Specificity of squamous cell carcinoma antigen (SCCA)-IgM detection in patients with HCV infection and rheumatoid factor seropositivity.

IgM antibodies bound to different cancer antigens have shown recently a higher diagnostic value, compared with the corresponding free molecule, giving rise to a new family of biomarkers. High or increasing levels of Squamous Cell Carcinoma Antigen (SCCA)-IgM immune complexes were associated with more advanced liver disease and increased risk of development of HCC. Rheumatoid factor (RF) represents a long-standing problem of interference for immunometric assays. The aim of the present study was to examine the specificity of SCCA-IgM in relation to the presence of RF reactivity in patients infected with hepatitis C virus (HCV). Sera of 73 patients with cirrhosis, infected with HCV, (mean age ± SD: 66 ± 13 years; M/F: 45/28), including 21 patients with HCC, were studied. SCCA-IgM immune complexes levels were measured by a commercial ELISA. To evaluate the possible interfering effect of RF, the standard calibrator, positive for SCCA-IgM, was spiked with serial dilutions of a RF positive or negative serum. SCCA-IgM immune complexes were positive in 35 out of 73 (48%) patients, while RF activity was found in 10 out of 73 (14%) patients. Patients with cirrhosis with RF activity had significantly higher levels of SCCA-IgM, compared to RF negative cases; however, no significant correlation between SCCA-IgM and RF values was observed. In samples created artificially the same results in terms of reactivity for SCCA-IgM were obtained, regardless of the presence of RF activity. These findings support the lack of correlation between the two parameters found in sera of patients infected with HCV.

Different biochemical correlates for different neuropsychiatric abnormalities in patients with cirrhosis.

UNLABELLED: The diagnosis of hepatic encephalopathy (HE) relies on clinical, neurophysiological, psychometric and laboratory variables. The relationships between such tests remain debated. The aim of this study was to determine the laboratory correlates/prognostic value of neurophysiological/psychometric abnormalities in patients with cirrhosis. Seventy-two patients and 14 healthy volunteers underwent EEG and paper-and-pencil psychometry (PHES). Blood was obtained for C reactive protein (CRP), interleukin 6 (IL6), tumor necrosis factor (TNF)α, ammonia and indole/oxindole. Patients were followed prospectively for a median of 22 months in relation to the occurrence of death, transplantation and HE-related hospitalizations. Thirty-three patients had normal PHES and EEG, 6 had abnormal PHES, 18 abnormal EEG and 13 abnormal PHES and EEG. Patients with abnormal PHES had higher CRP (17 ± 22 vs 7 ± 6, P < 0.01), IL6 (32 ± 54 vs 12 ± 13, P < 0.05) and TNFα (17 ± 8 vs 11 ± 7, P < 0.001) levels than those with normal PHES. Patients with abnormal EEG had higher indole (430 ± 270 vs 258 ± 255, P < 0.01) and ammonia (66 ± 35 vs 45 ± 27, P < 0.05) levels than those with normal EEG. Psychometric test scores showed significant correlations with CRP, TNFα and IL6; EEG indices with ammonia and IL6. CRP and TNFα concentrations were independent predictors of abnormal PHES, ammonia and indole of abnormal EEG on multivariate analysis. Seven patients were lost to follow-up; of the remaining 65, 20 died and 14 underwent transplantation; 15 developed HE requiring hospitalization. PHES and EEG performance were independent predictors of HE and death (P < 0.05). CONCLUSION: PHES and EEG abnormalities in patients with cirrhosis have partially different biochemical correlates and independently predict outcome.

SERPINB3 modulates TGF-beta expression in chronic liver disease.

Transforming growth factor-beta1 (TGF-beta1) is the master cytokine in the pathogenesis of liver fibrosis. TGF-beta1 and extent of fibrosis were correlated recently to the serpin SERPINB3 in idiopathic pulmonary fibrosis, a chronic disease recalling liver cirrhosis. The aim of this study was to assess the relation between SERPINB3, TGF-beta1 and fibrosis in chronic liver diseases and to determine the effect of this serpin on TGF-beta1 expression using in vitro models. SERPINB3 and TGF-beta1 were evaluated in liver biopsies of 94 patients with chronic liver disease. The effect of SERPINB3 on TGF-beta1 expression was determined in primary human hepatocytes, HepG2 and Huh7 cells transfected with intact SERPINB3 human gene or with reactive site loop deleted mutants. A significant correlation between TGF-beta1 and SERPINB3 at the protein level was observed in liver biopsies, confirmed by a positive correlation at mRNA level. Both proteins were correlated to the extent of liver fibrosis. All transfected cells showed increased TGF-beta1 mRNA and protein production and the integrity of the reactive site loop of the serpin was crucial to achieve this effect. In conclusion, chronically damaged hepatocytes produce SERPINB3 and TGF-beta, and the anti-protease activity of this serpin might be implicated in TGF-beta1 induction.

Experimental validation of specificity of the squamous cell carcinoma antigen-immunoglobulin M (SCCA-IgM) assay in patients with cirrhosis.

BACKGROUND: Squamous cell carcinoma antigen-immunoglobulin M (SCCA-IgM) is a useful biomarker for the risk of development of hepatocellular carcinoma (HCC) in patients with cirrhosis due to its progressive increase associated to HCC evolution. In patients with cirrhosis, other assays have been affected by interfering reactivities of IgM. In this study, the analytical specificity of the SCCA-IgM assay was assessed by evaluating SCCA-IgM measurement dependence on different capture phases, and by measuring the recovery of SCCA-IgM reactivity following serum fractionation. METHODS: Serum samples from 82 patients with cirrhosis were analyzed. SCCA-IgM was measured using the reference test (Hepa-IC, Xeptagen, Italy) that is based on rabbit oligoclonal anti-squamous cell carcinoma antigen (SCCA) and a dedicated ELISA with a mouse monoclonal anti-SCCA as the capture antibody. RESULTS: SCCA-IgM concentrations measured with the reference assay (median value=87 AU/mL) were higher than those measured with the mouse monoclonal test (median value=78 AU/mL). However, the differences in the SCCA-IgM distribution were not statistically significant (p>0.05). When SCCA-IgM concentrations measured with both tests were compared, a linear correlation was found (r=0.77, p<0.05). Fractionation of the most reactive sera by gel-filtration chromatography showed that total recovery of SCCA-IgM reactivity was seen only in the fractions corresponding to components with a molecular weight higher than IgM and SCCA (>2000 kDa) with both tests. CONCLUSIONS: The equivalence of both SCCA-IgM assays and the absence of reactivity not related to immune complexes support the analytical specificity of SCCA-IgM measurements. The results validate the assessment of SCCA-IgM for prognostic purposes in patients with cirrhosis.

SERPINB3 is associated with longer survival in transgenic mice.

The physiological roles of the protease inhibitor SERPINB3 (SB3) are still largely unknown. The study was addressed to assess the biological effects of this serpin in vivo using a SB3 transgenic mouse model. Two colonies of mice (123 transgenic for SB3 and 148 C57BL/6J controls) have been studied. Transgenic (TG) mice showed longer survival than controls and the difference was more remarkable in males than in females (18.5% vs 12.7% life span increase). In TG mice decreased IL-6 in serum and lower p66shc in the liver were observed. In addition, TG males showed higher expression of mTOR in the liver. Liver histology showed age-dependent increase of steatosis and decrease of glycogen storage in both groups and none of the animals developed neoplastic lesions. In conclusion, the gain in life span observed in SB3-transgenic mice could be determined by multiple mechanisms, including the decrease of circulating IL-6 and the modulation of ageing genes in the liver.

Increased Th1 immune response in SERPINB3 transgenic mice during acute liver failure.

Acute liver failure (ALF) is characterized by severe neurological complications, known as acute hepatic encephalopathy, where brain ammonia and inflammatory processes play a dominant role. In experimental models of acute liver failure SERPINB3 was found significantly increased in microglia, the intrinsic immune cells of the central nervous system. The aim of the present study was to investigate the extent of brain tissue damage and the inflammatory milieu in experimental acute liver failure using a SERPINB3-transgenic mouse model. C57BL/6J wild-type and transgenic mice were inoculated with acetaminophen or phosphate-buffered saline and sacrificed 20 h postinjection. Proliferation and apoptotic activity were analyzed in brain tissue by immunohistochemistry and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling technique. The expression of cytokines was analysed in brain and liver tissue by real time polymerase chain reaction and in the corresponding serum samples using a Bio-Plex system. Acetaminophen induced a significantly lower body temperature and shorter survival in transgenic than in wild-type mice, despite liver function was similar in both groups. The brain of transgenic mice, expressing SERPINB3 positivity in microglia, showed increased glial cell number, associated to significant lower apoptotic death events, compared with wild-type mice. In mice injected with acetaminophen, remarkably higher values of cytokines mRNA were observed in the liver of both groups, with a trend toward higher values in transgenic animals. In brain tissue similar increase of tumor necrosis factor-α was detected in transgenic and wild-type mice, while IL-10 mRNA increased only in the wild-type group. A remarkable increase of circulating Th1 cytokines was detected in serum of transgenic mice, while in the wild-type group they remained rather unchanged. These figures were associated with lower levels of granulocyte macropage colony-stimulating factor, despite similar increase of IL-10 values in both groups. In conclusion, in acute liver failure SERPINB3 determines an enhanced inflammatory background, mainly mediated by higher levels of Th1 proinflammatory cytokines.

IgM-linked SerpinB3 and SerpinB4 in sera of patients with chronic liver disease.

BACKGROUND: Epidemiological studies indicate that a growing number of cirrhotic patients will develop hepatocellular carcinoma (HCC) in the next decade. Recent findings have demonstrated that Squamous cell carcinoma antigen 1 (SCCA1) and 2 (SCCA2) isoforms, now classified as serpinB3 and serpinB4, are over-expressed in HCC, but not in normal liver. As reported, high levels of circulating SCCA-IgM immunocomplexes in patients with cirrhosis are significantly associated with HCC development. AIM: To ascertain whether IgM-linked SCCA isoforms circulate in patients with chronic liver disease, compared to total SCCA-IgM levels. METHODOLOGY AND FINDINGS: 79 patients with chronic liver disease were studied, including 17 patients with chronic hepatitis, 36 patients with cirrhosis and 26 with HCC. 28 blood donors were used as control. Monoclonal antibodies against serpinB3 and serpinB4 were used as catcher antibodies to set up specific ELISA assays, while total SCCA-IgM immunocomplexes were detected by commercially available ELISA assay. Overall, the results revealed a better diagnostic sensitivity of total SCCA-IgM assay, compared to both serpinB3 and serpinB4 IgM-linked assays. SerpinB4-IgM median values obtained with SCC103 antibody were moderately higher in patients with cirrhosis than in those with HCC, median values: 0.168 (IQR 0.140-0.427) vs. 0.140 (IQR 0.140-0.278), (p = 0.177). A trend toward decreasing serpinB4-IgM/serpinB3-IgM median ratio was observed in patients with advanced liver disease, being 1.08 in patients with HCC, 1.10 in patients with cirrhosis and 1.40 in patients with chronic hepatitis (p = 0.079). CONCLUSIONS: IgM-linked SCCA isoforms in serum of patients with chronic liver diseases were quantified for the first time. Although the number of patients was limited, this preliminary study reveals that the relative balance of the two serpin isoforms is altered in HCC and it is characterized by a lower serpinB4-IgM/serpinB3-IgM ratio, determined by lower serpinB4 levels.

Role of squamous cell carcinoma antigen-1 on liver cells after partial hepatectomy in transgenic mice.

Squamous Cell Carcinoma Antigen-1 (SCCA1) overexpression has been observed in tumours of epithelial origin and in hepatocellular carcinoma. Previous data indicate that this serpin inhibits apoptosis, while its proliferative activity was only recently proposed. The aim of this study was to evaluate the effect of SCCA1 on liver cells in a transgenic mouse model after partial hepatectomy. Twenty-one C57BL/6J mice (11 transgenic for human SCCA1, 10 wild-type) underwent partial hepatectomy and were sacrified after one week. Apoptosis and proliferation markers were determined in the liver at sacrifice, while a cytokine panel was measured in serum. Transgenic mice showed a relative liver weight significantly higher than wild-type mice at sacrifice (mean +/- SD, 5.38+/-0.50% vs 4.84+/-0.29%, p=0.0221), while no difference (p=0.2403) was observed in two untreated control groups (6 transgenic, 6 wild-type mice). Active caspase-3 was significantly lower in transgenic mice than in wild-type mice (p=0.0047). The transgenic mouse group showed overall higher proliferative activity at sacrifice, compared to wild-type mice, with increased proliferation parameters. Cytokine analysis revealed a remarkable and opposite sex-dependent behaviour of interleukin (IL)-6 after hepatectomy. At variance with wild-type mice, a significant IL-6 increase was documented only in transgenic females (p=0.0313), even more relevant than that observed in wild-type males. In conclusion, transgenic mice expressing SCCA1 showed higher liver regenerative potential compared to wild-type mice, supporting the dual role of this serpin as an anti-apoptotic and pro-proliferative stimulus for liver cells in vivo.

Longitudinal evaluation reveals a complex spectrum of virological profiles in hepatitis B virus/hepatitis C virus-coinfected patients.

Hepatitis B virus (HBV) and hepatitis C virus (HCV) coinfection is often associated with severe forms of liver disease. However, comprehensive studies are lacking, and scant information is available regarding the virological behavior over time in coinfected patients. This study enrolled 133 untreated HBV/HCV-positive patients (male/female = 102/31; median age 51 years [range: 22-83 years]) who were longitudinally followed up for 1 year with bimonthly evaluation of HBV/HCV viremia levels and liver biochemistry. Thirty of these patients had triple infection with hepatitis Delta virus (HDV), while 103 patients were HDV-negative. In the HDV-negative group, active infection with both HBV and HCV was revealed in 24 cases, inactive infection by both viruses was seen in 15 cases, active HBV/inactive HCV was seen in 15 cases, and inactive HBV/active HCV was seen in 49 cases. However, 32 subjects (31%) presented dynamic virological profiles characterized by fluctuation of HBV and/or HCV viremia levels that at different time points were over or under the cutoff limits. Consequently, a correct diagnosis could be performed in these subjects only by serially repeating the virological tests 1 year apart. Similarly, 15 of the 30 HDV-positive subjects showed active HBV and/or HCV infection, with fluctuating virological patterns in 8 cases. In conclusion, this study showed that the virological patterns in HBV/HCV coinfection are widely divergent and have dynamic profiles. A careful longitudinal evaluation of the viremia levels of both viruses is essential for making a correct diagnosis and tailoring the appropriate therapeutic schedule in coinfected patients.

Progressive increase of SCCA-IgM immune complexes in cirrhotic patients is associated with development of hepatocellular carcinoma.

About 3-4% of cirrhotic patients develop primary liver cancer every year. Specific serologic markers have not yet been identified for screening of high risk patients. The serpin squamous cell carcinoma antigen (SCCA) is overexpressed in liver cancer and circulating SCCA-IgM complexes have been described in patients with hepatocellular carcinoma (HCC). The aim of the present study was to assess the behavior of SCCA-IgM in relation to HCC development in patients with cirrhosis. A retrospective, longitudinal study was conducted in a cohort of prospectively followed cirrhotic patients. Two groups with similar clinical profile at presentation were studied : group A included 16 patients who developed HCC during a median follow up of 4 years; group B included 17 patients who did not develop HCC during the same time interval. Circulating SCCA-IgM immune complexes were determined using a recently standardized ELISA assay. At presentation similar levels of SCCA-IgM complexes [mean +/- SD: 267.40 +/- 382.25 U/ml vs. 249.10 +/- 446.90 U/ml, p = 0.9006] and of alpha-fetoprotein [AFP; 24.11 +/- 59.04 IU/ml vs. 10.91 +/- 23.34 IU/ml, p = 0.3995] were detected in group A and in group B. The increase over time (phi) of SCCA-IgM, assessed within at least one year before clinical diagnosis of HCC, was remarkably higher in group A than in group B (mean +/- SD = 280.05 +/- 606.71 (U/ml)/year vs. -37.92 +/- 95.94 (U/ml)/year, p = 0.0408), while AFP increase was not significantly different (11.89 +/- 23.27 (IU/ml)/year vs. 3.67 +/- 11.46 (IU/ml)/year, p = 0.2179). Receiver operating characteristic (ROC) curves were plotted for the rate of change in the levels of both markers and the diagnostic accuracy measured as AUROC was higher for SCCA-IgM phi (0.821) than for AFP phi (0.654). In conclusion, the progressive increase of SCCA-IgM over time was associated with liver tumor development, suggesting that monitoring the behavior of SCCA-IgM might become useful to identify cirrhotic patients at higher risk of HCC development.

Profiles of HCV core protein and viremia in chronic hepatitis C: possible protective role of core antigen in liver damage.

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.