Expression Of Ficolin-2during The Course Of Diseases In Chronic Hepatitis C And B Patients And The Relationship With Prognosis

ObjectiveFicolin-2is a kind of human serum complement lectin with a structure similar to mannan-binding lectin (MBL), which is mainly produced in the liver and secreted into the plasma. Ficolin-2has the abiliy of recognizing conserved PAMPs on the surface of invading pathogens and initiating the innate immune response. Ficolin-2plays an important role in innate immunity through activation of complement and opsonophagocytosis. Hepatitis C virus (HCV) and hepatitis B virus (HBV) are major causes of chronic liver disease in the world which induce liver ascites, liver cirrhosis, liver cancer and other serious consequences. The main transmission method of HCV and HBV is blood transfusion. The continuous replications of the virus can induce host produce antibody, antigen-antibody complexes and complement complex material, which not only show anti-virus results but also do some damage to the liver cells. Our previous study indicated that Ficolin-2could bind to HCV envelope glycoprotein Eland E2which might help to resist HCV infection; Our previous study also showed the concentrations of serum Ficolin-2in CHC patients were higher than that of healthy control group. However the relationship of Ficolin-2and chronic hepatitis B and C (CHB, CHC) remain elusive. The purpose of this study is to define the dynamics of Ficolin-2in CHC patients and CHB patients.MethodMore than50CHC patients and CHB patients’sero-samples were collected at different time point in the whole disease course.49CHC patients with no therapy include24hepatitis C patients with high alanine aminotransferase (ALT) level (>40IU/L),25hepatitis C carrier with normal alanine aminotransferase (ALT) levels (<40IU/L), another28CHC patients received antiviral treatment for2weeks, that including16patients who received a month or longer treatment, and42healthy human sera were collected as control group.31CHB patients showed alanine aminotransferase (ALT) increased (>40IU/L), HBV-DNA copy number> 500cps/ml and HBeAg was positive. All31CHB patients were received observation for12months. The sera were collected every3months, the sera of23CHB carriers with normal ALT level (≤40IU/L) and57healthy blood donors were collected as control group in the study.The sandwich enzyme-linked immunosorbent assay (ELISA) method was used to measure the concentration of serum Ficolin-2according to methods described in previous publications. Briefly,96-well ELISA plates were coated with100ml rabbit anti-Ficolin-2polyclonal antibody. After incubation at room temperature (RT) for one hour, the solution was removed and the plates were rinsed. After washing three times with0.1%Tween-20in phosphate buffered solution (PBS), the plates were washed three times and blocked with5%bovine serum albumin (BSA) overnight. After washing three times with0.1%Tween-20in phosphate buffered solution (PBS),100ml fresh sera (within6h after harvest) from each sample or100ml different concentrations of recombinant Ficolin-2protein were added and incubated at37℃for2h. After washing three times with0.1%Tween-20in phosphate buffered solution (PBS). Then, mouse monoclonal anti-human Ficolin-2GN5(1:1000dilution)(HyCult Biotechnology b.v.) was added to each well and incubated at37℃for1h. The plates were washed three times and incubated with100ml horseradish peroxidase (HRP) conjugated goat anti-mouse IgG (1:1000dilution). Color development was achieved by adding100ml/well of tetrame-thylbenzidine (TMB) chromogen-substrate (Sigma). The reaction was stopped by adding100ml of2M H2SO4, and the absorbance at OD450nm was measured using an ELISA reader. Ficolin-2concentrations were determined using a Ficolin-2standard curve made from different concentrations of Ficolin-2recombinant protein. Data shown are from at least three independent experiments. All statistical data shown represent the mean±SEM.Results1. We found that the concentrations of sero-Ficolin-2were significantly higher in CHC patients with abnormal ALT values than that in CHC patients with normal ALT values and healthy controls. But the sero-Ficolin-2concentrations of chronic hepatitis C carriers with normal ALT values were similar to that of the healthy controls.2. Ficolin-2concentrations in CHC patients with abnormal ALT values were positively correlated with ALT levels; Ficolin-2concentrations were positively correlated with HCV-RNA copies in CHC patients with abnormal ALT values when HCV-RNA levels were less than107copies/ml, but not correlated with HCV-RNA levels when RNA levels were more than107copies/ml.3. After therapy, the concentrations of Ficolin-2decreased accompany with ALT and HCV-RNA levels in the CHC patients.4. Ficolin-2concentrations in the rapid viral response (RVR) group decreased significantly, while in the non-RVR group, Ficolin-2decreased slightly in the CHC patients.5. Ficolin-2concentrations were significantly higher in CHB patients (ALT>40IU/L) than that of hepatitis B virus carrier (ALT<40IU/L) and healthy control. But the Ficolin-2concentrations of CHB carriers (ALT<40IU/L) were similar to that of the healthy controls.6. The sero-concentrations of Ficolin-2from31CHB patients were significantly decreased after12months’observation.7. The CHB patients with high ficolin-2concentrations (>4.8μg/ml) in early follow-up serum samples obtained favorable outcome of HBV-DNA decrease. The CHB patients with favorable outcome had the decreased ficolin-2concentrations accompanied with the decreased ALT and HBV-DNA values.8. The mean concentrations of Ficolin-2were significantly decreased accompany with decreased ALT value, HBV-DNA level and HBeAg sero-conversation.9. The hepatitis B patients with higher Ficolin-2concentration were easy to have HBeAg seroconversion.Conclusions.Our findings suggest that early increased Ficolin-2concentrations are highly correlated with hepatic inflammation and rapid viral response; High ficolin-2concentrations in early follow-up serum samples are associated with favorable outcome of HBV-DNA decreased. The hepatitis B patients with higher Ficolin-2 concentration are easy to have HBeAg seroconversion...