Marco Lenzi

The NAFLD fibrosis score: a noninvasive system that identifies liver fibrosis in patients with NAFLD.

Patients with nonalcoholic fatty liver disease (NAFLD) and advanced liver fibrosis are at the highest risk for progressing to end‐stage liver disease. We constructed and validated a scoring system consisting of routinely measured and readily available clinical and laboratory data to separate NAFLD patients with and without advanced fibrosis. A total of 733 patients with NAFLD confirmed by liver biopsy were divided into 2 groups to construct (n = 480) and validate (n = 253) a scoring system. Routine demographic, clinical, and laboratory variables were analyzed by multivariate modeling to predict presence or absence of advanced fibrosis. Age, hyperglycemia, body mass index, platelet count, albumin, and AST/ALT ratio were independent indicators of advanced liver fibrosis. A scoring system with these 6 variables had an area under the receiver operating characteristic curve of 0.88 and 0.82 in the estimation and validation groups, respectively. By applying the low cutoff score (−1.455), advanced fibrosis could be excluded with high accuracy (negative predictive value of 93% and 88% in the estimation and validation groups, respectively). By applying the high cutoff score (0.676), the presence of advanced fibrosis could be diagnosed with high accuracy (positive predictive value of 90% and 82% in the estimation and validation groups, respectively). By applying this model, a liver biopsy would have been avoided in 549 (75%) of the 733 patients, with correct prediction in 496 (90%). Conclusion: a simple scoring system accurately separates patients with NAFLD with and without advanced fibrosis, rendering liver biopsy for identification of advanced fibrosis unnecessary in a substantial proportion of patients. (HEPATOLOGY 2007;45:846–854.)

Early occurrence and recurrence of hepatocellular carcinoma in HCV-related cirrhosis treated with direct-acting antivirals

BACKGROUND & AIMS Hepatocellular carcinoma (HCC) represents a serious complication of HCV-related cirrhosis. New direct-acting antivirals (DAA) cure HCV infection in over 90% of patients. The aim of this study was to evaluate the early occurrence and recurrence of HCC in cirrhotic patients treated with DAA. METHODS We analysed 344 consecutive cirrhotic patients, without HCC, who were treated with DAA, and followed for 24weeks. Fifty-nine patients had previous HCC. RESULTS DAA therapy induced sustained virological response in 91% of patients. During 24-week follow-up, HCC was detected in 26 patients (7.6%, 95% CI: 4.99-10.84): 17 of 59 patients (28.81%, 95% CI: 17.76-42.07) with previous HCC and 9 of 285 patients (3.16%, 95% CI: 1.45-5.90) without previous HCC. Child-Pugh Class B, more severe liver fibrosis, lower platelet count, and previous HCC were significantly associated with HCC development, at univariate analysis. At multivariate analysis, Child-Pugh class (p=0.03, OR: 4.18, 95% CI: 1.17-14.8) and history of HCC (p<0.0001, OR: 12.0, 95% CI: 4.02-35.74) resulted independently associated with HCC development. Among the 59 patients with previous HCC, younger age and more severe liver fibrosis were significantly associated with HCC recurrence, both at univariate and at multivariate analysis. CONCLUSIONS In patients with HCV-related cirrhosis, DAA-induced resolution of HCV infection does not seem to reduce occurrence of HCC, and patients previously treated for HCC have still a high risk of tumour recurrence, in the short term. For these reasons, all cirrhotic patients should be closely monitored and followed during and after antiviral therapy. LAY SUMMARY New direct-acting antivirals are able to eradicate HCV infection in over 90% of patients with advanced liver disease. Unfortunately, the occurrence of liver cancer is not reduced in effectively treated cirrhotic patients. In addition, patients previously treated for HCC have still a high risk of tumour recurrence in the short term, despite DAA treatment.

A randomized controlled trial of rituximab for the treatment of severe cryoglobulinemic vasculitis

OBJECTIVE To conduct a long-term, prospective, randomized controlled trial evaluating rituximab (RTX) therapy for severe mixed cryoglobulinemia or cryoglobulinemic vasculitis (CV). METHODS Fifty-nine patients with CV and related skin ulcers, active glomerulonephritis, or refractory peripheral neuropathy were enrolled. In CV patients who also had hepatitis C virus (HCV) infection, treatment of the HCV infection with antiviral agents had previously failed or was not indicated. Patients were randomized to the non-RTX group (to receive conventional treatment, consisting of 1 of the following 3: glucocorticoids; azathioprine or cyclophosphamide; or plasmapheresis) or the RTX group (to receive 2 infusions of 1 gm each, with a lowering of the glucocorticoid dosage when possible, and with a second course of RTX at relapse). Patients in the non-RTX group who did not respond to treatment could be switched to the RTX group. Study duration was 24 months. RESULTS Survival of treatment at 12 months (i.e., the proportion of patients who continued taking their initial therapy), the primary end point, was statistically higher in the RTX group (64.3% versus 3.5% [P < 0.0001]), as well as at 3 months (92.9% versus 13.8% [P < 0.0001]), 6 months (71.4% versus 3.5% [P < 0.0001]), and 24 months (60.7% versus 3.5% [P < 0.0001]). The Birmingham Vasculitis Activity Score decreased only after treatment with RTX (from a mean ± SD of 11.9 ± 5.4 at baseline to 7.1 ± 5.7 at month 2; P < 0.001) up to month 24 (4.4 ± 4.6; P < 0.0001). RTX appeared to be superior therapy for all 3 target organ manifestations, and it was as effective as conventional therapy. The median duration of response to RTX was 18 months. Overall, RTX treatment was well tolerated. CONCLUSION RTX monotherapy represents a very good option for severe CV and can be maintained over the long term in most patients.

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.

Frequency and significance of anti-gliadin and anti-endomysial antibodies in autoimmune hepatitis.

Celiac disease has been associated withautoimmune disorders, but its frequency in autoimmunehepatitis is unknown. Sera from 157 patients with type1 autoimmune hepatitis, 24 patients with type 2autoimmune hepatitis, 62 patients with primary biliarycirrhosis, 30 patients with chronic hepatitis B, and 80patients with chronic hepatitis C were tested forimmunoglobulin A anti-endomysial antibodies by indirect immunofluorescence and immunoglobulin A and Gantibodies to gliadin by enzyme immunoassay. Duodenalbiopsy evaluation was recommended in patientsseropositive for immunoglobulin A anti-endomysialantibodies. Immunoglobulin A anti-endomysial antibodieswere present in eight of the 181 patients withautoimmune hepatitis (4%), including six with type 1disease (4%) and two with type 2 disease (8%).Immunoglobulin A antibodies to gliadin were found in six ofthese eight patients, but they were also present in twoothers, including one patient with chronic hepatitis C.Five of the eight patients with immunoglobulin A antiendomysial antibodies, including threepatients with no gastrointestinal symptoms, had duodenalbiopsies and subtotal villous atrophy was present in allof them. No patient with primary biliary cirrhosis or chronic viral hepatitis had antiendomysialantibodies. The presence of celiac disease in autoimmunehepatitis is high (at least one in 36 patients) and itis predominantly asymptomatic. Screening with anti-endomysial and anti-gliadin antibodiesshould be performed and results confirmed withintestinal biopsy.

Prevalence of non-organ-specific autoantibodies and chronic liver disease in the general population: a nested case-control study of the Dionysos cohort

BACKGROUND Several retrospective and prospective studies report an increased prevalence of non-organ-specific autoantibodies (NOSAs) in patients with hepatitis C virus (HCV) related chronic liver disease (CLD). Some of the data so far available are controversial and the true prevalence of NOSAs in the general population is still not known. AIM To explore the prevalence of NOSAs, their relation to different HCV genotypes, and the presence and severity of CLD in the general population of Northern Italy. PATIENTS All 226 anti-HCV positive and 87 hepatitis B surface antigen (HBsAg) positive patients of the Dionysos cohort study were analysed and compared with sex and age matched cases (226) negative for both anti-HCV antibody and HBsAg selected from the same cohort. METHODS Sera tested for the presence of NOSAs (anti-nuclear antibody (ANA), anti-smooth muscle antibody (SMA), and anti-liver/kidney microsomes type 1 antibody (LKM1)) were screened by indirect immunofluorescence at a 1:40 serum dilution. HCV RNA and HCV genotypes were also determined by nested polymerase chain reaction (PCR) of the 5′ non-coding region and by PCR amplification of the core region with type specific primers. RESULTS The overall prevalence of NOSA reactivity was significantly higher in anti-HCV positive subjects than in both normal and pathological controls (25%v 6% and 7% respectively, p<0.05). ANA, SMA, and LKM1 occurred in 16, 10, and 1.3% of cases respectively. No specific association between NOSAs and a specific HCV genotype was found. NOSAs were found more often associated with more than one genotype (35.7%) and with untypable genotypes (34.6%), although the association was not statistically significant. NOSAs were associated with HCV RNA and CLD but not with the presence of cirrhosis and/or hepatocellular carcinoma. On univariate analysis, NOSA reactivity was independently associated with abnormal alanine aminotransferase (p<0.01) and γ-glutamyltranspeptidase levels (p<0.05). The risk for the presence of NOSAs was 5.1 times higher in anti-HCV subjects than in controls. CONCLUSIONS In the general population the prevalence of NOSAs is higher in anti-HCV positive subjects than in normal or disease controls. Moreover NOSAs are associated with CLD and with a more active disease in terms of alanine aminotransferase activity.

Characterization and clinical impact of antinuclear antibodies in primary biliary cirrhosis.

OBJECTIVES:The clinical impact of antinuclear antibodies in primary biliary cirrhosis is uncertain. We analyzed in detail the antinuclear antibodies reactivity of primary biliary cirrhosis patients and correlated the fine specificities observed with clinical, biochemical, and immunologic parameters.METHODS:A total of 96 consecutive primary biliary cirrhosis patients and 283 pathologic controls were studied. To dissect the fine antinuclear antibodies specificities we used different techniques, such as indirect immunofluorescence on cryostat tissue sections and cell culture (HEp-2 cells), counterimmunoelectrophoresis with thymus and spleen extracts, ELISA assays with recombinant Sp100 and purified gp210 and Lamin B receptor, and immunoblot with several recombinant nuclear and cytoplasmic antigens.RESULTS:Antinuclear antibodies were detected in 53% of patients, with the following hierarchy of specificities: 27% anti-Sp100, 16% ldquo;multiple nuclear dots,” 16% anti-gp210, 16% anti-centromere, 7% XR1, 6% anti-lamin B receptor, 5% anti–SS-A/Ro, 5% anti-ribonucleoprotein, 4% XR2, 2% anti–SS-B/La, 2% perinuclear antineutrophil cytoplasmic antibodies, and 1% anti–double-stranded deoxyribonucleic acid. Several patients showed multiple specificities. The “multiple nuclear dots” pattern was detected more often in antimitochondrial antibodies negative patients. In particular, primary biliary cirrhosis specific antinuclear antibodies (anti-Sp100, anti-gp210, and anti-lamin B receptor) were detected in nine of 13 antimitochondrial negative primary biliary cirrhosis cases. Anti-gp210 was more frequent in patients with more pronounced cholestasis and more impaired liver function.CONCLUSIONS:Antinuclear antibodies reactivities are present in more than half of primary biliary cirrhosis patients and target diverse autoantigens located in distinct subnuclear structures. Anti-gp210 identifies a subgroup of primary biliary cirrhosis patients with more serious liver disease. Positivity for anti-Sp100, anti-gp210, and anti-lamin B receptor, either alone or in combination, may act as a serologic marker of antimitochondrial antibodies negative primary biliary cirrhosis.

A multifaceted imbalance of T cells with regulatory function characterizes type 1 autoimmune hepatitis

Immunotolerance is maintained by regulatory T cells (Tregs), including CD4+CD25hi, CD8+CD28−, γδ, and CD3+CD56+ [natural killer T (NKT)] cells. CD4+CD25hi cells are impaired in children with autoimmune hepatitis (AIH). Little is known about Tregs in adults with AIH. The aim of this study was to investigate the frequency and function of Treg subsets in adult patients with AIH during periods of active disease and remission. Forty‐seven AIH patients (16 with active disease and 31 in remission) and 28 healthy controls were studied. Flow cytometry was used to evaluate surface markers and function‐related intracellular molecules in γδ, CD8+CD28−, NKT, and CD4+CD25hi cells. CD4+CD25hi T cell function was determined by the ability to suppress proliferation and interferon gamma (IFN‐γ) production by CD4+CD25− target cells. Liver forkhead box P3–positive (FOXP3+) cells were sought by immunohistochemistry. In AIH patients, particularly during active disease, CD4+CD25hi T cells were fewer, expressed lower levels of FOXP3, and were less effective at inhibiting target cell proliferation versus healthy controls. Moreover, although the numbers of CD8+CD28− T cells were similar in AIH patients and healthy controls, NKT cells were numerically reduced, especially during active disease, and produced lower quantities of the immunoregulatory cytokine interleukin‐4 versus controls. In contrast, γδ T cells in AIH patients were more numerous versus healthy controls and had an inverted Vδ1/Vδ2 ratio and higher IFN‐γ and granzyme B production; the latter was correlated to biochemical indices of liver damage. There were few FOXP3+ cells within the portal tract inflammatory infiltrate. Conclusion: Our data show that the defect in immunoregulation in adult AIH is complex, and γδ T cells are likely to be effectors of liver damage. (HEPATOLOGY 2010)

Liver/kidney microsomal antibody type 1 and liver cytosol antibody type 1 concentrations in type 2 autoimmune hepatitis

Background—Liver/kidney microsomal antibody type 1 (LKM1) and liver cytosol antibody type 1 (LC1) are the serological markers of type 2 autoimmune hepatitis (AIH). Aims—Since LKM1 and LC1 react against two distinct liver specific autoantigens (cytochrome P450IID6 (CYP2D6) and a 58 kDa cytosolic polypeptide respectively), the aim was to see whether LKM1 and LC1 concentrations correlate with liver disease activity. Patients—Twenty one patients with type 2 AIH were studied. Methods—All sera were tested by indirect immunofluorescence, counterimmunoelectrophoresis, and immunoblotting visualised by enhanced chemiluminescence. To evaluate LKM1 and LC1 levels, the 50 kDa microsomal reactivity (corresponding to CYP2D6) and the 58 kDa cytosolic reactivity were quantified by densitometric analysis. Results—Seven patients were positive for LKM1, nine for LC1, and five for both. Serial serum samples at onset and during immunosuppressive treatment were analysed in 13 patients (four positive for LKM1, six positive for LC1 and three positive for both). During remission, LKM1 concentration remained essentially unchanged in six of seven patients, and decreased in only one. Conversely, in two of nine patients, LC1 was completely lost, and, in the remaining seven, LC1 concentration was reduced by more than 50%. After immunosuppression tapering or withdrawal, flare ups of liver necrosis ensued with increasing LC1 concentration, but not LKM1. Conclusions—LC1 concentration, at variance with that of LKM1, parallels liver disease activity, and its participation in the pathogenic mechanisms of liver injury can be hypothesised.

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.