G. Ballardini

Antibodies to hepatitis C virus in autoimmune liver disease: evidence for geographical heterogeneity

To resolve conflicting reports about the occurrence of antibodies against hepatitis C virus (HCV) in patients with autoimmune chronic active hepatitis (AI-CAH), sera from UK and Italian patients were tested with the original anti-HCV assay (Ortho) and a novel anti-HCV assay (UBI) based entirely on synthetic HCV peptides. 28 (60%) of 47 Italian patients with type-1 AI-CAH were anti-HCV-positive by Ortho ELISA, 25 of whom were also strongly positive by the UBI assay. 15 (60%) of 25 UK patients with type-1 AI-CAH were HCV-positive by Ortho ELISA but only 2 were positive by the UBI assay. Similarly, 29 (88%) of 33 Italian patients with type-2 AI-CAH, but 0 of 10 UK patients, were very strongly anti-HCV-positive with the UBI assay. Italian patients with AI-CAH appear to have a high frequency of genuine exposure to HCV, whereas seropositivity by the Ortho HCV ELISA in UK patients is likely to represent a false-positive result. These findings indicate important geographical and/or genetic influences in autoimmune liver disease among different populations.

Desmin and actin in the identification of Ito cells and in monitoring their evolution to myofibroblasts in experimental liver fibrosis

SummaryIt has been reported that myofibroblasts contain actin and that Ito cells are positive for desmin. The distribution of desmin and actin detected by immunofluorescence, of vitamin A autofluorescence and of Sudan III staining of lipid droplets has been evaluated in sequential stages of experimental liver fibrosis induced in rats by intraperitoneal injections of swine serum. In the normal rat liver Ito cells were positive for desmin and weakly positive for actin. Prior to the development of hepatic fibrosis a clearcut increase in number and desmin staining of lobular Ito cells was observed in treated rats, but the overall actin pattern was unchanged. In the fibrotic rat livers, highly cellular septa contained large numbers of strongly desmin-positive, actin-weakly positive Ito cells and strongly desmin-and actin-positive myofibroblasts. These observations indicate that both Ito cells and myofibroblasts are positive for desmin, but only myofibroblasts contain large amounts of actin. Visualization of actin and desmin using relatively simple techniques, allows the monitoring of Ito cells proliferation, the accumulation of these cells in fibrous septa and their evolution into myofibroblasts as characterized by their increased desmin and actin content; it also allows an indirect evaluation of the process of fibrogenesis.

Characterization and Isolation of Ductular Cells Coexpressing Neural Cell Adhesion Molecule and Bcl-2 from Primary Cholangiopathies and Ductal Plate Malformations

It has recently been shown that reactive bile ductules display neuroendocrine features, including immunoreactivity for the neural cell adhesion molecule (NCAM). In this study we have compared the immunohistochemical expression of NCAM with that of HEA-125 (biliary specific) and LKM-1 (hepatocyte specific) and other markers relevant to morphogenesis (Bcl-2, EMA) and cell proliferation (Ki-67) in cryostat sections from different chronic liver diseases and from fetal livers at different gestational ages. In parallel, viable NCAM-positive ductular cells were purified from collagenase digests of cirrhotic livers by immunomagnetic separation and characterized by immunocytochemistry and transmission electron microscopy. We demonstrated that reactive ductules with atypical morphology coexpressed NCAM and Bcl-2 and were found mainly in congenital diseases associated with ductal plate malformation and in primary cholangiopathies. On the contrary, reactive ductules with typical morphology were negative for NCAM/Bcl-2 and positive for EMA. Reactive ductules coexpressing NCAM/Bcl-2 were negative for the proliferation marker Ki-67 and appeared to be directly connected with periportal hepatocytes. In fetal livers NCAM/Bcl-2 was transiently expressed during the early developmental stages of ductal plate (10-16 weeks) and started to disappear as the ductal plate began duplicating. NCAM-positive ductal plate cells were Ki-67 negative, becoming positive in duplicated segments. Thus the histogenesis of ductular reactive cells seems to recapitulate the early stages of biliary ontogenesis. In primary cholangiopathies and ductal plate malformations, these cells do not appear to maturate further, and thus abundant ductular structures coexist with vanishing mature ducts. These NCAM-positive ductular cells were immunopurified from patients with chronic cholestatic liver diseases and showed ultrastructural features consistent with a less differentiated phenotype than mature cholangiocytes. These isolated cells represent a useful model for in vitro studies.

Interferon therapy in liver/kidney microsomal antibody type 1-positive patients with chronic hepatitis C

The association between liver/kidney microsomal antibody type 1 and adult cases of hepatitis C virus-related chronic liver disease has been firmly established. In the presence of both markers, evidence of autoimmunity (liver/kidney microsomal antibody type 1) and actual viremia (serum HCV RNA), the therapeutic dilemma arises between steroids, which are beneficial to autoimmune but deleterious to viral diseases, and interferon-alpha, which may exacerbate an autoimmune disorder. Six patients with liver/kidney microsomal antibody type 1 and serum HCV RNA were given interferon-alpha: three showed a response pattern similar to that observed in autoantibody-negative chronic hepatitis C cases; the other three developed a sharp transaminase peak, which was not followed by HCV RNA clearance. Considering the brisk flare-up of liver cell necrosis, interferon-alpha treatment proved to be dangerous in the above three liver/kidney microsomal antibody type 1/HCV RNA positive cases. Subsequent steroid administration reduced alanine aminotransferase peaks, but may be harmful in viral infections. Therapeutic alternatives are needed: they will probably include pure antivirals (exerting no immunostimulatory effects) with or without immunosuppressive drugs.

Liver transplantation for hepatocellular carcinoma: Further considerations on selection criteria

The selection criteria in liver transplantation for HCC are a matter of debate. We reviewed our series, comparing two periods: before and after 1996, when we started to apply the Milan criteria. The study population was composed of patients with a preoperative diagnosis of HCC, confirmed by the pathological report and with a survival of >1 year. Preoperative staging as revealed by radiological imagining was distinguished from postoperative data, including the variable of tumor volume. After 1996 tumor recurrences significantly decreased (6 out of 15 cases, 40% vs. 3 out of 48, 6.3%, P < .005) and 5‐year patient survival improved (42% vs. 83%, P < .005). Not meeting the Milan criteria was significantly related to higher recurrence rate (37.5% vs. 12.7%, P < .05) and to lower 5‐year patient survival (38% vs. 78%, P < .005%) in the preoperative analysis, but not in the postoperative one. The alfa‐fetoprotein level of more than 30 ng/dL and the preoperative tumor volume of more than 28 cm3 predicted HCC recurrences in the univariate and mutivariate analysis (P < .005 and P < .05, respectively). The ROC curve showed a linear correlation between preoperative tumor volume and HCC recurrence. Milan criteria significantly reduced tumor recurrences after liver transplantation, improving long‐term survival. In conclusion, the efficacy of tumor selection criteria must be analyzed with the use of preoperative data, to avoid bias of the postoperative evaluation. Tumor volume and alfa‐fetoprotein level may improve the selection of patients. (Liver Transpl 2004;10:1195–1202.)

Effect of ischemic preconditioning in whole liver transplantation from deceased donors. A pilot study.

The effect of ischemic preconditioning (IPC) in orthotopic liver transplantation (OLT) has not yet been clarified. We performed a pilot study to evaluate the effects of IPC in OLT by comparing the outcomes of recipients of grafts from deceased donors randomly assigned to receive (IPC+ group, n = 23) or not (IPC− group, n = 24) IPC (10‐min ischemia + 15‐min reperfusion). In 10 cases in the IPC+ group and in 12 in the IPC− group, the expression of inducible nitric oxide synthase (iNOS), neutrophil infiltration, and hepatocellular apoptosis were tested by immunohistochemistry in prereperfusion and postreperfusion biopsies. Median aspartate aminotransferase (AST) levels were lower in the IPC+ group vs. the IPC− group on postoperative days 1 and 2 (398 vs. 1,234 U/L, P = 0.002; and 283 vs. 685 U/L, P = 0.009). Alanine aminotransferases were lower in the IPC+ vs. the IPC− group on postoperative days 1, 2, and 3 (333 vs. 934 U/L, P = 0.016; 492 vs. 1,040 U/L, P = 0.008; and 386 vs. 735 U/L, P = 0.022). Bilirubin levels and prothrombin activity throughout the first 3 postoperative weeks, incidence of graft nonfunction and graft and patient survival rates were similar between groups. Prereperfusion and postreperfusion immunohistochemical parameters did not differ between groups. iNOS was higher postreperfusion vs. prereperfusion in the IPC− group (P = 0.008). Neutrophil infiltration was higher postreperfusion vs. prereperfusion in both groups (IPC+, P = 0.007; IPC−, P = 0.003). Prereperfusion and postreperfusion apoptosis was minimal in both groups. In conclusion, IPC reduced ischemia/reperfusion injury through a decrease of hepatocellular necrosis, but it showed no clinical benefits. Liver Transpl 12:628–635, 2006.

Dimethylnitrosamine-induced cirrhosis: Evidence for an immunological mechanism

The present study is concerned with the early events associated with the development of cirrhosis induced by dimethylnitrosamine (DMN). The antigenic expression of MHC class II components (Ia) and of some intermediate filament proteins (vimentin and desmin) have been studied by immunohistochemistry and the findings correlated with ultrastructural data. Micronodular cirrhosis developed after 3 weeks of treatment with DMN but enhanced expression of Ia antigen on macrophages and on infiltrating lymphocytes was observed after 1 week, before the formation of septa, suggesting that immune-mediated mechanisms are involved in the response to DMN-induced liver injury. The expression of vimentin and of desmin also increased at an early stage and at 3 weeks the septa were outlined by cellular elements showing positivity for both intermediate filament proteins. In keeping with these observations, ultrastructural data showed active division of macrophages in situ, infiltration of the parenchyma by T and B lymphocytes, activation of lipocytes (Ito cells) showing evidence of mitosis, and the presence of transitional elements between lipocytes, myofibroblasts and fibroblasts. This experimental model may be helpful in understanding the relationship between immune-mediated response to liver injury and development of hepatic fibrosis.

Increased risk of hepatocellular carcinoma development in patients with cirrhosis and with high hepatocellular proliferation

The immunohistochemical determination of the accessory protein of DNA-polymerase delta (PCNA), a marker of an early S-phase of the cell cycle, was used to evaluate cell proliferation retrospectively in formalin-fixed, paraffin-embedded liver biopsy sections in a group of patients with cirrhosis of similar age and duration of follow up, and with no evidence of hepatocellular carcinoma (41), including 17 patients with and 24 without hepatocellular carcinoma appearance during follow up. Proliferation was expressed as total (PCNA-TOT) and strongly (PCNA-STRO) positive nuclei per 1000 hepatocytes. The presence of dysplasia was also recorded. Histological findings and biochemical data, at the time of liver biopsy, were compared in the two groups. While total PCNA positivities were not significantly different in the two groups, strong reactivity was significantly higher in patients who eventually developed hepato-cellular carcinoma (median 0.7 vs 2.6). Univariate analysis of histological and biochemical data at the time of biopsy, followed by a stepwise regression study, showed that the significant parameters for a time-dependent disease-free state were, in decreasing order: cholesterol, PCNA-STRO, PCNA-TOT and alpha foeto-protein. Other clinical, biochemical and histological parameters, including dysplasia, provided no further information. From these data, hepatocellular proliferation can be evaluated in patients with cirrhosis with a currently available technique. Patients with high cell proliferation are at increased risk of developing hepatocellular carcinoma and may require differentiated follow up.

Liver Transplantation with the Meld System: A Prospective Study from a Single European Center

The efficacy of the Meld system to allocate livers has never been investigated in European centers. The outcome of 339 patients with chronic liver disease listed according to their Meld score between 2003 and 2005 (Meld era) was compared to 224 patients listed during the previous 2 years according to their Child score (Child era). During the Meld era, hepatocellular carcinomas (HCCs) had a ‘modified’ Meld based on their real Meld, waiting time and tumor stage. The dropouts were deaths, tumor progressions and too sick patients. The rate of removals from the list due to deaths and tumor progressions was significantly lower in the Meld than in the Child era: 10% and 1.2% versus 16.1% and 4.9%, p < 0.05. The 1‐year patient survival on the list was significantly higher in the Meld era (84% vs. 72%, p < 0.05). The prevalence of transplantation for HCC increased from 20.5% in the Child to 48.9% in the Meld era (p < 0.001), but between HCCs and non‐HCCs of this latter era the dropouts were comparable (9.4% vs. 14.9%, p = n.s.) as was the 1‐year patient survival on the list (83% vs. 84%, p = n.s.). The Meld allocation system improved the outcome of patients with or without HCC on the list.

Anti-actin antibodies: a new test for an old problem.

Smooth muscle antibodies with anti-actin specificity are commonly regarded as markers of autoimmune liver disease. However, there are interpretational problems because different techniques have been used for their identification and therefore the results are difficult to compare. The present paper reports the results of a new method for the identification of anti-actin antibodies (indirect immunofluorescence on cryostat sections of liver from rats chronically injected with phalloidin). The results have been compared with those obtained by four other techniques: demonstration by immunofluorescence of kidney peritubular reactivity (SMAT), of anti-microfilament antibodies (on HEp-2 cells and vinblastine-treated peripheral blood mononuclear cells) and counterimmunoelectrophoresis with purified muscle actin as antigen. The new method proved to be the most sensitive and specific. Furthermore, its reproducibility was found to be high, the interpretation easy and the cost low. The clinical significance of anti-actin antibodies in patients with chronic liver disease is also discussed.