E. Arosio

High rate of positive anti-tissue transglutaminase antibodies in chronic liver disease: Role of liver decompensation and of the antigen source

Background: Since the recognition of tissue transglutaminase (tTG) as the target antigen of anti-endomysium antibodies, several ELISA assays using either guinea pig or human recombinant tTG have been developed. The aim of the study was to compare the behaviour of anti-tTG and anti-endomysium antibodies assays in coeliacs and in patients with chronic liver disease. Methods: 34 patients (24 women, 34.9 ± 12.5 years) with coeliac disease and 41 with chronic liver disease (14 women, 57 ± 11.2 years), including 19 cirrhotics, were evaluated for anti-endomysium antibodies by indirect immunofluorescence and for anti-tTG IgA antibodies by ELISA, using guinea pig liver or human recombinant transglutaminase. Results: The prevalences of anti-tTG and anti-endomysium antibodies were 100% in patients with coeliac disease at diagnosis, 75% and 64.3% in patients on a gluten-free diet. All liver disease patients were negative for anti-endomysium antibodies, while 11 (26.8%) were positive for anti-tTG. All these patients had liver cirrhosis and represented 57.9% of all cirrhotics. The presence of anti-tTG was associated with higher Child-Pugh scores. The use of human transglutaminase determined a reduction in the rate of positive results; however, the rate of positive anti-tTG was still 17.1% in all liver disease patients and 31.6% in cirrhotics. Conclusions: Our data confirm that anti-tTG have a similar sensitivity compared with anti-endomysium antibodies assay in coeliacs. However, a high prevalence of positive anti-tTG results is observed in cirrhotic patients, even when human recombinant tTG is used. The high prevalence of positive results among cirrhotic patients is associated with more advanced liver disease.

Increased expression of vascular endothelial growth factor in small hepatocellular carcinoma.

Summary.  Vascular endothelial growth factor (VEGF) is involved in both development and progression of several epithelial tumours, but its role in hepatocellular carcinoma (HCC) is unclear. Assessment of liver and blood levels of VEGF may provide further insights on angiogenesis in HCC. Tissue mRNA of VEGF‐165, VEGF‐189 and their receptor KDR was assessed by a semi‐quantitative retro‐transcriptase polymerase chain reaction, and expressed as target transcript/beta‐actin ratio, in 29 patients with HCC, 26 with cirrhosis and 15 with chronic hepatitis. VEGF‐165 was also measured by ELISA in plasma samples obtained from both hepatic and femoral veins in additional 58 patients, including 15 with HCC. The liver expression of mRNA of VEGF‐165, VEGF‐189 and KDR was higher in HCC than in chronic liver diseases (1.54 ± 0.89 vs 0.62 ± 0.47, P < 0.0001; 1.09 ± 0.65 vs 0.64 ± 0.54, P = 0.003; 1.30 ± 1.09 vs 0.69 ± 0.72, P = 0.014). VEGF‐165 was higher in HCC tissue than in extra‐tumoural tissues (1.44 ± 0.31 vs 1.03 ± 0.21, P = 0.0009) and in the cirrhotic tissue of HCC patients than in HCC‐free cirrhosis (1.03 ± 0.23 vs 0.45 ± 0.45, P = 0.0002). Tissue VEGF‐189 mRNA inversely correlated with tumour size and degree of tumour cell proliferation. The hepatic and femoral vein levels of VEGF‐165 protein were significantly higher in HCC patients than in cirrhotic patients (66.7 ± 57.1 vs 24.2 ± 16.4 pg/mL, P = 0.0001 and 37.1 ± 42.2 vs 13.5 ± 9.6 pg/mL, P = 0.001). There was a gradient of VEGF‐165 between hepatic and femoral veins in both HCC and cirrhosis. In conclusion, VEGF appears to be involved in the development of HCC and it could be a predictor of HCC development in patients with cirrhosis.

Automatic cell count in digital images of liver tissue sections

A method for identifying and counting cells and biological structures in histological liver tissue sections is presented. The method uses several image processing operators. The final classification results from contextual fusion of different clues. The overall flow of information among the active components of the cell description procedure (CDP) is outlined. The CDP is constructed and validated by a sequence of exploration and confirmation phases. Exploration takes place when a set of known images that is progressively improved until the results are satisfactory is worked out through the CDP. The confirmation phase may then start, working out a new set of images (of the same type but not analyzed before) and proceeding until acceptable sets of agents are generated. The completion of a first exploration phase has identified some major fields of improvement such as the identification of revision criteria for the refinement of the F-nuclei class.<<ETX>>

Prospective study of natural killer cell phenotype in recurrent hepatitis C virus infection following liver transplantation.

BACKGROUND/AIMS Graft re-infection invariably occurs after liver transplantation (OLT) for chronic hepatitis C and disease progression is unpredictable. We prospectively examined peripheral blood mononuclear cells (PBMC) subsets and natural killer (NK) cell receptors (NKRs) in patients with recurrent hepatitis C post-OLT. METHODS PBMC were obtained at baseline and at different time points after OLT. NKRs were identified using monoclonal antibodies by flow cytometry. RESULTS The proportions of NK, natural T (NT), total and gammadelta T cells were significantly reduced (p<0.01) 7 days post-transplant, probably as a result of graft repopulation. NKG2D+ NK cells were significantly higher compared with healthy controls (p<0.01), declined post-OLT and subsequently returned to baseline values. This, together with a progressive increase in the proportion of CD94/NKG2C+ NK cells over time (p< or = 0.01), appeared to be related to hepatitis C recurrence. There was a statistically significant correlation between expression of the natural cytotoxicity receptors (NCRs) and ALT (p<0.05), supporting the hypothesis that NK cells participate in the necroinflammatory process. CONCLUSIONS The data are compatible with homing of immune cells to the liver allograft after surgery, most of which return to pre-OLT levels. HCV recurrence may cause variations in selected NKRs expression akin to other viral infections.

Proliferating cell nuclear antigen assessed by a computer-assisted image analysis system in patients with chronic viral hepatitis and cirrhosis

BACKGROUND Assessment of liver cell proliferation by immunodetection of proliferating cell nuclear antigen may predict regenerative potential and survival of liver and hepatocellular carcinoma risk in patients with chronic viral hepatitis. AIM To evaluate proliferating cell nuclear antigen status and its clinical significance in a large cohort of patients with chronic viral hepatitis and different degree of liver damage by a computer assisted imaging analysis system. MATERIALS Liver biopsies from 358 patients with chronic hepatitis (259 males, 49 years, 63% with hepatitis C infection, 27% with hepatitis B virus, 10% with multiple infections) were studied. METHODS Proliferating cell nuclear antigen was localised by immunoperoxidase on microwave oven pre-treated formalin-fixed, paraffin embedded sections using PC10 monoclonal antibody. Proliferating cell nuclear antigen labelling index was calculated by an automated imaging system (Immagini e Computers, Milan, Italy). RESULTS Mean proliferating cell nuclear antigen labelling index ranged from 0.1% for patients with minimal changes to 3.6% for those with cirrhosis and hepatocellular carcinoma. Overall, proliferating cell nuclear antigen labelling index was higher in males, in older patients, in multiple infections and in hepatitis C virus compared to hepatitis B virus related cases. By linear regression analysis, proliferating cell nuclear antigen labelling index correlated with older age, male gender; higher transaminase levels, hepatitis C virus, higher histological gradIng and staging: by multivariate analysis male gender, hepatitis C virus, higher grading and staging resulted as independent variables. Both hepatitis C virus or hepatitis B virus cirrhotics had similar liver cell proliferation rate but those with hepatitis B virus had higher prevalence of liver cell dysplasia with respect to those with hepatitis C virus. CONCLUSIONS Proliferating cell nuclear antigen labelling index was a reliable assay for assessing liver cell proliferation rate in patients with chronic viral hepatitis and correlated with liver disease severity

Interferon-alpha suppresses liver cell proliferation in patients with chronic hepatitis C virus infection.

Summary.  Interferon (IFN) therapy has been shown to reduce the risk of hepatocellular carcinoma (HCC) in patients with chronic hepatitis C, including virological nonresponders (NR). Whether IFN suppresses liver cell proliferation, i.e. the relevant risk factor for HCC, is unknown. The aim of the study was to evaluate the effect of IFN therapy on liver cell proliferation in chronic hepatitis C.

Primary biliary cirrhosis is associated with specific changes in liver IgG-bearing cell subpopulations

BACKGROUND/AIMS The density of total IgG-bearing cells and the distribution of their subclasses were studied in the liver of patients with primary biliary cirrhosis. METHODS Immunohistochemistry and computerized image analysis were used to compare liver specimens from 18 patients with primary biliary cirrhosis and 28 with chronic hepatitis of different etiology. RESULTS The density of total IgG-bearing cells was similar in the two groups. However, in patients with primary biliary cirrhosis the proportion of IgG3-positive cells was significantly higher than in patients with chronic hepatitis (53 +/- 7% vs. 7.5 +/- 2.4%) (p < 10(-8)). Conversely, IgG1-positive cells were significantly less prevalent in patients with primary biliary cirrhosis than in chronic hepatitis patients (27 +/- 6.9% vs. 68 +/- 7.2%, p < 10(-8)). Stratification of patients with primary biliary cirrhosis according to histology did not show any difference in the distribution of IgG subclasses associated with the progression of disease. CONCLUSION These data suggest a pathogenetic role of local IgG3-bearing cells in primary biliary cirrhosis.

Serum autoantibodies against cytochrome P450 2E1 (CYP2E1) predict severity of necroinflammation of recurrent hepatitis C.

We previously reported that autoantibodies against cytochrome P4502E1 (CYP2E1) are frequent in patients with chronic hepatitis C. As autoimmune reactions are increasingly detected after orthotopic liver transplantation (OLT), this study investigates prevalence and significance of anti‐CYP2E1 autoantibodies in 46 patients with post‐OLT recurrent hepatitis C.

Might rapid virological response be used as a stopping rule in liver transplant recipients treated with pegylated interferon plus ribavirin

We read with interest the article by Hanouneh et al. on the prediction of pegylated interferon plus ribavirin (P/R) response in liver transplant (LT) recipients with histologically proven recurrent hepatitis C. As highlighted by the authors, the management of problematic patients such as graft recipients can be eased by early kinetics of hepatitis C virus (HCV), that is, HCV-RNA clearance at week 4 [rapid virological response (RVR)] and a 2-log drop of HCV-RNA at week 12 [early virological response (EVR)], which predict treatment outcome. However, although in LT patients EVR has an absolute negative predictive value (NPV) for P/R treatment outcome, the NPV and positive predictive value (PPV) of RVR are far from being absolute, even in non-LT patients. We were therefore surprised by Hanouneh et al.’s findings of RVR having 100% PPV and 88% NPV in LT patients receiving P/R. We think that the RVR accuracy discrepancies between this study and other studies might reflect differences in the performance of the studies; for example, many patients (38%) in the present series were not tested for serum HCV-RNA at week 4 of treatment. We treated with P/R 46 patients transplanted for end-stage HCV with inclusion criteria similar to those investigated by Hanouneh et al. The treatment was intended for 48 weeks, being independent of the genotype or virological response to either peginterferon alfa-2a or peginterferon alfa-2b weekly, whereas the ribavirin dosing was less than that in Hanouneh et al.’s study (400-600 versus 1000-1200 mg/day). Serum HCV-RNA levels were assessed at baseline and at 4 weeks, 12 weeks, 48 weeks, and 6 months after the completion of therapy, and most patients received growth factors. The 2 cohorts were similar for clinicovirological features but differed in terms of the prevalence of non-Caucasians and body mass index (lower in our cohort). Cyclosporine A (CSA)–based immunosuppression was higher in our patients than in the American cohort (48% versus 17%). The rates of SVR were 41% and 35%, respectively, with a preference for genotypes 2 and 3 in both (76% versus 87%). The SVR rates in the 2 cohorts are in line with those reported by recent studies on P/R-treated HCV recipients ranging from 30% to 45%. In our patients, RVR had 86% PPV and 78% NPV (see Table 1), that is, midway between the data of Hanouneh et al. and other data. The results indicate that RVR is a good predictor of treatment outcome in LT patients with recurrent hepatitis C; however, it is not accurate enough to become a stopping rule of P/R therapy. In our cohort, CSA-based immunosuppression was independently associated with SVR in multivariate analysis (odds ratio 6.65, 95% confidence interval 1.79-24.7, P 0.005), and this supports the synergic anti-HCV effect of CSA and interferon already demonstrated in vitro and in vivo. This was not investigated by Hanouneh et al. because the majority of their patients received tacrolimusbased immunosuppression. The higher dose of ribavirin in the American patients with respect to ours might have been beneficial in terms of SVR, too.

In situ identification of immune competent cells in gastrointestinal mucosa: an evaluation by immunoelectronmicroscopy.

Thein situ identification of lymphocyte subpopulations by means of immunopathological techniques using specific monoclonal antibodies provides a tool for the study of the gastrointestinal-associated lymphoid tissue (GALT) in health and disease. In this field, monoclonal antibodies have been applied previously using light microscopy and either immunofluorescence or immunoperoxidase; however, these techniques are not sensitive enough to allow precise evaluation of localization of labelling. We describe an immunoelectronmicroscopic method, which defines labelling specificity, since it allows the identification of cells by immunophenotype labelling and ultrastructural markers simultaneously. This in turn allows a better evaluation of the labelled cells and of the relationship between labelled and unlabelled cells. The main features of the method are the use of fresh tissue samples, fixing in paraformaldehyde CaCl2, and the coupling of the immune reaction to an amplification system (avidin-biotin-peroxidase complex). The technique yields a good preservation of cellular ultrastructure, together with a strong and specific immunolabelling. Our results confirm the high specificity of monoclonal antibodies when applied to immunopathology techniques. We confirm the pattern of distribution of various lymphocyte subsets in the jejunal mucosa described by other authors by light microscopy.