F. Cassani

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.

Non-organ-specific autoantibodies in nonalcoholic fatty liver disease: prevalence and correlates.

Eighty-four consecutive subjects with nonalcoholic fatty liver disease (NAFLD) were tested for non-organ-specific autoantibodies (NOSA) by indirect immunoflorescence. Indices of insulin resistance and biochemical and anthropometric parameters were assessed. The overall prevalence of anti-nuclear-antibodies (ANA), smooth muscle antibodies (SMA) and anti-mitochondrial-antibodies (AMA) was 35.7% (30/84), 18 subjects (21.4%) being positive for ANA, 4 (4.7%) for SMA, 6 for ANA and SMA, and 2 for AMA. NOSA-positive subjects were older (P < 0.01) and mostly females (63.3%). No significant difference was found in the age-corrected parameters studied, except for copper and ceruloplasmin, which was more elevated in NOSA-positive patients. The subset of high titer (≥1:100) ANA-positive patients had significantly (P < 0.05) greater insulin resistance than ANA-negative patients. In contrast, SMA-positive patients had higher gammaglobulin and significantly lower insulin resistance as compared to high-titer ANA-positive patients. In 3 NOSA-positive but not in NOSA-negative patients, liver biopsy disclosed features of overlapping NASH with autoimmune hepatitis, partially responding to diet combined with steroid treatment. In conclusion, NOSA positivity in NAFLD is more prevalent than in the general population. High-titre ANA but not SMA positivity is associated with insulin resistance.

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.

IgA antiendomysial antibody test. A step forward in celiac disease screening.

Serum IgA antiendomysial antibodies (EmA) were found in 61 (87%) of 70 adults and children with untreated celiac disease, whereas IgA antigliadin antibodies (AGA) and IgA R1-antireticulin antibodies (R1-ARA) were positive in 71% and 47%, respectively, of the same patients. Two of the nine untreated celiacs negative for IgA EmA showed positivity for IgA AGA. While IgA AGA and R1-ARA disappeared in all the celiacs, tested one year after gluten-free diet, IgA EmA persisted at low titer in seven (18%) of these 38 subjects, although the jejunal biopsy showed a complete regrowth of jejunal villi. All the disease control patients as well as the blood donors tested were always negative for the three IgA antibodies. Our results state that the search for both IgA EmA and AGA gives the best results in the screening of celiac disease, since the positivity for at least one of these two antibodies allows identification with a 100% specificity of the 90% of untreated celiac patients.

Antinuclear Antibodies and Patterns of Nuclear Immunofluorescence in Type 1 Autoimmune Hepatitis

To determine the significance of antinuclearantibodies and their patterns of indirectimmunofluorescence in type 1 autoimmune hepatitis, serafrom 99 patients were evaluated. Patients withantinuclear antibodies had a lower frequency of livertransplantation (6% vs 22%, P = 0.04) than seronegativepatients. They were also more commonly HLA-DR4- positivethan seronegative patients (56% vs 30%, P = 0.05) and normal subjects (56% vs 30%, P = 0.004).The 42 patients with antinuclear antibodies and adiffuse pattern of indirect immunofluorescence hadhigher serum titers of ANA (serum titers ≥1:500, 71%vs 14%, P < 0.0001) and SMA (serum titers ≥1:500,69% vs 27%, P = 0.003) than the 22 patients withantinuclear antibodies and a speckled pattern. Thesepatients, however, were otherwise not distinguished byclinical features and treatment response. Patients witha speckled pattern had A1-B8-DR3 more frequently thanpatients with a diffuse pattern (65% vs 23%, P = 0.005)and normal subjects (65% vs 13%, P < 0.0001), but they had no other salient features. We concludethat patients with antinuclear antibodies have a betterlong-term prognosis than seronegative patients, and theyhave HLA-DR4 more commonly. The patterns of indirect immunofluorescence associated withANA positivity have no practical clinicalimplications.

Autoimmune hepatitis in Italy: The Bologna experience

BACKGROUND/AIMS Autoimmune hepatitis affects mainly women. It is subdivided into type 1 and type 2 according to the autoantibody profile and without immunosuppression usually evolves to cirrhosis and end-stage liver failure. METHODS We evaluated clinical, biochemical, immunological and genetic features and treatment response of 163 consecutive Italian patients with autoimmune hepatitis. RESULTS At diagnosis, type 1 autoimmune hepatitis showed more inflamed liver histology and more pronounced cholestasis, whereas type 2 was more common in children. Male and female patients shared similar clinical, biochemical and immunological features. Of 89 patients with 5-year follow-up or longer, 23 patients irrespective of presenting clinical, biochemical and immunological features achieved complete remission (normal transaminases and gammaglobulin levels) which was maintained with minimal steroid dosage; attempt at treatment withdrawal led to disease exacerbation. Complete responders had more often HLA DRB1*0401 (p = 0.011) and their risk of disease progression was lower (p < 0.0001). CONCLUSIONS Type 1 and type 2 autoimmune hepatitis is one and the same disease. Autoimmune hepatitis has similar features in male and female patients. HLA DRB1*0401 positive patients are more likely to achieve complete remission. Continuous low-dose steroids are necessary to maintain remission, significantly reducing the risk of disease progression.

‘True’ antimitochondrial antibody-negative primary biliary cirrhosis, low sensitivity of the routine assays, or both?

Anti‐mitochondrial antibody (AMA) is considered the serological hallmark of primary biliary cirrhosis (PBC), but may be missing in a proportion of these patients. We assessed sensitivity and specificity of the currently available techniques for AMA detection in a large series of PBC patients and controls, and analysed their clinical and immunological features according to the AMA status. By indirect immunofluorescence on rat tissue sections and HEp‐2 cells, Western immunoblot with bovine submitochondrial particles, and two ELISAs with AMA‐specific recombinant proteins, we evaluated the presence of AMA in 127 PBC patients, 166 patients with type 1 autoimmune hepatitis and 100 with non alcoholic fatty liver disease. In PBC patients Western immunoblot detects AMA significantly more often than indirect immunofluorescence on HEp‐2 cells (85%versus 72%, P = 0·02) or rodent tissue sections (71%, P = 0·01); both ELISAs are only slightly less sensitive than Western immunoblot (81% and 78%). Ten patients with non alcoholic fatty liver disease were AMA‐positive by indirect immunofluorescence, but none recognized AMA‐specific epitopes in Western immunoblot or in ELISAs. Twelve patients with type 1 autoimmune hepatitis were AMA‐positive by indirect immunofluorescence, but only 6 (3·6%) reacted by Western immunoblot and ELISAs. Western immunoblot or ELISA should be regarded as first‐line assay for the detection of AMA. Up to 15% of PBC patients are consistently AMA‐negative, yet they share the same clinical, biochemical and histological features of AMA‐positive PBC. Detection of AMA in type 1 autoimmune hepatitis might identify a subset of patients at risk of developing a hepatitic/cholestatic syndrome.

Antibodies to filamentous actin (F-actin) in type 1 autoimmune hepatitis

Aims: To evaluate the diagnostic significance of anti-filamentous actin antibodies (A-FAA) assessed with a commercial ELISA in comparison with immunofluorescence reactivity and patterns of anti-smooth muscle antibodies (SMA); and to correlate A-FAA positivity with clinical, immunogenetic, laboratory, and histological features in patients with autoimmune hepatitis type 1 (AIH-1). Methods: We studied 78 consecutive untreated AIH-1 patients and 160 controls: 22 with autoimmune hepatitis type 2 (AIH-2), 51 with hepatitis C, 17 with coeliac disease (CD), 20 with primary biliary cirrhosis (PBC) and 50 blood donors. SMA was evaluated by indirect immunofluorescence (IIF) on frozen sections of rat tissues, and A-FAA with a modified commercial ELISA. Results: SMA was detected by IIF in 61 (78%) of 78 AIH-1 patients, of whom 47 (60%) had the SMA-T/G and 14 (18%) the SMA-V pattern. Of the pathological controls, 32 (20%) had the SMA-V pattern (25 with hepatitis C, 2 with AIH-2, 2 with PBC, 3 with CD). A-FAA were present in 55 AIH-1 patients (70.5%; 46 with SMA-T/G, 7 with SMA-V, and 2 SMA-negative), and in 10 controls (6%), of whom five had hepatitis C, two AIH-2, two PBC and one CD. The association between A-FAA and the SMA-T/G pattern was statistically significant (p<0.0001). A-FAA levels were higher in SMA-T/G positive than SMA-V positive AIH-1 patients and controls (p<0.0001). A-FAA positivity was significantly associated with higher γ-globulin and IgG levels, but did not correlate with other considered parameters. Conclusion: The modified A-FAA ELISA strictly correlates with the SMA-T/G pattern and is a reliable and operator independent assay for AIH-1. Detection of A-FAA, even if devoid of prognostic relevance, may be useful when interpretative doubts of standard IIF arise.