV RNA (P = 0.01). The estimated probability of hepatitis relapse by 4 years after therapy was 53% in viremic patients and 0% in patients negative for HCV RNA (P< 0 001). Conclusion: Patients with chronic hepatitis C should be tested for serum HCV RNA 1 year after a sustained biochemical response to interferon-[alpha] therapy to determine whether the response is complete and permanent., Patients infected with hepatitis C virus (HCV) who maintain normal liver functioning after treatment but who test positive for HCV at one year or less appear likely to relapse. Patients remaining virus-free at one year appear to be cured. Researchers periodically tested 107 patients who had maintained normal liver function for 12 months after hepatitis C treatment for the presence of HCV RNA in blood plasma. One-quarter of the group tested positive for HCV RNA, of whom half tested positive at the end of therapy and half became positive in the first year. All HCV-negative patients maintained normal liver function whereas 30% of the HCV-positive patients relapsed. The probability of relapse within four years was calculated to be 53% in patients testing positive within one year and 0% for patients remaining negative. The data suggest that all hepatitis C patients maintaining normal liver function should be tested at one year. Patients testing HCV-positive should be monitored for relapse.

Hepatitis is disease that causes inflammation of the liver. There are at least four different strains of hepatitis virus: A, B, C and D. When a person is infected with hepatitis virus, their blood will contain viral antigens (specific virus proteins) and viral DNA (genetic material). This is called the replicative or active stage of infection. After the infection subsides, antibodies (produced during active infection to attack the virus) are still present in the blood, but HBV DNA replication no longer takes place. This is known as the inactive stage of infection. Liver disease occurs in patients with active hepatitis B virus (HBV) infections. However, some patients in the inactive stage of HBV infection continue to have liver disease. The reason for this is unknown. Hepatitis C virus (HCV) has been identified as a leading cause of liver disease in patients who do not have active hepatitis A or hepatitis B infections. To examine the possibility that HCV may be responsible for the liver damage observed in patients who are in the inactive stage of HBV infection, blood samples from patients with HBV infection were tested for the presence of HCV. Antibodies against HCV were found in the blood of 11 (8 percent) of 136 patients with active HBV infection, and 7 (35 percent) of 20 patients with chronic inactive HBV infection. However, antibodies to HCV were not found in blood samples of patients who were 'healthy' carriers of HBV (people who have HBV antibodies, but no history of symptoms of hepatitis infection). These findings demonstrate that HCV is present in some patients who have HBV infection, and that HCV may be an important cause of liver damage in patients who are in the inactive stage of HBV infection. (Consumer Summary produced by Reliance Medical Information, Inc.)

Non-alcoholic steato-hepatitis (NASH) is a severe disease characterised by liver inflammation and progressive hepatic fibrosis, which may progress to cirrhosis and hepatocellular carcinoma. Clinical evidence suggests that in hepatitis C virus patients steatosis and NASH are associated with faster fibrosis progression and hepatocellular carcinoma. A safe and reliable non-invasive diagnostic method to detect NASH at its early stages is still needed to prevent progression of the disease. We prospectively enrolled 91 hepatitis C virus-positive patients with histologically proven chronic liver disease: 77 patients were included in our study; of these, 10 had NASH. For each patient, various clinical and serological variables were collected. Different algorithms combining squamous cell carcinoma antigen-immunoglobulin-M (SCCA-IgM) levels with other common clinical data were created to provide the probability of having NASH. Our analysis revealed a statistically significant correlation between the histological presence of NASH and SCCA-IgM, insulin, homeostasis model assessment, haemoglobin, high-density lipoprotein and ferritin levels, and smoke. Compared to the use of a single marker, algorithms that combined four, six or seven variables identified NASH with higher accuracy. The best diagnostic performance was obtained with the logistic regression combination, which included all seven variables correlated with NASH. The combination of SCCA-IgM with common clinical data shows promising diagnostic performance for the detection of NASH in hepatitis C virus patients.

Iron overload results in cellular toxicity, tissue injury, organ fibrosis and increased risk of neoplastic transformation. SerpinB3 is a serine protease inhibitor overexpressed in the liver in oxidative stress conditions, able to induce fibrosis and increased risk of malignant transformation. Aim of the present study was to assess the effect of iron overload on SerpinB3 expression in the liver using in vivo and in vitro models.The expression of Serpinb3 was assessed in the liver of hemojuvelin knockout mice (Hjv−/−), an established model of hereditary hemochromatosis, and of wild type control mice, following dietary or pharmacological iron manipulation. To assess the direct effect of iron in vitro, cell lines were treated with different concentration of hemin or with an iron chelator.Hepatic Serpinb3 mRNA and protein were highly expressed in Hjv−/− mice, but not in wild type controls fed with a standard diet. Serpinb3 became detectable in wild type mice fed with a high iron diet or injected with iron dextran; these treatments further induced Serpinb3 expression in Hjv−/− mice. Livers expressing Serpinb3 showed a positive staining also for HIF-2α in the same areas. Hemin promoted induction of SerpinB3 mRNA in HeLa and HA22T/VGH cells, but a mild stimulation of SerpinB3 promoter activity in HeLa and Huh7 cells. In conclusion, Serpinb3 is strongly induced by iron in the mouse liver. The molecular link between iron, ROS and SerpinB3 seems to be HIF-2α, which is induced by iron overload and was previously found capable of up-regulating SerpinB3 at the transcriptional level.

Background Cirrhosis represents the end stage of chronic liver disease, characterized by high mortality and morbidity. The prevalence of liver disease is difficult to assess, given its clinical latency up to the late stage.Objective We aimed to assess the prevalence of unrecognized chronic liver disease and cirrhosis using surrogate indicators from medical records of family physicians.Methods Medical records of 139,104 subjects, collected from 99 family physicians of the Veneto region, were used. Persistently high transaminases were used as indicators of occult chronic liver disease; thrombocytopenia, unrelated to haematological malignancies, was used as indicator of occult cirrhosis. Diagnosis of chronic liver disease and cirrhosis was assessed using ICD9-CM-1997 codes.Results Alteration of transaminases was found in 32.7% of the subjects, and among them only one-third had an already diagnosed liver disease. Patients with diagnosis of cirrhosis were 0.3%, while thrombocytopenia, indicator of occult cirrhosis, was detected in 1.3% of the remaining population. Patients with overt and occult cirrhosis showed a higher metabolic profile, with significantly higher prevalence of arterial hypertension, obesity and diabetes than the general population.Conclusion A large proportion of patients with chronic liver disease is still undiagnosed. Surrogate biochemical indicators might be useful for disease recognition.

Aim: this study evaluates the analytical and clinical performances of a new technology, CompleXima HCC Biochip, for the simultaneous serum measurement of alpha-fetoprotein-IgM (AFP-IgM) and squamous cell carcinoma antigen-IgM (SCCA-IgM). Methods: AFP- and SCCA-IgM were measured by both ELISA and CompleXima HCC Biochip in 39 blood donors and in 174 patients (102 liver cirrhosis – LC – and 72 hepatocellular carcinoma – HCC). Results: the intra-assay coefficients of variation were lower than 12% and the inter-assay variations comprised between 14% and 21%. The linearity interval for the CompleXima HCC Biochip was 50–300 AU mL−1for AFP-IgM and SCCA-IgM. The comparison between the prototype and the ELISA test was studied by using the Bland–Altman method and Passing–Bablok regression analyses. Passing–Bablok showed that the Biochip under-estimated AFP-IgM (Intercept A: −165.06; 95% CI: −313.11 to −51.32) and overestimated SCCA-IgM (Intercept A: 26.83; 95% CI: 14.47–35.86) with respect to ELISAs. Both biomarkers were higher in LC and HCC with respect to controls (p< 0.001) with no difference between LC and HCC (p= 0.864 for AFP-IgM and p= 0.214 for SCCA-IgM). The thresholds for AFP-IgM and SCCA-IgM were calculated by means of ROC curves, fixing the specificity at 95%. The sensitivity of AFP-IgM and SCCA-IgM associated with CompleXima in detecting patients with liver diseases was 47% and 46%, respectively. The combined evaluation of macrocomplexes with CompleXima in diagnosing HCC with respect to LC was associated with a sensitivity of 51.4% and a specificity of 48%. Conclusions: AFP-IgM and SCCA-IgM increase in chronic liver disease. The prototype CompleXima HCC Biochip allows their measurement with a good analytical reproducibility.

Purpose Germline nonsense and frameshift mutations in the adenomatous polyposis coli (APC) gene are found in approximately 90% of individuals affected by familial adenomatous polyposis (FAP) and a genotype-phenotype relationship has been observed. Missense mutations have also been found in a few cases, even if their role in FAP is still unknown. An association between a missense mutation, APC I1307K, and the risk of sporadic colorectal cancer (CRC) has been reported. In order to improve the knowledge about the genetic effect of APC I1307K on the phenotype, we tried a new approach using matrix-assisted laser desorption/ionization mass spectrometry (MALDI/MS).Experimental design An APC mutation (I1307K) was found in an index case of a non-Jewish woman and her son with attenuated familial adenomatous polyposis (A-FAP) and no family history of cancer. In order to evaluate whether the presence and abundance of the ionic species are related to the presence of cancer or the presence of mutation, comparative analyses of 11 healthy clean-colon subjects, 59 patients with CRC (stage II n=19, stage III n=23, stage IV n=17) without polyps, and 9 FAP patients, carriers of a nonsense mutation in the APC gene, were evaluated.Results Comparative analysis of serum protein profiles of the index patient and her healthy son, FAP and sporadic CRC patients, and subjects with preneoplastic lesions showed a characteristic abundance of ionic species at m/z 905, which was not present in healthy controls. Two peptides were identified from MALDI/MS/MS spectra of m/z 905 belonging to the kininogen-1 precursor and the human forkhead box protein 01A (FOXO1A). FOXO1A was present in only two subjects carrying I1307K, but not in other patients.Conclusions Our findings seem to suggest a relationship between m/z 905, FOXO1A and the development and growth of colorectal cancer. FOXO1A fragment determination in serum with MALDI/MS might be a promising approach for early detection of colon carcinoma or for the development of targeted therapies.

Background Hepatitis C virus (HCV) is believed to exert a suppressive effect on hepatitis B virus (HBV) in most HBV/HCV-coinfected patients; once HCV is cured by interferon-based therapy, these patients may show HBV reactivation. However, recent evidence revealed that the virological status in HBV/HCV-untreated individuals may vary over time and may show fluctuating profiles.Methods To evaluate the behaviour of apparently inactive HBV infection in patients under treatment for a concurrent HCV infection, we performed a prospective study that evaluated nine consecutive patients (eight males with a median age of 45.9 years, and one female aged 62 years) longitudinally followed-up with bi-monthly evaluation of HBV/HCV viraemia levels and liver biochemistry during a 1-year treatment with interferon plus ribavirin.Results In seven cases the HBV infection maintained its inactive status independently of the HCV response to therapy. By contrast, two non-responder cases with persistently high HCV RNA levels showed HBV DNA flairs during the follow-up, indicating a status of active HBV infection with fluctuating virological profiles.Conclusions This study suggests that the HBV behaviour may be independent of the HCV activity during anti-HCV therapy in HBV/HCV-coinfected patients, and that the HBV virological profile should be monitored to recognize possible reactivations that might lead to more proper therapeutic choices or adjustments.

The relation between HCV core antigen and HCV RNA has been confirmed in patients with chronic hepatitis C and a parallel behavior of the two markers has been described in early kinetics analysis during antiviral therapy. Variations of the core antigen to HCV RNA ratio have been reported in individual patients and the existence of nucleocapsid particles, not always associated with viral genomes, have been described. To assess the characteristics of HCV core antigen reactivity in relation to viremia in patients with different clinical profiles, 233 patients with chronic hepatitis C were studied serially. Group A included 54 asymptomatic HCV carriers, group B included 8 viremic patients with biochemical long‐term response after antiviral therapy, while group C was composed of 171 patients with chronic liver disease and 75 were treated with combination therapy. Core antigen levels were not significantly different in the three groups of patients and a wide range of antigenic reactivity was observed in individual patients. A close relationship was observed between core antigen and HCV RNA, although their ratio was significantly higher in biochemical long‐term responders (group B), compared to the other groups (P< 0.05). Physicochemical characterization of core antigen reactivity by equilibrium CsCl density gradient identified two distinct peaks migrating at 1.08–1.12 g/ml CsCl density and at 1.18–1.31 CsCl density, respectively. The first one, corresponding to the lipid‐associated fraction, contained higher amounts of core antigen reactivity and was associated with clinical remission of liver damage, while the second peak, corresponding to naked nucleocapsids, was observed mainly in sera with active disease. In conclusion, a close relationship between core and HCV RNA was documented both in treated and untreated patients. The finding of an excess of lipid‐associated core particles in a subset of viremic patients without biochemical activity of liver disease suggests their protective effect in liver cell damage. J. Med. Virol. 76:55–60, 2005. © 2005 Wiley‐Liss, Inc.

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 α-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.

Background & Aims:The NS5A and the E2 proteins of hepatitis C virus (HCV)-1b can bind and inhibit in vitro the interferon (IFN)-induced cellular kinase PKR. The role of such interaction in modulating the antiviral effect of IFN is still controversial. We have analyzed the E2 and the NS5A sequences in HCV-1b–infected patients treated with IFN to assess whether and how different combinations of wild-type and mutant proteins correlated with early and long-term virological response. Methods:In 30 patients, sequences of pretreatment and on-treatment E2-PePHD and NS5A-PKR binding domain (including the putative ISDR) were analyzed in parallel by sequencing cDNA–polymerase chain reaction products and up to 25 independent clones. Results:The E2-PePHD sequence was highly conserved with a homogeneous quasispecies and was identical in 29 of 30 cases with no association with the pattern of response and no evidence of evolution during therapy. Patients with a mutated NS5A-ISDR had a higher rate of early virological response (67%) than cases with wild-type ISDR (17%). This association was lost in long-term responders (33% vs. 17%). Conclusions:Although the highly conserved E2-PePHD motif might contribute to reduce IFN responsiveness, variations within this region do not seem to play a role in modulating IFN sensitivity. The NS5A-ISDR sequence influenced the early, but not the sustained response, to IFN, suggesting that other factors may be more important for the long-term outcome of therapy.

ABSTRACTAlpha interferon (IFN-a) treatment is effective on a long-term basis in only 15 to 25% of patients with chronic hepatitis C. The results of recent trials indicate that response rates can be significantly increased when IFN-a is given in combination with ribavirin. However, a large number of patients do not respond even to combination therapy. Nonresponsiveness to IFN is characterized by evolution of the hepatitis C virus (HCV) quasispecies. Little is known about the changes occurring within the HCV genomes when nonresponder patients are retreated with IFN or with IFN plus ribavirin. In the present study we have examined the genetic divergence of HCV quasispecies during unsuccessful retreatment with IFN or IFN plus ribavirin. Fifteen nonresponder patients with HCV-1 (4 patients with HCV-1a and 11 patients with HCV-1b) infection were studied while being retreated for 2 months (phase 1) with IFN-a (6 MU given three times a week), followed by IFN plus ribavirin or IFN alone for an additional 6 months (phase 2). HCV quasispecies diversification in the E2 hypervariable region-1 (HVR1) and in the putative NS5A IFN sensitivity determining region (ISDR) were analyzed for phase 1 and phase 2 by using the heteroduplex tracking assay and clonal frequency analysis techniques. A major finding of this study was the relatively rapid evolution of the HCV quasispecies observed in both treatment groups during the early phase 1 compared to the late phase 2 of treatment. The rate of quasispecies diversification in HVR1 was significantly higher during phase 1 versus phase 2 both in patients who received IFN plus ribavirin (P= 0.017) and in patients who received IFN alone (P= 0.05). A trend toward higher rates of quasispecies evolution in the ISDR was also observed during phase 1 in both groups, although the results did not reach statistical significance. However, the NS5A quasispecies appeared to be rather homogeneous and stable in most nonresponder patients, suggesting the presence of a single well-fit major variant, resistant to antiviral treatment, in agreement with published data which have identified an IFN sensitivity determinant region within the NS5A. During the entire 8 months of retreatment, there was no difference in the rate of fixation of mutation between patients who received combination therapy and patients who were treated with IFN alone, suggesting that ribavirin had no major effects on the evolution of the HCV quasispecies after the initial 2 months of IFN therapy.

There is limited information about the long-term efficacy of prolonged therapy (more than 6 months) with interferon a in hemophilic patients with chronic hepatitis C who are not coinfected with the human immunodeficiency virus (HIV-1). One hundred and seven hemophiliacs were randomly assigned to 3 million U of interferon a2b three times weekly for 12 months or no therapy. The patients were followed up for at least 12 months posttreatment. Response was assessed by both serial alanine aminotransferase (ALT) levels and hepatitis C virus (HCV)-RNA measured by reverse transcribed polymerase chain reaction (RT-PCR) method. Before treatment, serum levels of HCV-RNA were measured quantitatively by second-generation branched-DNA assay and the HCV genotype was determined by RT-PCR. Serum HGV-RNA, a marker of infection with the hepatitis G virus, was also measured by RT-PCR. Normalization of ALT was sustained and serum HCV-RNA was cleared in 6 of 45 treated patients, compared with none of the 50 untreated controls (13% v 0% P < .01). Low pretreatment viremia was the only feature that was associated with an increased likelihood of sustained response (P < .01). This study shows that multitransfused hemophiliacs with chronic hepatitis C not coinfected with HIV-1 respond at low rates to prolonged interferon therapy.

The pattern of display of envelope proteins on HBV indicates that the preS domains have an important role in virus assembly and secretion. Furthermore sites of interaction with the membrane of hepatocytes and of other cells which are susceptible to infection have been identified within the preS1 and preS2 protein domains. These observations support the concept that these sequences represent the virus attachment sites for the host cell membrane. The nature of the cellular receptors involved in this binding has not yet been fully clarified. Several B and T cell epitopes, which evoke antibody and cellular response in humans, have been identified within the preS1 and preS2 protein domains. The immune response to some of these determinants seems to be particularly relevant in virus neutralization and in termination of virus replication. Their exact characterization is needed for possible inclusion in recombinant HB vaccines, to further increase their efficacy. The immune response to preS may also be involved in the pathogenesis of liver disease, as preS1 and preS2 epitopes are recognized by cytotoxic T lymphocytes during chronic infection and after HB vaccination. These findings need to be extended further and may provide new important information on the pathogenesis of HBV infection.

During a prospective study of acute symptomatic viral hepatitis, started in 1978, 664 consecutive adult patients, including 223 drug abusers, fulfilled the diagnostic criteria (anti-HBc IgM positivity) for acute type B hepatitis. In order to evaluate the outcome of the disease, 443 patients were followed for up to 12 months after the onset. 2.4% of the infections became chronic; the rate did not significantly differ between drug addicts and non-drug abusers, suggesting that chronic hepatitis is a rare complication of acute symptomatic hepatitis type B. Ongoing liver damage after clearance of HBsAg from serum was observed in drug abusers only (14% of the cases). Clinical, biochemical and virological features of the acute phase in patients with ongoing infection were compared with those of uncomplicated cases. Anicteric hepatitis and lower transaminase values were significantly (p<0.05) associated to a chronic evolution of the disease, as well as a higher prevalence of HBV-DNA, DNA polymerase and HBcAg positivity in serum. Testing HBV-DNA and DNA polymerase early in the course of the infection appeared to be of high predictive value for the subsequent outcome of the illness. Im Verlauf einer 1978 begonnenen, prospektiven Studie über die akute symptomatische B-Virus-Hepatitis wurde die Diagnose bei 664 Patienten gesichert (anti-HBc IgM-positiv); in dieser Gruppe waren 223 Drogensüchtige und 441 Nicht-Drogensüchtige. Verlaufsbeobachtungen erfolgten bei 443 der Patienten bis zu 12 Monate nach Ausbruch der Krankheit. 2,4% der Infektionen nahmen einen chronischen Verlauf, dabei bestanden zwischen den Drogensüchtigen und Nicht-Drogensüchtigen keine signifikanten Unterschiede. Dies deutet darauf hin, daß die akute symptomatische B-Virus-Hepatitis nur selten in die chronische Form übergeht. Ein Fortschreiten der Leberschädigung nach dem Verschwinden von HBsAg aus dem Serum wurde nur bei Drogensüchtigen (14% der Fälle) beobachtet. Die klinischen, biochemischen und virologischen Parameter der akuten Krankheitsphase wurden bei Patienten mit persistierender HBV-Infektion und bei Patienten mit unkompliziertem Verlauf verglichen. Eine anikterische Hepatitis und niedrigere Transaminasenwerte sowie eine höhere Prävalenz der HBV-DNA, DNA-Polymerase und HBcAg-Positivität waren signifikant mit einem chronischen Verlauf assoziiert (p<0,05). Die Testung der HBV-DNA und DNA-Polymerase in der Frühphase der Infektion hat sich als sehr wertvoll für die Prognose des weiteren Krankheitsverlaufes erwiesen.

To analyze interleukin-2-dependent immunoregulatory function in hepatitis B virus infection and in other forms of viral hepatitis, levels of soluble interleukin-2 receptors (sIL-2R) were measured by an enzyme-linked assay in sera from patients with acute and chronic viral hepatitis of different etiology. Increased sIL-2R levels were detected in the early phase of acute hepatitis type A and type B, but not during acute non-A, non-B hepatitis. Among 46 patients with chronic hepatitis B virus infection, levels of sIL-2R were significantly increased only in cases with chronic active hepatitis, while they were about normal in chronic persistent hepatitis or in healthy carriers of the infection. These differences were independent of virus replication, being maintained when patients were stratified according to HBeAg/anti-HBe status and to serum HBV-DNA. Nine patients with chronic active hepatitis type B and high sIL-2R levels at presentation were followed prospectively for two to eight years, and in HBeAg -positive patients, the behavior of receptor levels closely paralleled disease activity. These results, which may reflect increased shedding of IL-2R by activated T lymphocytes in patients with active destruction of HBV infected hepatocytes, indicate the usefulness and potential prognostic importance of serum slL-2R determination in patients with chronic viral hepatitis. Patients with chronic non-A, non-B hepatitis had much lower sIL-2R levels, although their liver disease was similar to hepatitis B cases, suggesting that different pathogenetic mechanisms operate in these patients.

A double-blind, randomized, controlled trial has been undertaken to evaluate treatment of chronic hepatitis type B with levamisole. Ten patients received levamisole (150 mg/day, 3 days/wk) and 10 received placebo until seroconversion to antibody to hepatitis B e antigen eventually occurred, or for a maximum of 18 mo. Final evaluation at 24 mo after starting treatment revealed that 60% of the patients in the levamisole group had become hepatitis B e antigen negative, 90% were hepatitis B virus—deoxyribonucleic acid negative in serum, and 8 of 9 (89%) patients had cleared hepatitis B core antigen from the liver. On the other hand, in the placebo group only 4 of the 10 subjects (40%) were hepatitis B e antigen and hepatitis B virusdeoxyribonucleic acid negative in serum and 3 of 8 (37.5%) of them became hepatitis B core antigen free in the liver. Moreover, in 8 patients of the treated group and in 4 of the control cases aminotransferase activities fell into the normal range. A liver biopsy specimen was obtained after treatment in 17 patients and 7 of 9 levamisole recipients showed marked improvement in hepatic histology, compared with 3 of 8 placebo recipients. These data show that patients treated with long-term levamisole therapy have a tendency toward normalization of aminotransferase activities and suppression of hepatitis B virus replication, suggesting that the drug may be of benefit in chronic hepatitis B e antigenpositive hepatitis.

A great deal of information on the molecular heterogeneity of hepatitis C virus (HCV) has been achieved since its discovery in 1989. However, little is known about the clinical significance of these variations.Based on the degree of sequence variation, HCV has been classified into six major groups or types, differing by 31–34% at the nucleotide level over the entire virus genome. Each type is divided into several subtypes that differ by 20–23% in nucleotide sequence. Viruses within the same subtype are up to 10% divergent and, within infected individuals, vary by up to 1.5%. Genotype distributions are not homogeneous around the world and may reflect both historical and recent parenteral routes of transmission. The clinical implication of these genomic variations are not yet fully elucidated: genotype 1b has been associated with end-stage liver disease, including liver cirrhosis and hepatocellular carcinoma, but this finding might rather reflect its earlier introduction to the populations studied. Consistent evidence exists that types 2 and 3 have a higher response rate to interferon treatment than type 1, although the interplay between genotype and viral load in determining the response is still unclear. Immunohistochemical studies indicate a stronger activation of the endogenous interferon system in the liver of patients infected with type 1 compared to those infected with types 2 and 3, explaining, at least in part, its low responsiveness to exogenous interferon treatment. Biological, sequence-dependent variations of genotypes have been poorly investigated to date, but differential efficiency of translation activity of the 5′ non-coding region has been reported. The availability of ‘in vitro’ systems for evaluating pathogenetic aspects and neutralization mechanisms will improve the present knowledge on this world-wide infectious disease and on the clinical usefulness of distinguishing between genotypes.

The aim of this study was to ascertain prevalence and natural history of hepatitis C virus (HCV) infection in a large cohort of patients cured of childhood leukemia who had been followed prospectively for liver disease for at least 10 years since chemotherapy withdrawal: 114 consecutive patients entered the study. Liver function tests and ultrasonography were used to assess presence of liver disease. Patients were tested for antibody to HCV and for serum HCV-RNA at the end of chemotherapy and at the end of follow-up. At chemotherapy withdrawal, 56 patients (49%) were HCV-RNA positive, often without detectable anti-HCV, and in these cases, transaminase levels were more elevated during (P = .08) and after (P = .04) chemotherapy compared with HCV-RNA negative cases. Patients were then followed-up 13 to 27 years (mean, 17) after chemotherapy withdrawal. During this period, 38 initially anti-HCV negative patients seroconverted to anti-HCV and 17 initially anti-HCV positive cases lost reactivity. Forty patients were persistently HCV-RNA positive in serum, while 16 initially viremic patients became HCV-RNA negative during follow-up. At the end of the observation period, a persistent transaminase elevation was detected only in four HCV-RNA positive and anti-HCV positive cases, while no patient developed signs or symptoms of decompensated liver disease. Thus, hepatitis C was a frequent finding in long-term survivors after chemotherapy. It was associated with an atypical serologic profile and did not cause severe liver impairment over a period of 13 to 27 years.

Recombinant DNA techniques have recently contributed a great deal of informations on hepatitis B virus (HBV) infection. Serum HBV-DNA appeared as the most sensitive marker of viral replication activity both in hepatitis Beantigen (HBeAg)-positive and in anti-HBe-positive patients. In the latter group, a significant correlation between serum viral DNA positivity and liver disease activity was present. In our experience, more than 50% of anti-HBe-positive cases with chronic liver disease showed circulating HBV-DNA, while none of healthy HBsAg chronic carriers was found positive for serum HBV-DNA. In type B acute hepatitis, viral nucleic acid sequences were detectable only in a small number of uncomplicated cases, but were observed in all the patients who progressed to chronic hepatitis. HBV-DNA represents therefore an early and useful prognostic parameter in acute infection. Several epidemiological studies have established a striking correlation between HBV infection and development of hepatoma. Using molecular hybridization techniques, viral DNA has been identified in liver cancer cells. Finally, HBV-DNA has also been identified in the pancreas, kidney, skin, bile ducts and in cells of the vascular system. In addition, the presence of viral genome has been recently identified in circulating lymphocytes of patients with acute or chronic HBsAg-positive hepatitis. These findings add further informations to the understanding of viral biology and of virus-host interactions in the natural history of the infection and associated liver disease.

The envelope gene of hepatitis B virus (HBV) consists of a large open reading frame which codes 3 different polypeptides by the variable use of 3 contiguous regions: the pre-S1, pre-S2 and S regions. The protein coded by the entire sequence (L protein) is identified by its unique pre-S1 epitopes and has a role in virus assembly and secretion. Pre-S1 antigens are expressed preferentially on virions and are less abundant, but present, on subviral particles. The pre-S2 sequence contains a species-specific receptor for polyalbumin, which has been implicated in virus binding to hepatocytes. Methods have been recently developed to measure pre-S1 and pre-S2 levels in serum and available data indicate that these assays may be useful in prognostic assessment of acute hepatitis B and to measure virus replication in chronic infection, although further studies are certainly needed to define specificity and sensitivity compared to conventional HBV markers. Both pre-S1 and pre-S2 are highly immunogenic and elicit anti-pre-S antibodies. Methods for detection of anti-pre-S in human sera have proved difficult to be developed due to unspecific reaction of serum components with pre-S sequences and epitope variability of the immune response in humans. Anti-pre-S2 seems a marker of recovery from acute infection, while evidence of its involvement in liver damage is weak. Both anti-pre-S2 and anti-pre-S1, but we have limited information on the latter, may be involved in virus neutralization. These issues are particularly relevant for future HB vaccine development, as it is proposed that inclusion of strategic pre-S sequences could increase efficacy.

Polymerized human serum albumin (pHSA) binding activity due to antibodies was evaluated by a sensitive and specific enzyme-linked immunosorbent assay (ELISA) in well characterized cases of acute (AVH) and chronic active (CAH) viral hepatitis. Moreover, hepatitis B virus (HBV) infection serum samples were tested for the presence of HBsAg-associated pHSA receptors detected by radioimmunoassay. Specificity of ELISA in detecting pHSA binding activity due to antibodies was assayed by testing IgG fractions from positive serum samples and purified HBsAg particles. A very high prevalence (mean=87%) of antibody response to pHSA was detected in serum samples of patients with type B, A and post-transfusion non-A, non-B (NANB) AVH and CAH. IgM antibodies were found in 75% of type B AVH, in 77% of type A AVH and in none of the NANB cases at the onset of illness. In CAH, IgM antibodies were found in 37.5% of HBsAg-positive cases and in 44% of NANB cases. In HBV-related AVH and CAH no correlation was found between anti-pHSA antibodies and HBsAg-associated pHSA receptors or the HBeAg/anti-HBe status. The antibody response detected by ELISA showed a cross-reactivity with monomeric human albumin and heterologous polymeric and monomeric albumins. However, in HBV infection the antibody binding was significantly higher for pHSA than for monomeric human albumin or albumins from other species, thus suggesting that the antigenic sites of pHSA evoking the antibody response may differ in relation to the etiology of viral hepatitis.

In this report we studied a 35-year-old male who developed an abnormal expansion of granular lymphocytes (GL) which spontaneously regressed over a period of 2 years. The immunological and molecular characterizations of expanded cells showed the CD3+,CD8+,HNK-1+ phenotype, a polyclonal organization of the T-cell receptor ß-chain gene and normal natural killer activity. At the time of presentation, spot and blot hybridization techniques revealed the presence of viral hepatitis B virus (HBV) DNA sequences only in highly enriched CD4+ T cells, while proliferating GL were negative. With this as a background, we addressed the question of whether in our case the polyclonal GL proliferation represented an immunoreactive response against CD4+ infected cells. In particular, we tested the possibility that expanded GL could be cytotoxic against autologous infected CD4+ cells. At the time of the first determination, when several of the CD4+ cells harbored HBV, GL showed a minimal degree of cytotoxicity against51Cr-labeled CD4+ cells; 2 years later, when GL became capable of lysing these targets, the appearance of the specific cytotoxicity was concomitant with the disappearance of the HBV-infected CD4+ cells and with the recovery of granular lymphocytosis. Taken together, our data suggest that in this case GL proliferation could represent an immunoreactive process against CD4+ cells.

The detection of serum hepatitis B virus (HBV) DNA in 32 chronic HBsAg carriers was performed by spot hybridization technique using both biotinylated and radiolabeled probes, in order to compare their specificity and sensitivity. Our results show that both assays are specific since neither evidence of cross-hybridization between HBV DNA and sera from patients with chronic non-A, non-B (NANB) hepatitis was found, nor HBV DNA was detected both in patients with chronic anti-HBs/anti-HBc-positive hepatitis and in patients negative for all HBV markers. An agreement between the two assays was observed in 94% of the tested sera. Even though in 5 serum samples (6%) low levels of HBV DNA (0.1–1 pg/100 µl) remained undetected using the biotin-labeled probe, the lower detection limit of the two assays (0.1 pg of HBV DNA) was the same using purified Dane particles as control. This study indicates that the enzymatic detection of HBV DNA is suitable for routine and rapid monitoring of HBV replication in both HBeAg-and anti-HBe-positive patients, as well as for a semiquantitative analysis of serum HBV DNA.

Severe liver damage revealed by a sharp transaminase elevation may be seen in patients with leukemia. This may be due to several possible causes, including viral hepatitis, chemotherapy-induced hepatotoxicity and leukemic infiltration. HCV infection may be suspected to play a relevant role as these patients are often heavily transfused after the onset of their hematologic disorder. We have therefore examined the role of HCV in 15 children with leukemia who developed severe liver damage shortly after the diagnosis of leukemia. All patients were tested for HCV-RNA by the polymerase chain reaction at the time of peak SGPT elevation and for anti-HCV on serial serum samples taken thereafter. Only one patient (6.6%) showed hepatitis C viremia and none developed confirmed anti-HCV positivity during follow-up, suggesting that HCV had not played a major role in causing these severe episodes of liver necrosis. This is in agreement with observations made in non-immunocompromised patients in whom fulminant hepatitis is only exceptionally due to HCV.

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. J. Med. Virol. 54:7–11, 1998.© 1998 Wiley‐Liss, Inc.

Introduction Biomarkers for early hepatocellular carcinoma (HCC) detection are still a clinical need. Recent data indicate that cancer-associated antigens lead to the formation of circulating IgM-linked immune complexes as the result of natural IgM production by the innate immune response. The SCCA-IgM immune complex has already been described for primary liver cancer detection. Osteopontin (OPN) is a member of the SIBLING family of proteins recently shown to be related to tumorigenesis, progression and metastasis in various cancer types.Aim To evaluate the serum levels of the OPN-IgM complex in comparison to the SCCA-IgM complex in patients with HCC.Patients A total of 256 patients were analyzed including 151 patients with HCC (M/F 115/36; mean age ± SD: 67 ± 12 years) and 106 patients with cirrhosis (M/F 68/38; mean age ± SD: 62 ± 12 years). HCC, tested before any therapeutic approach, was mainly of viral etiology (58% HCV, 11% HBV), while alcohol abuse (22%) was the main risk factor for the remaining cases. A similar distribution was found in patients with cirrhosis (46% HCV, 15% HBV, 38% alcohol).Methods Serum levels of OPN-IgM were measured using a homemade ELISA assay with a polyclonal anti-human OPN antibody (Biodesign Int, USA). SCCA-IgM was detected in serum using an ELISA assay kit (Hepa-IC, Xeptagen).Results OPN-IgM was positive in 64/151 (42%) patients with HCC and in 45/106 (42%) patients with cirrhosis (specificity 58%), while the reference biomarker SCCA-IgM showed 35% sensitivity (49/139) and 71 % specificity. When patients were stratified on the basis of etiology, the sensitivity values for OPN-IgM and SCCA-IgM were 51% vs 44% in HCV patients, and 36% vs 23% in the other patients, while the specificity was lower for OPN-IgM (55% vs 69% in HCV patients; 62% vs 72% in the other patients). Combination of the two biomarkers resulted in an increase in sensitivity to 63% for viral etiology and to 40% for nonviral etiology.Conclusion Different IgM-linked biomarkers are detectable in primary liver cancer. OPN-IgM and SCCA-IgM showed a similar behavior, while the combination of these biomarkers increased the diagnostic performance for primary liver cancer.

Introduction SCCA1 and SCCA2 belong to the family of serine protease inhibitors (SERPIN). Both SCCA isoforms have been found to be increased in patients with squamous cell carcinoma (SCC) of different organs. Recent findings have demonstrated that SCCA-IgM immune complexes are a novel class of biomarkers for primary liver cancer detection. An increase in SCCA-IgM levels in patients with cirrhosis is associated with the development of liver cancer.Aims and methods To evaluate the diagnostic accuracy of different isoforms of SCCA-IgM compared with a commercial assay for SCCA-IgM in patients with hepatocellular carcinoma (HCC). Twenty-six patients with HCC (M/F 1 9/7; mean age ± SD: 65 ± 13 years) were analyzed. Serum samples from 28 blood donors (M/F 17/11; mean age ± SD: 38 ± 9 years) were used as control. Two monoclonal antibodies, SCC 111 and SCC 1 04 (CanAg Diagnostics, Gothenburg, Sweden) were used as catcher antibodies to perform specific ELISA for SCCA1-lgM and SCCA2-lgM. SCCA-IgM immune complexes were also detected in serum by a commercial ELISA kit (Hepa-IC, Xeptagen, Marghera, Italy) according to the manufacturer's instructions.Results Circulating SCCA1-IgM was detectable in 6 out of 26 patients (sensitivity 23%), while SCCA2-lgM ELISA was positive in 9 of 26 patients (sensitivity 35%). When SCCA-IgM levels were measured using the commercial assay the diagnostic sensitivity increased to 58% (15/26). The SCCA1-IgM, SCCA2-lgM and SCCA-IgM tests had 100% specificity in healthy controls.Conclusions The commercial assay allowed the detection of both isoforms of SCCA forming the IgM immune complexes, providing a better diagnostic sensitivity score than that obtained with the SCCA1-IgM or SCCA2-lgM test.

Background Survivin, also known as BIRC5, is the smallest member of the mammalian IAP family and is a well recognized inhibitor of apoptosis with an important role in cell-cycle regulation. It is detected in fetal and neoplastic adult tissue, but not in normal tissues. Survivin acts on cancer promotion not only by the inhibition of apoptosis but also by acceleration of the proliferative activity of cancer cells and several papers have suggested the involvement of this protein in hepatocarcino-genesis. It has been reported that the detection of HCC biomarkers circulating as IgM immune complexes (ICs) improved the diagnosis and prognosis of HCC. The aim of this study was to compare the expression of survivin as an IgM immune complex in serum from healthy controls and of patients with cirrhosis and patients with HCC to identify a novel biomarker for the monitoring of liver diseases.Methods Serum levels of survivin-IgM from 1 97 individuals, including 39 healthy subjects, 94 patients with cirrhosis and 64 with HCC, were measured by ELISA and the relationship with clinical parameters was evaluated.Results Survivin-IgM was almost undetectable in sera from healthy subjects, high in patients with cirrhosis, and moderately lower in patients with HCC. The survivin-IgM assay was positive in 62 of 94 patients with cirrhosis (66%) and in 28 of 64 patients with HCC (43.7%) using a cut-off of 264.89 AU/mL (specificity of 94%). Statistical analysis showed that IC values were significantly different between groups (cirrhosis versus healthy control group, p<0.001; HCC versus cirrhosis group, p<0.001). Circulating survivin-IgM ICs in patients with HCC were lower than in patients with cirrhosis; in fact, the statistical significance in comparison with healthy subjects was lost. On the other hand, the concentration of circulating survivin-IgM ICs was found to increase with progression of Child score.Conclusions The high expression of survivin-IgM in sera from patients with cirrhosis and the values of sensitivity indicate that survivin-IgM could be a novel candidate biomarker for cirrhotic disease. Furthermore, the increase in ICs with the progression of Child score seems to promote the survivin-IgM immune complex as marker of liver failure. Survivin-IgM was found to be lower in sera from HCC patients. Follow-up studies are in progress to monitor patients with cirrhosis and to validate the association of the downregulation of this marker with progression towards hepatocellular carcinoma.

Background and aim Circulating immune complexes of alpha-fetoprotein (AFP) and SCCA (squamous cell carcinoma antigen) with IgMs (AFP-IgM and SCCA-IgM, respectively) represent a promising new class of serum markers with diagnostic and prognostic value in the management of patients with hepatocellular carcinoma (HCC). The enhancement of diagnostic indexes for cancer detection achieved with combination of biomarkers by linear logistic regression has been demonstrated in several simulation studies on multiple diagnostic assays. The aim of the study was to evaluate the improvement of the diagnostic accuracy by combination of SCCA-IgM and AFP-IgM compared with the diagnostic accuracy of free AFP in sera of patients with HCC and cirrhosis.Patients and methods Serum samples from 81 patients with HCC (mean age ± SD: 63 ± 8 years; male patients 73%; HCV infected 52%; without any therapeutic treatment 50%) and 82 patients with cirrhosis (mean age ± SD: 53 ± 9 years; male patients 66%; HCV infected 68%) were collected. The logistic regression parameters were calculated on a previously published data set of 50 patients with HCC and 50 patients with cirrhosis, where the distribution of serum levels of SCCA-IgM and AFP-IgM in HCC patients was significantly different from that in patients with cirrhosis (Mann-Whitney U test, p<0.01). Serum levels of SCCA-IgM and AFP-IgM were assessed in parallel using the Hepa-IC and Hepa AFP-IC kits and AFP was determined by an automatic analyzer (ADVIA Centaur®, Siemens Diagnostics, Italy).Results The patients were stratified according to sex (male), HCV infection (positive) and therapeutic treatment (negative) to apply a linear logistic regression model using regression parameters calculated from a data set of a reference population. A subgroup of 30 patients with HCC and 41 patients with cirrhosis was analyzed. The diagnostic accuracy measured as the area under the ROC curve (AUC) for the SCCA-IgM and AFP-IgM assays was roughly the same, with a weak supremacy of AFP-IgM (AUC = 0.62), and was comparable with that of free AFP (AUC = 0.64). The gain in diagnostic accuracy achieved with the combination of SCCA-IgM and AFP-IgM levels by linear logistic regression was 14% (AUC = 0.71) compared with the diagnostic accuracy of AFP-IgM and 11% compared with that of free AFP. With the estimation of the partial AUC (pAUC0.3), an alternative measurement of AUC that takes into consideration only specificity rates with clinical relevance (specificity >70%), the highest improvement in accuracy for HCC detection was obtained, with an increase of up to 62% compared to pAUC0.3 of AFP-IgM and up to 23% compared to pAUC0.3 of free AFP.Conclusion The results demonstrate that the combination by linear logistic regression of biomarkers-IgM immune complexes improves the diagnostic accuracy for HCC detection using regression parameters calculated in a reference population with defined clinical characteristics. These results support the usefulness of devices based on a panel of non-overlapping biomarkers-IgM complexes, such as multi-marker biochips, increasing the sensitivity and maintaining a high specificity for HCC diagnosis compared with conventional single-marker assays.

Background and aim An increasing number of clinical data suggests the importance of the assessment of serum levels of the squamous cell carcinoma antigen (SCCA)-lgM immune complex for the diagnosis of hepatocellular carcinoma (HCC). In addition, monitoring of SCCA-IgM immune complexes has been described as a useful prognostic approach in patients with cirrhosis since the progressive increase of SCCA-IgM over time is associated with a higher risk of HCC development. Because other assays in patients with cirrhosis have been affected by interfering IgMs, the aim of the present study was to assess the specificity of SCCA-IgM reactivity in cirrhotic patients by evaluating SCCA-IgM detection dependence on different capturing phases and by measuring the recovery of SCCA-IgM reactivity after serum fractionation.Patients and methods Serum samples from 82 patients with cirrhosis (M/F ratio 3/1; mean age ± SD: 56 ± 9 years) were collected at the Liver Unit of the Department of Clinical and Experimental Medicine, University of Padua, according to the approved institutional procedures. Serum levels of SCCA-IgM were measured using two different ELISA tests: the reference assay based on a rabbit oligoclonal anti-human SCCA antibody (Hepa IC, Xeptagen, Italy) and a dedicated ELISA with a mouse monoclonal anti-SCCA as capture antibody.Results Serum levels of SCCA-IgM measured with the reference assay (median value 87 AU/mL) were higher than levels measured with the mouse monoclonal test (median value 78 AU/mL), but the differences were not statistically significant (Mann-Whitney U test, p>0.05). When SCCA-IgM levels measured with both tests were compared, a linear correlation was found (r = 0.77, p < 0.001). An in silico analysis using available structural data of SCCA showed the presence of up to three putative antigenic sites localized on the SCCA surface, thus providing evidence that the test with the mouse monoclonal anti-SCCA antibody could underestimate the total circulating immune complexes due to the steric hindrance of the enormous mass of IgM (900 kDa) that could mask on the SCCA (45 kDa) surface the binding site recognized by the monoclonal antibody. To show that the SCCA-IgM assay was not affected by interfering IgMs, we measured the recovery of SCCA-IgM reactivity after serum fractionation of ten of the most reactive samples in both assays. Total recovery of SCCA-IgM reactivity was obtained with both assays in the fractions corresponding to components with higher molecular weight than IgM and SCCA (>2000 kDa).Conclusions The results of this study indicate that the reactivity measured in cirrhotic patients is related only to SCCA-IgM immune complexes and is not affected by other serum components, supporting the analytical specificity of the SCCA-IgM assay and validating the importance of SCCA-IgM as a risk biomarker for HCC development.

In an assessment of the clinical relevance of serum hepatitis B virus (HBV) DNA testing in chronic HBV infection, changes in the presence of this marker were investigated by spot hybridization in 138 hepatitis B surface antigen (HBsAg)-positive patients with chronic liver disease who were followed up for one to eight years. Forty-one patients were treated with steroids, often with evidence of potentiation of viral replication, whereas 92 patients remained untreated and had no evidence of δ agent infection during follow-up. Data analysis in these patients allowed us to determine the significance of testing for hepatitis B e antigen and for HBV DNA in the natural history of the infection. The findings indicate that sequential testing for serum HBV DNA may be of great importance in HBsAg chronic carriers with liver disease for adequate evaluation of HBV replication and for the contribution of HBV DNA to the clinical assessment of chronic hepatitis.