Francesco B. Bianchi

Simplified criteria for the diagnosis of autoimmune hepatitis

Diagnosis of autoimmune hepatitis (AIH) may be challenging. However, early diagnosis is important because immunosuppression is life‐saving. Diagnostic criteria of the International Autoimmune Hepatitis Group (IAIHG) were complex and purely meant for scientific purposes. This study of the IAIHG aims to define simplified diagnostic criteria for routine clinical practice. Candidate criteria included sex, age, autoantibodies, immunoglobulins, absence of viral hepatitis, and histology. The training set included 250 AIH patients and 193 controls from 11 centers worldwide. Scores were built from variables showing predictive ability in univariate analysis. Diagnostic value of each score was assessed by the area under the receiver operating characteristic (ROC) curve. The best score was validated using data of an additional 109 AIH patients and 284 controls. This score included autoantibodies, immunoglobulin G, histology, and exclusion of viral hepatitis. The area under the curve for prediction of AIH was 0.946 in the training set and 0.91 in the validation set. Based on the ROC curves, two cutoff points were chosen. The score was found to have 88% sensitivity and 97% specificity (cutoff ≥6) and 81% sensitivity and 99% specificity (cutoff ≥7) in the validation set. Conclusion: A reliable diagnosis of AIH can be made using a very simple diagnostic score. We propose the diagnosis of probable AIH at a cutoff point greater than 6 points and definite AIH 7 points or higher. (HEPATOLOGY 2008.)

Coeliac disease hidden by cryptogenic hypertransaminasaemia

BACKGROUND Hypertransaminasaemia of unknown, cryptogenic, origin occasionally has been found to be the only sign of coeliac disease. Raised concentrations of transaminases, or aminotransferases, have been retrospectively observed in about a half of patients with coeliac disease who are on a gluten-containing diet. We aimed to establish the overall prevalence of coeliac disease among patients with cryptogenic hypertransaminasaemia. METHODS Of the 600 consecutive patients referred to our outpatient clinic for liver disease due to raised serum transaminases from September, 1995, to June, 1997, 55 were classified as having cryptogenic hypertransaminasaemia after the exclusion of every known cause of liver disease. These patients were tested by indirect immunofluorescence for IgA to endomysium and for IgA and IgG to gliadin. FINDINGS Five patients were positive for both IgA to endomysium and IgG to gliadin, whereas IgA to gliadin was only found in four patients. IgG to gliadin was also present in another patient who was not positive for antibodies to endomysium. The six antibody-positive patients had duodenal biopsy that showed a subtotal villous atrophy consistent with coeliac disease in the five patients with antibodies to endomysium. The patient with only IgG to gliadin had a normal small-intestine mucosa. None of the five patients with coeliac disease had gastrointestinal symptoms. Liver biopsy samples were taken from three of the five patients with flat mucosa and showed a histological picture of nonspecific reactive hepatitis. Transaminase concentrations reverted to normal within 6 months in four patients with coeliac disease who followed a strict gluten-free diet. INTERPRETATION Our results show that about 9% of patients with cryptogenic hypertransaminasaemia are affected by symptom-free coeliac disease. Gluten-sensitive enteropathy and antibody screening for coeliac disease by means of antibodies to endomysium and gliadin should be considered in these patients.

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.

Cytochrome P4502D6(193-212): A new immunodominant epitope and target of virus/self cross-reactivity in liver kidney microsomal autoantibody type 1-positive liver disease

Cytochrome P4502D6 (CYP2D6), target of liver kidney microsomal autoantibody type 1 (LKM1), characterizes autoimmune hepatitis type 2 (AIH2) but is also found in patients with chronic hepatitis C virus (HCV) infection. To provide a complete linear epitope B cell map of CYP2D6, we tested peptides spanning the entire sequence of CYP2D6. In addition to confirming previously described antigenic sites, we identified four new epitopes (193–212, 238–257, 268–287, and 478–497). CYP2D6193–212 is immunodominant and was the target of 12 of 13 (93%) patients with AIH2 and 5 of 10 (50%) HCV/LKM1-positive patients. Because LKM1 is present in both AIH2 and a viral infection, we tested whether Abs to CYP2D6193–212 arise through cross-reactive immunity between virus and self. We identified a hexameric sequence “RLLDLA” sharing 5 of 6 aa with “RLLDLS” of HCV2985–2990 and all 6 aa with CMV130–135. Of 17 CYP2D6193–212-reactive sera, 11 (7 AIH and 4 HCV) reacted by ELISA with the HCV homologue, 8 (5 AIH and 3 HCV) with the CMV homologue, and 8 (5 AIH and 3 HCV) showed double reactivity. Autoantibody binding to CYP2D6193–212 was inhibited by preincubation with HCV2977–2996 or CMV121–140. Recombinant HCV-nonstructural protein 5 and CMV-UL98 proteins also inhibited Ab binding to CYP2D6193–212. Affinity-purified CYP2D6193–212-specific Ab inhibited the metabolic activity of CYP2D6. The demonstrated similarity and cross-reactivity between CYP2D6193–212 and two unrelated viruses suggests that multiple exposure to viruses mimicking self may represent an important pathway to the development of autoimmunity.

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.

High prevalence of celiac disease in Italian general population.

The worldwide increase of celiac disease prompted us to assess its prevalence in the Italian general population. The 3483 inhabitants of Campogalliano were tested for immunoglobulin A anti-endomysial antibodies. Twenty subjects showed antibody positivity and duodenal biopsy detected typical mucosal lesions of celiac disease in 17 of them; the remaining three cases had a normal villous architecture, but the finding of increased γ/δ intraepithelial lymphocytes in all and the heterodimer DQA1*0501, DQB1*0201 in two of them was consistent with potential celiac disease. Only one patient had an overt malabsorption syndrome, characterized by diarrhea, weight loss, and severe weakness. In eight subjects atypical symptoms of celiac disease, such as dyspepsia and depression, were present, whereas the remaining subjects were silent. Celiac disease was more frequent in younger age groups. Our cross-sectional design study demonstrates that celiac disease prevalence in the Italian general population is 4.9 per 1000 (95% CI 2.8–7.8), increasing up to 5.7 per 1000 (95% CI 3.5–8.8) with the inclusion of potential cases.

Celiac disease in autoimmune cholestatic liver disorders

OBJECTIVES:In this study, serological screening for celiac disease (CD) was performed in patients with autoimmune cholestasis to define the prevalence of such an association and to evaluate the impact of gluten withdrawal on liver disease associated with gluten sensitive enteropathy.METHODS:Immunoglobulin A endomysial, human and guinea pig tissue transglutaminase antibodies, and immunoglobulin A and G gliadin antibodies were sought in 255 patients with primary biliary cirrhosis, autoimmune cholangitis, and primary sclerosing cholangitis.RESULTS:Immunoglobulin A endomysial and human tissue transglutaminase antibodies were positive in nine patients (seven primary biliary cirrhosis, one autoimmune cholangitis, and one primary sclerosing cholangitis), whose duodenal biopsy results showed villous atrophy consistent with CD. Two of these patients had a malabsorption syndrome, and one had iron-deficiency anemia. Clinical and biochemical signs of cholestasis did not improve after gluten withdrawal in the three patients with severe liver disease. A longer follow-up of the six celiac patients with mild liver damage is needed to clarify whether gluten restriction can contribute to slow down the progression of liver disease.CONCLUSIONS:The high prevalence of CD (3.5%) in autoimmune cholestasis suggests that serological screening for CD should be routinely performed in such patients by immunoglobulin A endomysial or human tissue transglutaminase antibodies.

IgA anti-endomysial antibodies on human umbilical cord tissue for celiac disease screening. Save both money and monkeys.

Since celiac disease screening by traditional IgA anti-endomysial antibody test is limited by high costs of monkey esophagus commercial kits as well as by rising ethical problems related to the endangered species, the identification of an inexpensive and commonly available substrate for this antibody determination is urgently required. To achieve this goal, we compared the prevalence of IgA anti-endomysial antibodies detected on monkey esophagus with that on human umbilical cord. Fifty-seven (95%) of 60 untreated adult celiacs were positive for these antibodies on monkey esophagus as well as on human umbilical cord. IgA anti-endomysial antibodies, detected on both tissues, were negative in all 200 disease and healthy controls tested, displaying a 100% specificity for gluten-sensitive enteropathy. These data suggest that human umbilical cord can replace monkey esophagus for IgA anti-endomysial antibodies test. Human umbilical cord allows unlimited testing for celiac disease screening on wide series of high-risk subjects, permitting identification of greater numbers of asymptomatic celiac patients with a remarkable saving of money and bypassing the ethical problems related to killing monkeys.

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)