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Unlabelled: Nonalcoholic fatty liver disease (NAFLD) ranges from simple steatosis (SS) to nonalcoholic steatohepatitis (NASH). Though liver-related risk seems confined to NASH, it is currently unclear whether NASH has a higher risk of cardiovascular disease (CVD) and diabetes than SS as a result of the coexistence of obesity and other cardiometabolic confounders. Adipose tissue is an emerging modulator of liver disease in NAFLD and of cardiometabolic disease in the general population. We evaluated in SS and NASH (1) glucose homeostasis and cardiovascular risk profile and (2) the effect of adipose tissue dysfunction, assessed in fasting conditions and postprandially, on liver injury, glucose and lipoprotein metabolism, and markers of early atherosclerosis. Forty nonobese, nondiabetic, normolipidemic biopsy-proven NAFLD patients (20 with SS and 20 with NASH) and 40 healthy subjects, matched for overall/abdominal adiposity and metabolic syndrome, underwent an oral fat load test, with measurement of plasma triglyceride-rich lipoproteins, oxidized low-density lipoproteins, adipokines, and cytokeratin-18 fragments, and an oral glucose tolerance test with minimal model analysis to yield glucose homeostasis parameters. Circulating endothelial adhesion molecules were measured, and adipose tissue insulin resistance (adipose IR) index and visceral adiposity index were calculated. Despite similar fasting values, compared to SS, NASH showed a more atherogenic postprandial lipoprotein profile, an altered adipokine response (i.e., higher resistin increase and an adiponectin fall), and hepatocyte apoptosis activation after fat ingestion. Adipose IR index, endothelial adhesion molecules, and hepatic insulin resistance progressively increased across NAFLD stages. NASH, but not SS, showed an impaired pancreatic β-cell function. On multiple regression analysis, adipose IR index and postprandial adiponectin independently predicted liver histology and altered cardiometabolic parameters., Conclusion: Adipose tissue dysfunction, including a maladaptive adipokine response to fat ingestion, modulates liver injury and cardiometabolic risk in NAFLD., (Copyright © 2012 American Association for the Study of Liver Diseases.)

Genetic factors are critical in determining susceptibility to primary biliary cirrhosis (PBC), but there has not been a clear association with human leukocyte antigen (HLA) genes. We performed a multicenter case-control study and analyzed HLA class II DRB1 associations using a large cohort of 664 well-defined cases of PBC and 1992 controls of Italian ancestry. Importantly, healthy controls were rigorously matched not only by age and sex, but also for the geographical origin of the proband four grandparents (Northern, Central, and Southern Italy). After correction for multiple testing, DRB1*08 [odds ratio (OR), 3.3; 95% confidence interval (CI), 2.4-4.5] and DRB1*02 (OR 0.9; 95% CI 0.8-1.2) were significantly associated with PBC, whereas alleles DRB1*11 (OR 0.4; 95% CI 0.3-0.4) and DRB1*13 (OR 0.7; 95% CI 0.6-0.9) were protective. When subjects were stratified according to their grandparental geographical origin, only the associations with DRB1*08 and DRB1*11 were common to all three areas. Associated DRB1 alleles were found only in a minority of patients, whereas an additive genetic model is supported by the gene dosage effect for DRB1*11 allele and the interaction of DRB1*11,*13, and *08. Lastly, no significant associations were detected between specific DRB1 alleles and relevant clinical features represented by the presence of cirrhosis or serum autoantibodies. In conclusion, we confirm the role for HLA to determine PBC susceptibility and suggest that the effect of HLA is limited to patient subgroups. We suggest that a large whole-genome approach is required to identify further genetic elements contributing to the loss of tolerance in this disease.

More precise prognostic models are needed for prediction of survival in patients with primary sclerosing cholangitis (PSC), particularly for the selection of candidates for liver transplantation. The aim of this study was to develop a time-dependent prognostic model for the calculation of updated short-term survival probability in PSC. Consecutive clinical and laboratory follow-up data from the time of diagnosis were collected from the files of 330 PSC patients from 5 European centers, followed for a median of 8.4 years since diagnosis. Time-fixed and time-dependent Cox regression analyses, as well as the additive regression model, were applied. The reliability of the models was tested by a cross-validation procedure. Bilirubin (on a logarithmic scale), albumin, and age at diagnosis of PSC were identified as independent prognostic factors in multivariate analysis of both the time-fixed and the time-dependent Cox regression models. The importance of bilirubin was more pronounced in the time-dependent model (hazard ratio [HR], 2.84) than in the time-fixed analysis (hazard ratio, 1.51). The additive regression model indicated that once the patients survive beyond the first 5 years, the impact on prognosis of albumin at diagnosis ceases. The time-dependent prognostic model was superior to the time-fixed variant in assigning low 1-year survival probabilities to patients that actually survived less than 1 year. In conclusion, a time-dependent Cox regression model has the potential to estimate a more precise short-term prognosis in PSC compared with the traditional time-fixed models.

The kinetics of the immunoglobulin (Ig) M type antibody to the hepatitis D virus (IgM anti-HD) were investigated in hepatitis B surface antigen (HBsAg) carriers with chronic hepatitis D treated with interferon (IFN) and in patients with terminal hepatitis delta virus (HDV) cirrhosis who underwent liver transplantation. The IgM antibody disappeared in each of 8 patients who responded to IFN therapy with the persistent normalization of aminotransferases and with the clearance of serum HBsAg and HDV-RNA. The IgM reactivity did not decline in the 45 treated patients who did not respond to the cytokine or who experienced a relapse after responding while on therapy. The antibody rapidly disappeared from serum post-transplantation in each of 10 examined patients with HDV who underwent transplantation. In 5 patients who underwent transplantation and who became reinfected with HDV, the antibody remained undetectable during the early reinfection phase, as marked by HDV replication and by the absence of liver damage; however, it rapidly raised to pre-transplantation levels with the recurrence of hepatitis D (HD) in the liver graft. Monomeric 7S IgM anti-HD predominated over pentameric 19S antibody in each of the two patients examined for IgM anti-HD molecular species. The IgM antibody to HDV raises in response to HDV-induced damage and represents a valid surrogate marker of liver damage which is immunopathologically related to HDV infection. Besides providing diagnostic information, it provides the best predictor of impending resolution of chronic HDV disease, whether spontaneous or IFN-induced.

The detection of antimitochondrial antibodies (AMAs) is an important criterion for the diagnosis of primary biliary cirrhosis (PBC). During the last decade, the mitochondrial autoantigens have been cloned, sequenced, and identified as members of the 2-oxo-acid dehydrogenase pathway, including the E2 subunits of pyruvate dehydrogenase (PDC-E2), branched-chain 2-oxo-acid dehydrogenase (BCOADC-E2), and 2-oxo-glutarate dehydrogenase (OGDC-E2). We have developed a rapid and sensitive diagnostic test for use in PBC based on a triple hybrid recombinant molecule (r-MIT3) that contains the autoepitopes of PDC-E2, BCOADC-E2, and OGDC-E2. To help understand the frequency and antigen specificity of AMAs in an asymptomatic population and to identify patients with early disease, we investigated the prevalence of AMA, by enzyme-linked immunosorbent assay (ELISA), in a cohort of 1,530 people from northern Italy. Positive sera were further analyzed for immunoglobulin (Ig) isotypes, subclasses, and epitopes of AMA by a combination of ELISA and immunoblotting. In this cohort of 1,530 people, 9 (0.5%) reacted to r-MIT3 by ELISA. Of the 9 reactive sera, 2 recognized PDC-E2, 2 of 9 recognized BCOADC-E2, 1 of 9 recognized OGDC-E2, 2 of 9 recognized both PDC-E2 and BCOADC-E2, and 1 of 9 recognized PDC-E2 and OGDC-E2. AMA reactivity was primarily IgM and IgA. Epitope mapping revealed an AMA pattern of reactivity to PDC-E2 that differed from that found in patients with histologically proven PBC in most of the sera. However, 1 sera of a 72-year-old female with a normal alkaline phosphatase had an AMA profile identical to typical PBC. After a variable follow-up period (8-14 months), sera from 8 of 9 of these people were re-obtained for AMA and relative epitope mapping. Interestingly, the reactivity had a wider AMA pattern than before.

The intrahepatic accumulation of the c-myc protooncogene product was observed on immunofluorescence in each of six patients with chronic hepatitis delta virus infection who exhibited the hepatitis D antigen in their livers. The c-myc product was stained in the same nuclei that contained the hepatitis D antigen. C-myc was not observed in acute hepatitis D or in cases of chronic hepatitis delta virus infection without expression of the hepatitis D antigen. The protooncogene product was detected in only 1 of 32 viral and nonviral liver disorders unrelated to hepatitis delta virus. To confirm these observations, we transfected HBsAg-positive (PCL/PRF/5) and HBsAg-negative (HepG2) transformed liver cell lines with a plasmid containing a hepatitis delta virus cDNA trimer under the control of the SV40 early enhancer/promoter sequences. Whereas baseline c-myc expression was barely detectable in mock-transfected PLC/PRF/5 or HepG2 cells, strong c-myc nuclear fluorescence was observed when these same cells were transfected with the hepatitis D antigen expression vector. Similar results were obtained after infection of HeLa cells with a recombinant vaccinia virus expressing the hepatitis D antigen. Detection of c-myc mRNA sequences by means of in situ hybridization suggested that the c-myc product accumulation was not due to increased amounts of its mRNA. The c-myc protein accumulates selectively in the livers of patients with chronic hepatitis delta virus infection and in the same nuclei that contain the hepatitis D antigen. The expression of c-myc in hepatitis D antigen-containing cells does not require the presence of hepatitis B virus infection.

Eighty patients with chronic hepatitis C who completed a previously reported randomized controlled trial on the efficacy of interferon-alpha 2b were followed up for at least 36 mo after therapy discontinuation. Seventeen patients (21.2%) maintained normal ALT values throughout the follow-up; 63 (78.8%) either did not normalize the levels of ALT or relapsed during the follow-up. A significantly greater proportion of patients treated with 3 million units of interferon three times a week subcutaneously for 48 wk were long-term responders compared with patients treated for 24 wk. Sex, age, hepatitis C virus antibody status, source of infection and pretreatment levels of ALT were not predictive of long-term response. Cirrhosis was found to be an unfavorable predictive factor. After 3 yr of follow-up, clearance of viremia was observed in 58.9% of the 17 long-term responders but in none of the non-responders (p = 0.002). E2-NS1 antibody tested negative in 88.2% of long-term responders and in 14.3% of nonresponders (p = 0.001). Fifty-nine percent of long-term responders tested negative for C100-NS4 antibody compared with 14.3% of nonresponders (p = 0.031). No significant change was observed in other antibodies. Four long-term responders underwent liver biopsy 2 yr after discontinuation of therapy. All four patients had normal liver histology compared with baseline assessment of chronic active hepatitis in three and chronic persistent hepatitis in the other. Three of the four were negative for serum hepatitis C virus RNA.

To determine whether long-term therapy with recombinant interferon-alpha can improve the course of chronic delta hepatitis, 61 Italian patients with this disease were randomly assigned to receive either interferon-alpha-2b three times a week (5 MU/m2 for 4 mo and then 3 MU/m2 for another 8 mo) or no treatment. At the end of the 12-mo study, all patients were followed-up for 12 additional months. Normalization or decrease of more than 50% from baseline of serum ALT levels occurred in 42% of treated patients the fourth month of therapy, 26% the twelfth month and 3% the twenty-fourth month vs. 7%, 7% and 0%, respectively, in the control group. However, relapses occurred in 7 of 8 (87.5%) responders 1 to 10 mo (mean = 3.5 mo) after cessation of therapy. Liver biopsies were carried out at baseline and during the twelfth month of treatment. Histological improvement, mostly caused by decrease of portal inflammation, was observed in 57% of treated and 36% of untreated patients. Measures of antiviral activity (serum hepatitis delta virus RNA and intrahepatic hepatitis delta antigen) showed similar levels in treated and control patients. In treated patients the percentage of patients who were negative for HDV RNA never exceeded that of baseline. Although interferon-alpha in the dosage given in this study had no antiviral effect on patients with chronic hepatitis D, it reduced hepatic inflammation as measured by ALT levels. Whether a longer duration or reinstitution of interferon-alpha therapy would achieve long-term control of ALT levels and prevent chronic liver damage is not known.

Hepatitis D virus and hepatitis B virus nucleic acids were detected by Northern and Southern blot hybridization in the sera and livers of 85 chronic carriers of HBsAg and anti-hepatitis D followed up for a mean of 10 yr. We identified three subsets of patients: 13 with hepatitis D virus and hepatitis B virus viremia, 53 with serum hepatitis D virus RNA, but without hepatitis B virus DNA and 19 negative for both nucleic acids. Genomic and subgenomic forms of hepatitis D virus RNA were detected only in patients with hepatitis D virus and hepatitis B virus viremia. Histological findings and disease activity at admission were comparable in the three groups of patients, but the outcome was significantly worse in patients with active replication of both viruses; two of them died of terminal liver failure and hepatocellular carcinoma developed in two; the remaining patients had an uneventful course. These results suggest that active hepatitis B virus replication represents an important previously unrecognized determinant of severe liver damage in patients with chronic hepatitis D virus infection. Since hepatitis B virus provides the means for hepatitis D virus secretion and release from infected cells, active hepatitis B virus multiplication favoring the spread of hepatitis D virus from cell to cell may increase the pathogenetic potential of the defective agent.

Sixty-four heterosexual Italian carriers of HBsAg with chronic HBeAg and hepatitis B virus DNA-positive hepatitis were assigned randomly either to receive human lymphoblastoid interferon (injections of 5 million units per m2 three times per week for 6 months) or to serve as untreated controls. After 18 months of follow-up evaluation, 26 of the 33 treated patients (79%) had cleared hepatitis B virus DNA, 23 (70%) had lost HBeAg and 20 (61%) had seroconverted to anti-HBe. Fifteen of the 31 controls (48%) had cleared hepatitis B virus DNA (p = 0.01), 12 (39%) had lost HBeAg and nine (29%) had seroconverted to anti-HBe (p = 0.002). Eight treated patients but only one control had lost HBsAg and seroconverted to anti-HBs (24% vs. 3%, p = 0.01). Treated patients cleared hepatitis B virus markers after a mean interval of 4 months, compared with 8 months in the controls. All responders to interferon cleared intrahepatic HBcAg, and 50% showed histological improvement. The baseline hepatitis B virus DNA levels and the original histology were not predictive of a response to therapy; women appeared to respond better than men. Lymphoblastoid interferon provides an effective therapy in the heterosexual Italian patient with chronic hepatitis B.