Abstract: OBJECTIVES: Some patients with primary biliary cirrhosis (PBC) have antinuclear antibodies (ANAs). These ANAs include the "multiple nuclear dots" (MND) staining pattern, targeting promyelocytic leukemia protein (PML) nuclear body (NB) components, such as "speckled 100-kD" protein (Sp100) and PML. A new PML NB protein, designated as Sp140, was identified using serum from a PBC patient. The aim of this study was to analyze the immune response against Sp140 protein in PBC patients. METHODS: We studied 135 PBC patients and 157 pathological controls with type 1 autoimmune hepatitis, primary sclerosing cholangitis, and systemic lupus erythematosus. We used indirect immunofluorescence and a neuroblastoma cell line expressing Sp140 for detecting anti-Sp140 antibodies, and a commercially available immunoblot for detecting anti-Sp100 and anti-PML antibodies. RESULTS: Anti-Sp140 antibodies were present in 20 (15%) PBC patients but not in control samples, with a higher frequency in antimitochondrial antibody (AMA)-negative cases (53 vs. 9%, P<0.0001). Anti-Sp140 antibodies were found together with anti-Sp100 antibodies in all but one case (19 of 20, 90%) and with anti-PML antibodies in 12 (60%) cases. Anti-Sp140 positivity was not associated with a specific clinical feature of PBC. CONCLUSIONS: Our study identifies Sp140 as a new, highly specific autoantigen in PBC for the first time. The very frequent coexistence of anti-Sp140, anti-Sp100 and anti-PML antibodies suggests that the NB is a multiantigenic complex in PBC and enhances the diagnostic significance of these reactivities, which are particularly useful in AMA-negative cases.
Abstract: Anti nuclear (ANA) immunomorphological patterns such as multiple nuclear dots (MND) and rim-like/membranous (RL/M) are considered highly specific but little sensitive for primary biliary cirrhosis (PBC) diagnosis. To evaluate frequency and clinical significance of MND and RL/M in PBC patients when investigated at the level of immunoglobulin G isotypes. MND and RL/M pattern have been tested in 141 PBC sera and 230 pathological controls using HEp-2 cells as substrate and anti- total IgG and individual IgG subclasses (IgG1, IgG2, IgG3, IgG4) as specific antisera. One hundred and fourteen of 141 (80%) PBC patients had RL/M or MND pattern when IgG subclasses were used as revealing reagents (vs. 34% when anti total IgG were used, p < 0.0001). The prevalent isotype was IgG1 for RL/M, and IgG2 for MND pattern. None of controls was positive. No clinical differences in terms of severity and outcome of disease have been observed in PBC patients positive for MND and RL/M when investigated with IgG isotypes. The research for RL/M and MND pattern at level of IgG isotype determines a wide gain in terms of sensitivity without a loss of specificity. In Italian PBC patients MND and RL/M pattern did not seem to characterize any subgroup of patients with a poorer prognosis.
Abstract: 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.
Abstract: BACKGROUND/AIMS: Anti-liver/kidney microsomal antibody type 1 (anti-LKM1), a serological marker of type 2 autoimmune hepatitis, is also detected in a small proportion of patients with hepatitis C. This study aimed to evaluate clinical features and effect of antiviral therapy in patients with hepatitis C who are anti-LKM1 positive. METHODS: Sixty consecutive anti-LKM1 positive and 120 age and sex-matched anti-LKM1 negative chronic hepatitis C patients were assessed at diagnosis and during follow-up. Of these, 26 anti-LKM1 positive and 72 anti-LKM1 negative received antiviral therapy. Anti-LKM1 was detected by indirect immunofluorescence and immunoblot. Number of HCV-infected hepatocytes and intrahepatic CD8+ lymphocytes was determined by immunohistochemistry. RESULTS: At diagnosis anti-LKM1 positive patients had higher IgG levels and more intrahepatic CD8+ lymphocytes (p 0.022 and 0.046, respectively). Viral genotypes distribution and response to therapy were identical. Hepatic flares during antiviral treatment only occurred in a minority of patients in concomitance with anti-LKM1 positivity. CONCLUSIONS: Immune system activation is more pronounced in anti-LKM1 positive patients with hepatitis C, possibly representing the expression of autoimmune mechanisms of liver damage. Antiviral treatment is as beneficial in these patients as in anti-LKM1 negative patients, and the rare necroinflammatory flares are effectively controlled by corticosteroids, allowing subsequent resumption of antiviral therapy.
Abstract: Autoimmune hepatitis (AIH) is a chronic progressive hepatitis, characterized by interface hepatitis with lymphoplasmacellular infiltrates on liver biopsy, high serum globulin level and circulating autoantibodies. It is classified into two types, according to autoantibody profile: type 1 is characterized by anti-nuclear (ANA) and/or anti-smooth muscle (SMA) antibodies; type 2 by anti-liver kidney microsomal type 1 (anti-LKM-1) antibodies. AIH affects all ages, may be asymptomatic, frequently has an acute onset, and can present as fulminant hepatitis. The diagnosis of AIH is based on a scoring system codified by an international consensus. Corticosteroids alone or in conjunction with azathioprine is the treatment of choice in patients with AIH and results in remission induction in over 80% of patients. Alternative proposed strategies in patients who have failed to achieve remission on standard therapy or patients with drug toxicity include the use of cyclosporine, tacrolimus, budesonide or mycophenolate mofetil. Liver transplantation is the treatment of choice in managing decompensated disease, however AIH can recur or develop de novo after liver transplantation.
Abstract: OBJECTIVES: During the last decade patients with concomitant clinical, biochemical, immunoserological, and histological features of both autoimmune hepatitis (AIH) and primary biliary cirrhosis (PBC) were sporadically described, but definite diagnostic criteria and specific serological markers to support the diagnosis of AIH/PBC overlap syndrome (AIH/PBC OS) are still lacking. METHODS: Clinical, biochemical, and histological features, autoantibody profile, and treatment response of 15 patients with coexistent hepatitic and cholestatic liver damage, all fulfilling strict diagnostic criteria for both AIH and PBC, were compared with those of 120 patients with pure PBC and 120 patients with pure AIH. RESULTS: At diagnosis, the AIH/PBC OS patients' median age was 51 years, similar to that of the PBC patients (52 years, P=NS), but significantly higher than that of the AIH patients (40 years, P=0.04). Anti-dsDNA antibodies were detected in 60% of AIH/PBC OS patients, but only in 4% of PBC patients and 26% of AIH patients (P<0.0001 and 0.01, respectively). Double positivity for antimitochondrial antibodies (AMA) and anti-dsDNA was present in 47% of those with AIH/PBC OS, but only in 2% of the pathological controls (P<0.0001; specificity: 98; 95% confidence interval (CI): 97-99.2; positive likelihood ratio: 28; 95% CI: 9.8-79.4). Combined therapy (ursodeoxycholic acid (UDCA) plus steroids) achieved biochemical response in 77% of AIH/PBC OS patients. CONCLUSIONS: Concomitant AMA/anti-dsDNA seropositivity can be considered the serological profile of AIH/PBC OS. The combination of UDCA and steroids is effective in achieving persistent biochemical amelioration in most AIH/PBC OS patients.
Abstract: Autoimmune liver disease (ALD) includes a spectrum of diseases which comprises both cholestatic and hepatitic forms: autoimmune hepatitis (AIH), primary biliary cirrhosis (PBC), primary sclerosing cholangitis (PSC) and the so called "overlap" syndromes where hepatitic and cholestatic damage coexists. All these diseases are characterized by an extremely high heterogeneity of presentation, varying from asymptomatic, acute (as in a subset of AIH) or chronic (with aspecific symptoms such as fatigue and myalgia in AIH or fatigue and pruritus in PBC and PSC). The detection and characterization of non organ specific autoantibodies plays a major role in the diagnostic approach of autoimmune liver disease; anti nuclear reactivities (ANA) and anti smooth muscle antibodies (SMA) mark type 1 AIH, liver kidney microsomal antibody type 1 (LKM1) and liver cytosol type 1 (LC1) are the serological markers of type 2 AIH; antimitochondrial antibodies (AMA) are associated with PBC, while no specific marker is found in PSC, since anticytoplasmic neutrophil antibodies with perinuclear pattern (atypical p-ANCA or p-ANNA) are also detected in a substantial proportion of type 1 AIH cases. Treatment options rely on immunosoppressive therapy (steroids and azathioprine) in AIH and on ursodeoxycholic acid in cholestatic conditions; in all these diseases liver transplantation remains the only therapeutical approach for the end stage of liver disease.
Abstract: Antimitochondrial antibodies (AMA) are the serologic cornerstone in the diagnosis of primary biliary cirrhosis (PBC), even if they are not detectable in a proportion of patients, notwithstanding the most sensitive and sophisticated technologies used. To fill in the serologic gap in AMA-negative PBC, there is sound evidence to consider antinuclear antibody (ANA) patterns, such as anti-multiple nuclear dots and anti-membranous/rim-like, as PBC-specific surrogate hallmarks of the disease, and their detection can be considered virtually diagnostic. Furthermore, particular ANA specificities, such as anti-gp210, anti-p62, anticentromere antibodies, and anti-dsDNA, may provide additional diagnostic and prognostic information.
Abstract: 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.
Abstract: Hepatitis C virus (HCV) infection is characterized by a number of autoreactive manifestations, such as autoantibody production, cryoglobulinemia and thyroid disorders. We will analyse critically the mechanisms invoked, and partially documented, to explain such manifestations arising in genetically predisposed individuals exposed to HCV. In particular we will examine the available evidence implicating the virus in lowering the B cell activation threshold, in directly infecting lymphocytes and in inducing self-reactivity through a mechanism of molecular mimicry. We will then move to the HCV related clinical immunopathological manifestations, with a specific attention to the effects of antiviral treatment.
Abstract: Antibodies to liver/kidney microsome type 1 occur in Italian patients with hepatitis C, but rarely develop in North American patients. Our goals were to compare the frequencies of the HLA markers associated with autoimmune expression in Italian and North American patients with chronic hepatitis C and to determine genetic bases for regional differences in antibody production. HLA B8, DR3, DR4, DR7, DR11, DR13, DQ2, and the B8-DR3-DQ2 haplotype were determined by microlymphocytotoxicity and polymerase chain reaction in 105 Italian patients (50 with microsomal antibodies), 100 North American patients (none with microsomal antibodies), and Italian and North American healthy control subjects. Italian patients with microsomal antibodies differed from North American patients without these antibodies by having a higher frequency of HLA DR7 (54% vs. 27%, P=0.002). HLA DR7 occurred more frequently in seropositive Italian patients than in seronegative counterparts (54% vs. 11% P < 0.0001), Italian healthy control subjects (54% vs. 29%, P=0.0009), and North American healthy control subjects (54% vs. 19%, P < 0.0001). The frequency of HLA DR7 was similar in North American patients and controls (27% vs. 19%, P=0.2), but it was lower than in Italian controls (19% vs. 29%, P=0.059). Seropositive Italian patients had a lower frequency of HLA DR11 than seronegative Italian patients and Italian controls (18% vs. 34%, P=0.07, and 18% vs. 35%, P=0.02, respectively). In contrast to seropositive Italian patients, North American patients had HLA DR4 (30% vs. 12%, P=0.02), HLA DR13 (29% vs. 10%, P=0.01), and the B8-DR3-DQ2 haplotype (23% vs. 6%, P=0.01) more often. Similarly, HLA DR4 and the B8-DR3-DQ2 phenotype were more frequent in North American patients than in Italian controls (30% vs. 16%, P=0.005, and 23% vs. 7%, P=0.00002, respectively). HLA DR7 is associated with the development of microsomal antibodies in Italian patients with chronic hepatitis C. The lower frequency of HLA DR7 in North America could contribute to the rarity of these antibodies in this region. HLA DR11 may be protective against the development of microsomal antibodies in Italian patients, whereas HLA DR4, HLA DR13, and the B8-DR3-DQ2 haplotype may be protective in North American patients.
Abstract: BACKGROUND: Primary biliary cirrhosis (PBC) may be associated with various rheumatological disorders. AIM: To investigate the frequency and significance of 'rheumatological' antinuclear antibodies in the field of autoimmune chronic liver disease, with special regard to PBC. METHODS: We studied 105 patients with PBC, 162 autoimmune liver disease controls (type 1 and 2 autoimmune hepatitis, primary sclerosing cholangitis), 30 systemic lupus erythematosus and 50 blood donors. Sera were tested for the presence of antibodies to extractable nuclear antigens (anti-ENA) by counterimmunoelectrophoresis, enzyme-linked and immunoblot (IB) assay, and for the presence of anti-centromere antibodies (ACA) by indirect immunofluorescence on HEp-2 cells and IB. RESULTS: The overall prevalence of IB-detected anti-ENA in PBC (30%) was higher than in type 1 autoimmune hepatitis (2.5%, P < 0.0001), type 2 autoimmune hepatitis (0%, P < 0.0001) and primary sclerosing cholangitis (11.5%, P = 0.006) and lower than in systemic lupus erythematosus (53%, P = 0.03). The most frequent anti-ENA reactivity in PBC was anti-SSA/Ro-52kD (28%). ACA were detected by IB in 21% PBC patients and never in the other subjects (P < 0.0001). Anti-SS-A/Ro/52kD positive PBC patients had at the time of diagnosis a more advanced histological stage (P = 0.01) and higher serum levels of bilirubin (P = 0.01) and IgM (P = 0.03) compared with negative ones. CONCLUSIONS: In the autoimmune liver disease setting, anti-SS-A/Ro-52kD and ACA have a high specificity for PBC and can thus be of diagnostic relevance in anti-mitochondrial antibodies negative cases. If confirmed in further studies with adequate follow-up, anti-SS-A/Ro-52kD antibodies might identify PBC patients with a more advanced and active disease.
Abstract: After the discovery of HCV in 1989 a great amount of data has been produced in order to identify a possible aetiology for a number of idiopathic diseases, especially those with a suspected immune origin. Many associations have not been confirmed by prospective studies (as in the case of autoimmune hepatitis); other immune abnormalities, such as the emergence of non organ-specific autoantibodies and cryoglobulins, have been reported by many specific studies. To date, the link between HCV and autoreactivity is tentatively explained on the basis of sequence homologies shared by the HCV polyprotein and "self" proteins (such as CYP 2D6, target of anti-LKM1) (molecular mimicry mechanism); a second interpretation relies on the demonstration that the HCV - B lymphocyte interaction is able to induce a polyclonal B cell activation, an important cofactor for the development of clinically relevant B-lymphocyte autoimmune disorders. In this review we will focus on the major aspects of the autoimmune phenomena in HCV-infected patients, their clinical and therapeutical implications.
Abstract: 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 gamma-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.
Abstract: GM and KM allotypes-genetic markers of immunoglobulin (Ig) gamma and kappa chains, respectively-are associated with humoral immunity to several infection- and autoimmunity-related epitopes. We hypothesized that GM and KM allotypes contribute to the generation of autoantibodies to liver/kidney microsomal antigen 1 (LKM1) in hepatitis C virus (HCV)-infected persons. To test this hypothesis, we characterized 129 persons with persistent HCV infection for several GM and KM markers and for anti-LKM1 antibodies. The heterozygous GM 1,3,17 23 5,13,21 phenotype was significantly associated with the prevalence of anti-LKM1 antibodies (odds ratio, 5.13; P=0.002), suggesting its involvement in this autoimmune phenomenon in HCV infection.
Abstract: BACKGROUND: Serum antinuclear antibodies giving the 'multiple nuclear dots' or the 'rim-like/membranous' patterns are frequently detected by indirect immunofluorescence on HEp-2 cells in patients with primary biliary cirrhosis. AIM: To assess the accuracy of multiple nuclear dot and rim-like/membranous antinuclear antibodies for the diagnosis of primary biliary cirrhosis. METHODS: Sera from 4371 consecutive patients referred to our laboratory were analysed under code for antinuclear antibodies testing by indirect immunofluorescence on HEp-2 cells. RESULTS: Review of the clinical records of the 4371 patients allowed identification of 101 patients with antimitochondrial antibody-positive primary biliary cirrhosis and 22 with antimitochondrial antibody-negative variant. Multiple nuclear dot and/or rim-like/membranous patterns were found in 59 (1.3%) of the 4371 patients: 31 antimitochondrial antibody-positive primary biliary cirrhosis, 17 antimitochondrial antibody-negative primary biliary cirrhosis and 11 non-primary biliary cirrhosis. The specificity for primary biliary cirrhosis of both the antinuclear antibodies pattern was 99%. Positive predictive value and likelihood ratio for a positive test were 86% (95% CI: 72.7-94) and 221 (95% CI: 91.7-544) for multiple nuclear dot, 79% (95% CI: 62.2-90.1) and 132 (95% CI: 56.8-312.7) for rim-like/membranous, respectively. CONCLUSIONS: Multiple nuclear dot and rim-like/membranous antinuclear antibodies are rare findings. Their positivity strongly suggests the diagnosis of primary biliary cirrhosis, irrespective of antimitochondrial antibody status. The high specificity for primary biliary cirrhosis makes them a useful diagnostic tool especially in antimitochondrial antibody-negative patients.
Abstract: Anti-mitochondrial antibodies are the serological markers of primary biliary cirrhosis. We analysed the detailed anti-mitochondrial antibodies patterns to see whether the immunological specificities detected at the time of the diagnosis correlate with the histological, clinical and immunological expression of the disease. One hundred and thirty primary biliary cirrhosis patients were studied at the time of presentation/diagnosis. Anti-mitochondrial antibodies reactivity was dissected and evaluated by Western immunoblotting with bovine heart submitochondrial particles as antigenic source. Six different Western immunoblotting patterns have been identified with the following hierarchy: pattern A (anti-PDC-E2+anti-E3BP, 38.5%), pattern B (anti-PDC-E2+anti-E3BP+anti-OGDC-E2, 20.8%), pattern C (anti-PDC-E2+anti-E3BP+anti-BCOADC-E2+anti-OGDC-E2, 13.1%), pattern D (anti-PDC-E2+anti-E3BP+anti-BCOADC-E2, 6.9%), pattern E (anti-BCOADC-E, 6.1%) and pattern F (anti-mitochondrial antibodies negative primary biliary cirrhosis, 14.6%). The different patterns were neither associated with peculiar clinical, biochemical, histological and immunological features nor with the Mayo Risk Score. The anti-mitochondrial antibodies pattern at presentation is independent of the stage of the liver disease; therefore, the Western immunoblotting characterisation of anti-mitochondrial antibodies does not seem to be helpful in identifying the clinical, biochemical or histological expression of primary biliary cirrhosis at the time of the diagnosis.
Abstract: AIM: Our goals were to analyze the known genetic predispositions for autoimmune hepatitis (AIH) in AIH Italian population and to compare them with North American counterparts. METHODS: Human leukocyte antigens (HLA) B8, C7, DR3, DR4, DR7, DR11, DR13, DQ2 and the B8-DR3-DQ2 phenotype were determined by microlymphocytotoxicity and polymerase chain reaction in 74 Italian patients (57 with type 1 and 17 with type 2 AIH) and 149 North American patients with type 1 AIH, and in adequate controls. RESULTS: B8-DR3-DQ2 occurred more frequently in Italian patients with type 1 AIH than in Italian controls (30% vs 7%, P<0.0001), but less frequently than in North American counterparts (30% vs 48%, P = 0.02). DR4 occurred less frequently in Italian patients with type 1 AIH (23% vs 43%, P = 0.01) and in controls (16% vs 34%, P = 0.0003) than in North American counterparts. No differences were found in alleles' frequency between type 1 and type 2 Italian AIH patients. DR11 had a frequency lower in type 1 Italian AIH patients than controls (17% vs 35%, P = 0.01). CONCLUSION: HLA DR4 is not associated with AIH in Italy. The known HLA risk factors for AIH occur similarly in Italian patients with type 1 and type 2 AIH, and they are less frequent than in North American patients. B8-DR3-DQ2 is the predominant phenotype of type 1 AIH also in Italy, and HLA DR11 may be a regionally distinctive protective factor against type 1 AIH.
Abstract: BACKGROUND: Hepatitis C virus (HCV)-related chronic hepatitis is frequently associated with non-organ-specific autoantibodies (NOSAs), but available data about the relationship between NOSA positivity and the effect of antiviral therapy in persons with hepatitis C are few and controversial. Our aim was to evaluate the impact of NOSA positivity on the outcome of combined antiviral therapy in HCV-positive patients. METHODS: A total of 143 consecutive adult patients with hepatitis C were studied. Antinuclear antibody (ANA), anti-smooth muscle antibody (SMA), and anti-liver/kidney microsomal antibody type 1 (LKM1) were detected by indirect immunofluorescence. All patients were treatment naive and received combined antiviral therapy (interferon [IFN]-ribavirin) after enrollment in the study. Patients were classified as nonresponders if HCV RNA was detectable after 6 months of therapy, as relapsers if abnormal transaminase levels and reactivation of HCV replication were observed after the end of treatment, and as long-term responders if transaminase levels were persistently normal and HCV RNA was undetectable 6 months after the end of treatment. RESULTS: Thirty-seven patients (25%) were NOSA positive (SMA was detected in 19 patients, ANA in 10, ANA and SMA in 4, LKM1 in 3, and SMA and LKM1 in 1). The prevalence of long-term response was similar between NOSA-positive patients and NOSA-negative patients (48.6% vs. 56.6%; P=not significant). Compared with HCV genotype 1 (HCV-1), HCV genotypes other than 1 were more often associated with long-term response among NOSA-positive patients (93.3% vs. 30%; P=.0017). The overall rate of long-term response, irrespective of NOSA status, was 54.5%. Detection of HCV-1 and elevated gamma-glutamyl transpeptidase serum levels were independent negative prognostic factors of treatment response (P=.007 and P=.026, respectively). CONCLUSIONS: Combined antiviral treatment (IFN-ribavirin) is safe and effective in NOSA-positive patients with hepatitis C, even if long-term response is less likely in those infected with HCV-1.
Abstract: BACKGROUND: The usual onset of type 1 autoimmune hepatitis occurs at puberty or around menopause, whereas disease presentation in the advanced age is less often reported. AIM: To assess the clinical, immunological and histological features of Type 1 autoimmune hepatitis in elderly Italian patients. METHODS: We assessed, at diagnosis, the clinical and immunological features of 76 consecutive Italian patients with type 1 autoimmune hepatitis, focusing particularly on a subgroup of 20 patients presenting at > or = 65 years (females 95%, median age 72 years, range 65-82). RESULTS: In comparison with the younger group, at the time of autoimmune hepatitis diagnosis, elderly Italian patients are more often asymptomatic (25% vs. 7%; P = 0.04), are more frequently positive for antinuclear autoantibodies (95% vs. 52%; P = 0.0004) and HLA-DR4 (45% vs. 18%; P = 0.03); among the extra-hepatic manifestations, autoimmune thyroid disorders are prevalent in the elderly group (25% vs. 5%; P = 0.02). However, no difference was observed in the histological/biochemical expression of the liver disease and response to immunosuppression. CONCLUSIONS: In elderly Italian patients, autoimmune hepatitis has typical serological and genetic characteristics, is more frequently asymptomatic, although prognosis and response to therapy is similar to that of younger patients. As a concomitant autoimmune thyroid disorder is common, autoimmune hepatitis should be suspected and investigated in elderly patients with autoimmune thyroid disorder and abnormal liver function tests.
Abstract: BACKGROUND: Anti-Saccharomyces cerevisiae and perinuclear anti-neutrophil cytoplasmic autoantibodies are markers of Crohn's disease and ulcerative colitis respectively. AIM: To determine the prevalence of anti-S. cerevisiae and perinuclear anti-neutrophil cytoplasmic autoantibodies in a large series of coeliac disease patients before and after gluten free diet, and to correlate anti-S. cerevisiae-positivity with intestinal mucosal damage. METHODS: One hundred and five consecutive coeliac disease patients and 141 controls (22 ulcerative colitis, 24 Crohn's disease, 30 primary sclerosing cholangitis, 15 postenteritis syndrome, 50 blood donors) were tested for anti-S. cerevisiae by enzyme-linked immunosorbent assay and for perinuclear anti-neutrophil cytoplasmic autoantibodies by indirect immunofluorescence. RESULTS: In coeliac disease anti-S. cerevisiae (immunoglobulin G and/or immunoglobulin A) were slightly less frequent (59%) than in Crohn's disease (75%, P = 0.16) and significantly more frequent than in ulcerative colitis (27%), primary sclerosing cholangitis (30%), postenteritis syndrome (26%) and blood donors (4%) (P = 0.009, P = 0.0002, P = 0.025, P < 0.0001). No correlation was found between anti-S. cerevisiae and degree of mucosal damage. Perinuclear anti-neutrophil cytoplasmic autoantibodies were detected only in one coeliac. After gluten free diet the disappearance of anti-S. cerevisiae-immunoglobulin A (93%) was more frequent than that of immunoglobulin G (17%, P = 0.0001); perinuclear anti-neutrophil cytoplasmic autoantibodies disappeared in the only coeliac positive at diagnosis. CONCLUSION: More than half of untreated coeliacs are anti-S. cerevisiae-positive irrespective of the severity of mucosal damage. Differently from immunoglobulin A, anti-S. cerevisiae-immunoglobulin G persisted in more than 80% after gluten free diet. The high prevalence of anti-S. cerevisiae in coeliac disease suggests that they may be the effect of a non-specific immune response in course of chronic small bowel disease.
Abstract: The preliminary question regarding the clinical issue of the antiviral therapy in the HCV related chronic hepatitis patients is: is it mandatory the research for the autoantibodies in the eligible patients for the antiviral treatment? This issue is of particular interest at the light of the the reported cases of HCV positive patients with positivity for liver kidney microsome type 1 antibody who developed a hepatitic flare during the antiviral treatment. The data from literature about the efficacy and safety on the antiviral treatment in patients with autoantibodies are few and controversial, particularly if the ones regarding antiviral drugs and more recent treatment regimens are taking into account (peg-interferon, combined therapy of interferon and ribavirin). Large and prospective studies are needed for a thorough evaluation about the potential impact of autoantibodies reactivity on the therapeutic outcome. To date, it must be confirmed that a strict monitoring of hepatic parameters is to recommend during the whole treatment phase. This in the light of a potential appearance of significant flares of aminotransferases, particularly in subjects with anti LKM-1 autoantibodies, during interferon therapy.
Abstract: BACKGROUND: There is increasing evidence that hepatic steatosis contributes to the progression of liver fibrosis, whereas its impact on the efficacy of anti-viral treatment is still under investigation. AIM: To evaluate the effect of steatosis on the outcome of combined anti-viral treatment. METHODS: We studied 102 consecutive naive patients with chronic hepatitis C receiving combined anti-viral therapy (peg-interferon alpha-2b and ribavirin). RESULTS: Fifty (49%) of 102 patients had evidence of hepatic steatosis (29 grade 1, 16 grade 2 and 5 grade 3). Sustained virological response was similar in patients with and without steatosis (58% vs. 56%); moreover, the grade of steatosis did not affect the rate of sustained virological response (grade 1: 58%, grade 2: 56% and grade 3: 60%). Patients with steatosis had significantly higher serum levels of aspartate transaminase, alanine transaminase and gamma-glutamyltransferase (P = 0.007, 0.004 and 0.03, respectively), higher histological activity (P = 0.03), more advanced stage of fibrosis (P = 0.0394) and more often hepatitis C virus genotype 3 (P = 0.04). CONCLUSIONS: Our findings suggest that hepatic steatosis in chronic hepatitis C, irrespective of its grade, is not a negative prognostic factor of response to combined anti-viral therapy, even when the histological and biochemical profile of the disease is more aggressive.
Abstract: BACKGROUND & AIMS: Autoimmune thyroid disorders (AITDs) are reported, especially during interferon treatment, in chronic HCV infection, in which non-organ-specific autoantibodies (NOSAs) are common. We wondered whether seropositivity for NOSA is associated with susceptibility to AITDs. METHODS: We evaluated thyroid function and antithyroglobulin and antithyroperoxidase antibodies in 348 Italian patients with chronic hepatitis C (34% NOSA-positive), 196 patients (33% NOSA-positive) of whom received interferon treatment. RESULTS: At baseline, thyroid disorders were significantly more frequent in liver/kidney microsomal antibody type 1 (LKM1)-positive patients (29% vs 9%, P < .005). Similarly, on interferon therapy de novo autoimmune thyroid markers and/or symptomatic thyroid disorders appeared more often in LKM1-positive patients (50% vs 3%, P < .0001). Both female sex and LKM1 positivity were predictors of AITD, but only the latter remained significant after logistic regression analysis. Cross-reactivity to all 7 linear epitopes encoding homologous amino acid sequences shared by the HCV polyprotein, CYP2D6 (the LKM1 autoantigen), and thyroperoxidase was detected in 86% LKM1-positive HCV patients with clinical thyroid disorders, but in none of the LKM1-positive or negative HCV patients without thyroid disease, and none of an HCV-negative control group comprising subjects with LKM1-positive autoimmune hepatitis or AITD without liver disease ( P < .0001). CONCLUSIONS: Patients receiving interferon therapy for hepatitis C seropositive for LKM1 are susceptible to develop AITDs, in association with treatment. Molecular mimicry and epitope spreading are potential pathogenic mechanisms.
Abstract: Liver Kidney Microsomal autoantibody type 1(LKM1) directed to cytochrome P4502D6 (CYP2D6) characterises autoimmune hepatitis type-2 (AIH-2), but is also found in a proportion of chronic hepatitis C virus (HCV) infected patients, CYP2D6252-271 being a major B- cell autoepitope. Molecular mimicry and immunological cross-reactivity between CYP2D6252-271, HCV polyprotein and the infected cell protein 4 (ICP4) of herpes simplex virus type 1 (HSV-1) have been suggested as triggers for the induction of LKM1, but reactivity and cross-reactivity to the relevant sequences have not been investigated experimentally. CYP2D6252-271 and its viral homologues were constructed and tested by ELISA in the sera of 46 chronically infected HCV patients, 23 of whom were LKM1 positive. Reactivity to the E1 HCV and ICP4 HSV1 mimics was frequently found in HCV infected patients irrespectively of their LKM1 status; viral/self cross-reactivity (as indicated by inhibition studies), however, was present in the only 2 of the 23 LKM1 seropositive HCV patients, who possessed the HLA allotype B51. Our results indicate that in HCV infected patients virus/self cross-reactivity is dependent on a specific immunogenetic background, a finding awaiting confirmation by studies in larger series of patients.
Abstract: 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.
Abstract: Serum antibodies against human intracisternal A-type particle (HIAP) endogenous retrovirus have been found to be associated with various autoimmune pathologies. To evaluate the presence of serum antibody reactivity to HIAP proteins in systemic sclerosis, a Western blot analysis was performed on sera from 42 patients with systemic sclerosis, in comparison with 18 sera from patients with primary biliary cirrhosis and 52 healthy subjects. A positive Western blot was found in 55.5% of serum samples from patients with primary biliary cirrhosis and in 66.0% of patients with systemic sclerosis. None of the 52 healthy subjects showed positive results. Although this difference may be attributable either to an autoimmune response to antigenically related cellular proteins or to a specific antibody response to HIAP proteins expressed as an incidental consequence of attendant pathological processes, the high prevalence of antibodies against HIAP proteins demonstrated in patients with systemic sclerosis may be considered a hallmark of this disease.
Abstract: BACKGROUND: Autoimmune hepatitis (AIH) has three different presentations: chronic, acute and asymptomatic. Aim: To evaluate AIH presentation in Italian patients and investigate criteria that differentiate between acute-type AIH and acute viral hepatitis. DESIGN: Prospective observational study. METHODS: Eighty-six consecutive patients with type 1 AIH and 41 with acute viral hepatitis (controls) were studied. 'Acute' AIH was defined as recent-onset (<30 days) symptoms (jaundice and/or fatigue and/or fever) with marked alterations in serum liver tests; the 'asymptomatic' pattern as the occasional detection of liver abnormalities, and the 'chronic' pattern as the presence of signs and/or symptoms of long-lasting liver disease. RESULTS: Of 86 AIH patients, 59 (68%) presented with the chronic pattern, 22 (26%) with the acute pattern, and 5 (6%) were asymptomatic. 'Acute' patients had higher AST, ALT and bilirubin serum levels (p < 0.0001). No differences were detected with respect to age and serum levels of alkaline phosphatase, gamma-GT, albumin or gamma-globulin. All three groups had similar prevalences of moderate/severe (vs. mild) histological findings and liver cirrhosis. When compared with controls with acute viral hepatitis, 'acute' AIH patients were more often female (82% vs. 24%, p < 0.0001) and had higher serum gamma-globulin levels (26.9 vs. 13.4 g/l, p < 0.0001) and AST/ALT ratio (1.20 vs. 0.61, p < 0.0001). DISCUSSION: Although in Italy type 1 AIH patients usually present with a chronic pattern, some 25% have an acute presentation resembling that of viral hepatitis. 'Acute' AIH and viral hepatitis can be reliably differentiated by simple parameters such as gender, gamma-globulin serum levels and AST/ALT ratio.
Abstract: Anti-actin IgA antibodies have been found in sera of coeliacs. Our aim was to define the prevalence and clinical significance of anti-actin IgA in coeliacs before and after gluten withdrawal. One hundred and two biopsy-proven coeliacs, 95 disease controls and 50 blood donors were studied. Anti-actin IgA were evaluated by different methods: (a) antimicrofilament positivity on HEp-2 cells and on cultured fibroblasts by immunofluorescence; (b) anti-actin positivity by enzyme-linked immuosorbent assay (ELISA); and (c) presence of the tubular/glomerular pattern of anti-smooth muscle antibodies on rat kidney sections by immunofluorescence. Antimicrofilament IgA were present in 27% of coeliacs and in none of the controls. Antimicrofilament antibodies were found in 25 of 54 (46%) coeliacs with severe villous atrophy and in three of 48 (6%) with mild damage (P < 0.0001). In the 20 patients tested, antimicrofilaments IgA disappeared after gluten withdrawal in accordance with histological recovery. Our study shows a significant correlation between antimicrofilament IgA and the severity of intestinal damage in untreated coeliacs. The disappearance of antimicrofilament IgA after gluten withdrawal predicts the normalization of intestinal mucosa and could be considered a useful tool in the follow-up of severe coeliac disease.
Abstract: BACKGROUND: Bile duct injury is not a feature of classical autoimmune hepatitis (AIH), but it has been described in variant forms of the disease. AIMS: Our goals were to assess the similarity of AIH with bile duct injury to classical disease and to evaluate the possibility of concurrent primary biliary cirrhosis (PBC). METHODS: Fifteen patients with bile duct injury were compared with 151 patients with classical AIH. Patterns of nuclear immunofluorescence and the frequency and nature of autoantibodies associated with AIH and PBC were determined. RESULTS: Patients with bile duct injury had the same nuclear-staining patterns, frequency and nature of autoantibodies, and genetic risk factors as the comparison group. Features specific for PBC, including the multiple nuclear dot pattern of immunofluorescence and antibodies to the M2 antigens, Sp100 and nuclear pore complex antigen, gp210, did not distinguish them from classical disease. Remission and treatment failure occurred with similar frequencies in both groups. CONCLUSIONS: Patients with AIH and bile duct injury lack features of PBC, and they respond as well to corticosteroid therapy as patients with classical disease. Background bile duct changes should not alter the diagnosis or treatment of AIH.
Abstract: 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% "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.
Abstract: BACKGROUND/AIMS: The etiologic link between chronic hepatitis C virus (HCV) infection and mixed cryoglobulinemia is well established, while its prognostic significance within the context of HCV-related hepatitis is not as clear. Patients with an HCV-related cryoglobulinemic syndrome oft have mild liver disease, an aspect that can be influenced by an individual's Th1/Th2 orientation. Our goal was to document stigmata of differentiate cytokine production in this subgroup of patients. METHODS: Fifteen patients with chronic HCV-related liver disease (CLD) and a cryoglobulinemic syndrome (CRYO) were compared to age/sex matched CLD controls with negative cryocrit. Cultured monocytes were stimulated with either Staphylococcus aureus (SAC) or lipopolysaccharide (LPS). RESULTS: The protein concentrations of TNF-alpha and of the Th1-type cytokines interleukin (IL)-12 and IL-18 were significantly greater in the CRYO group, while IL-10 (a Th2 cytokine) levels were greater in the control group. CONCLUSIONS: The clinical distinctiveness of the two groups was reflected at the cytokine level. The cryoglobulinemic patients studied showed a greater Th1 polarization than their cryoglobulin-negative counterparts. This enhanced production of Th1-type cytokines is seemingly not able to rid the host of infection but may account for a milder course of liver disease.
Abstract: OBJECTIVE: To evaluate the clinical pattern and evolution of chronic hepatitis C in children with liver/kidney microsomal antibody type 1 autoantibodies (LKM1). STUDY DESIGN: A multicenter, retrospective study, including the following groups of children with hepatitis C virus infection: (1). 21 consecutive LKM1-positive patients, (2). 42 age- and sex- matched LKM1-negative patients, and (3). 4 interferon-induced LKM1-positive cases. LKM1 reactivity to human microsomes and recombinant cytochrome P450IID6 (CYP2D6) was assayed by immunoblotting. RESULTS: Clinical and biochemical features overlapped in LKM1-positive and LKM1-negative children, but a fibrosis score >3 (range 0-6) was significantly more frequent (P =.04) in the former. Reactivity to microsomal protein and CYP2D6 was significantly (P =.02) associated with LKM1 titers >or=1:320 and was found in 39% of patients, including severe cases and both children (of 4 treated) who achieved a sustained alanine aminotransferase (ALT) normalization after steroid treatment. Five of 7 LKM1-positive children treated with interferon had an ALT exacerbation. CONCLUSIONS: LKM1-positive hepatitis C in children is characterized by a wide spectrum of biochemical, serologic, and histologic features. Whether autoimmunity may contribute to liver damage in a subgroup of patients with more severe liver disease, high LKM1 titers, and reactivity to CYP2D6 is a question deserving further investigation.
Abstract: Antibodies to the baker's yeast Saccharomyces cerevisiae (ASCA), recently proposed as a serological marker of Crohn's disease, have also been detected in other autoimmune disorders. The aim of this study was to determine prevalence and clinical significance of ASCA in autoimmune liver disease. The presence of IgG and IgA ASCA was evaluated using a commercially available immunoassay in 215 patients with autoimmune liver disease (primary biliary cirrhosis, PBC, 123 cases; autoimmune hepatitis, AIH, 67 cases; primary sclerosing cholangitis, PSC, 25 cases), 48 with inflammatory bowel disease and 19 healthy blood donors. Anti neutrophil cytoplasmic antibodies with the perinuclear pattern (p-ANCA) were assessed by indirect immunofluorescence in PSC patients. The main clinical and biochemical parameters between ASCA-positive and negative patients were analysed and compared. ASCA are predominant in Crohn's disease (70%); among liver patients, PSC and AMA-negative PBC show the highest ASCA prevalence (53% and 44%). In PBC ASCA correlate with higher levels of circulating IgA (P < 0.05). In PSC the detection of either ASCA or p-ANCA is neither associated with any clinical or biochemical feature, nor with an underlying inflammatory bowel disease. ASCA can not be considered an additional serological marker of autoimmune liver disease, but the possibility of detecting such a reactivity in autoimmune liver disorders should be considered; their correlation with elevated IgA in PBC suggests that ASCA may be an indirect sign of enhanced mucosal immunity; in PSC patients neither ASCA nor p-ANCA predict the occurrence of a concomitant inflammatory bowel disease.
Abstract: We evaluated the prevalence and clinical significance of non-organ-specific autoantibodies (NOSAs) in 47 hepatitis C virus (HCV)-positive children with abnormal alanine transaminase levels and analyzed the association between NOSAs and virus level, genotype, human leukocyte antigen status, and interferon (IFN) response. Forty-two hepatitis B virus (HBV)-positive children and 25 age- and sex-matched healthy children served as control subjects. NOSAs were found in 34% of the HCV-positive children, 12% of the HBV-positive controls, and none of the healthy control subjects. Liver-kidney microsomal antibody type 1 (LKM1) was detected in 11% of the HCV-positive children but in none of the controls. The HCV load was significantly higher in NOSA-negative than in NOSA-positive children. HCV genotype distribution and human leukocyte antigen alleles were similar, irrespective of NOSA status. Long-term response to IFN therapy was achieved by 18% of the NOSA-positive and 55% of the NOSA-negative subjects. Two LKM1-positive children developed acute, self-limited hepatocellular necrosis while receiving IFN therapy. NOSAs are frequently present in children with hepatitis C, who are less likely to benefit from IFN therapy.
Abstract: 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). CYP2D6(193-212) 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 CYP2D6(193-212) arise through cross-reactive immunity between virus and self. We identified a hexameric sequence "RLLDLA" sharing 5 of 6 aa with "RLLDLS" of HCV(2985-2990) and all 6 aa with CMV(130-135). Of 17 CYP2D6(193-212)-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 CYP2D6(193-212) was inhibited by preincubation with HCV(2977-2996) or CMV(121-140). Recombinant HCV-nonstructural protein 5 and CMV-UL98 proteins also inhibited Ab binding to CYP2D6(193-212). Affinity-purified CYP2D6(193-212)-specific Ab inhibited the metabolic activity of CYP2D6. The demonstrated similarity and cross-reactivity between CYP2D6(193-212) and two unrelated viruses suggests that multiple exposure to viruses mimicking self may represent an important pathway to the development of autoimmunity.
Abstract: The significance of non-organ specific antibodies (NOSAs) in HCV-related chronic hepatitis is largely unclear. In this study we evaluated the prevalence of NOSAs in a non-selected population of HCV-infected subjects. One hundred and seventy anti-HCV positive and 192 anti-HCV negative sex and age-matched subjects (median age 64 years, range 7-91 years, female 68%) enrolled from the general population of a small Italian town were evaluated for NOSAs by indirect immunofluorescence on rat tissue sections and HEp-2 cells, and by counterimmunoelectrophoresis with thymus and spleen extracts as the antigen source. One hundred and sixty-three (96%) HCV-infected subjects had normal ALT serum levels and no evidence of liver disease. NOSAs were found in 31 out of 170 (18%) anti-HCV positive subjects and in 20 out of 192 (10%) controls (P = NS), with similar median titre (1:40) and range (1:40 to 1:160). Neither liver/kidney microsomal antibody type 1 nor antiactin reactivity were detected. No significant association between NOSAs and HCV genotypes was observed. In the general population, HCV-infected subjects and healthy controls have a similar prevalence of NOSAs. Without continuous liver damage HCV infection is unlikely to induce the appearance of NOSAs.
Abstract: Eukaryotically expressed CYP2D6 is the universal target of liver kidney microsomal Ab type 1 (LKM1) in both type 2 autoimmune hepatitis (AIH) and chronic hepatitis C virus (HCV) infection. In contrast, reactivity to prokaryotically expressed CYP2D6 protein and synthetic peptides is significantly lower in HCV infection than in AIH. The aim of the present study was to characterize LKM1 reactivity against a panel of eukaryotically expressed CYP2D6 constructs in the two conditions. LKM1-positive sera obtained from 16 patients with AIH and 16 with HCV infection were used as probes to perform a complete epitope mapping of CYP2D6. Reactivity to the full-length protein and 16 constructs thereof was determined by radioligand assay. We found that antigenicity is confined to the portion of the molecule C-terminal of aa 193, no reactivity being detectable against the aa sequence 1-193. Reactivity increases stepwise toward the C-terminal in both AIH and HCV, but the frequency of reactivity in the two conditions differs significantly between aa 267-337. To further characterize this region, we introduced a five and a three amino acid swap mutation selected from the homologous regions of CYP2C9 and HCV. This maneuver resulted in a substantial loss of LKM1 binding in both conditions, suggesting that this region contains a major epitope. Molecular modeling revealed that CYP2D6(316-327) is exposed on the surface of the protein, and may represent a key target for the autoantibody. These findings provide an initial characterization of the antigenic constitution of the target of LKM1 in AIH and HCV infection.
Abstract: Multiple nuclear dots pattern has been described in primary biliary cirrhosis and, less often, in rheumatological disorders. Sp100 is the major antigen of multiple nuclear dots. We evaluated prevalence and diagnostic significance of multiple nuclear dots and anti-Sp100 reactivity both in hepatic and rheumatological diseases. A series of 283 consecutive liver patients (89 primary biliary cirrhosis, 12 primary sclerosing cholangitis, 85 autoimmune hepatitis, 97 hepatitis C virus-related chronic liver disease) and of 89 consecutive rheumatological cases were evaluated. Presence of multiple nuclear dots was assessed by indirect immunofluorescence on HEp-2 cells, anti-Sp100 reactivity by ELISA with recombinant protein. Multiple nuclear dots were detected in 20 patients (7%) with liver disease (of whom 15 with primary biliary cirrhosis), and in eight patients (9%) with rheumatological disorders. Anti-Sp100 was detected in 45 liver patients (16%), of whom 30 with primary biliary cirrhosis, but in only two with rheumatological disorders (2%) (P =0.0004). The concordance between multiple nuclear dots and anti-Sp100 in liver and rheumatological patients was 90% and 25% (P=0.0018), respectively. Among 89 consecutive patients with primary biliary cirrhosis, multiple nuclear dots and anti-Sp100 were present in 17% and 34%, respectively (P=0.0152). Anti-Sp100 positivity was associated with older age and higher gamma-globulin levels. Multiple nuclear dots are similarly observed in liver and rheumatological patients. In contrast, anti-Sp100 is more frequent in liver patients and is significantly more often detected in primary biliary cirrhosis, of which it can be regarded as a highly specific serological marker. The antigenic target of multiple nuclear dots in most rheumatological patients is other than Sp100.
Abstract: The molecular characterization of the autoreactivities associated with autoimmune liver disease will improve their diagnosis and enhance understanding of their pathogenic mechanisms. Surprisingly, little is known about the nature of the major autoreactivities associated with type 1 AIH, including homogeneous ANA and antibodies to microfilaments [3]. Type 1 AIH is, however, the prototype of autoimmune liver disease [103].
Abstract: The presence of anti-endothelial cells (AECA), smooth muscle (SMA), antinuclear (ANA) and antimitochondrial (AMA) autoantibodies, and liver/kidney microsomal antibody type 1 (LKM1) was investigated retrospectively in sera of liver transplant patients and correlated with cytomegalovirus (CMV) infection as determined by the antigenemia test and with the appearance of acute or chronic allograft rejection. Indirect immunofluorescence analysis was carried out in sequential sera from 40 liver transplant patients. Ten out of 23 antigenemia-positive and none of the antigenemia-negative patients developed serum autoantibodies (P = 0.002, Fisher's exact test). Anti-endothelial cell autoantibodies were found in nine cases and SMA in four patients. Antinuclear antibodies were detected in one otherwise autoantibody-negative patient. All but one case of autoantibody positivity were observed in the high antigenemia group (P < 0.0001, Fisher's exact test). In all but one case, autoantibodies were detected in blood during the antigenemia phase and in most cases in coincidence with or after the antigenemia peak. Even though a statistically significant correlation was not found between autoantibody production and the development of acute or chronic allograft rejection, proportionally more acute rejection cases were observed in the autoantibody-positive than in the autoantibody-negative group. It has been speculated that CMV-induced endothelial damage may be a potent antigenic stimulus, which leads to the production of anti-endothelial cells autoantibodies. Anti-endothelial cell autoantibodies may represent not only a marker of cell injury but also contribute to the progression of the inflammatory response leading to the exposure of tissue-privileged self antigens and the induction of other autoantibodies such as SMA.
Abstract: BACKGROUND/AIM: Smooth muscle antibodies (SMA) characterize type 1 autoimmune hepatitis. Our aim was to evaluate sensitivity and specificity of different immunofluorescence substrates for the detection of SMA. METHODS: Sera from 55 patients with type 1 AIH 20 with primary biliary cirrhosis, 20 with HCV-related chronic hepatitis and 25 blood donors were studied for SMA and anti-microfilaments reactivity by immunofluorescence on rat tissue sections, cultured fibroblasts and commercially available HEp-2 cells (collectively revealing the so called anti-actin pattern), and for the XR1 system by counterimmunoelectrophoresis. SMA was classified on the basis of its immunofluorescence pattern (V--vessels, G--glomerular, T--tubular). As further control group, we studied 26 patients with a diagnosis other than AIH, selected on the basis of a SMA-non-T/XR1 positivity. RESULTS: In patients with AIH the SMA-T pattern on rodent tissue, and anti-MF on fibroblasts and on HEp-2 cells were present in 80, 82 and 80%, respectively. Five out of 11 SMA-non T positive AIH patients were anti-MF positive. None of the pathological and healthy controls was positive for SMA-T or anti-MF reactivity. XR1 system was present in 84% of AIH patients and in 5% of pathological controls (p = 0.01). Two out of 26 SMA-non-T/XR1 positive sera were positive for anti-MF by fibroblasts and HEp-2 cells. A significant correlation was found between SMA-T pattern and anti-MF reactivity; no correlation was found between XR1 system and SMA-T pattern or anti-MF reactivity. CONCLUSIONS: SMA-T pattern is highly sensitive and specific first diagnostic test for type 1 AIH; anti-MF can be used as additional tool for the diagnosis, particularly when, despite the absence of the SMA-T pattern, AIH is strongly suspected.
Abstract: 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.
Abstract: BACKGROUND: Patients with hepatitic and cholestatic autoimmune liver disease ("overlap syndrome") represent a diagnostic and therapeutic challenge. AIM: To evaluate the prevalence of the "hepatitic/cholestatic overlap" in a large series of consecutive patients with cholestatic autoimmune liver disease. METHODS: We re-evaluated the diagnosis of 235 patients with autoimmune liver disease, including 70 with type 1 autoimmune hepatitis (AIH), 142 with primary biliary cirrhosis (PBC), and 23 with primary sclerosing cholangitis (PSC), using the revised International Autoimmune Hepatitis Group (IAIHG) scoring system. Anti-mitochondrial, anti-nuclear, anti-smooth muscle, anti-liver kidney microsomal type 1, anti-liver cytosol type 1, perinuclear anti-neutrophil nuclear and anti-soluble liver antigen antibodies were evaluated in each patient. RESULTS: Ten patients (3 with a previous diagnosis of PBC and 7 of PSC) scored as "probable" or "definite" AIH. These patients did not have a specific autoantibody profile. CONCLUSIONS: Among patients with PBC, the occurrence of a PBC/AIH overlapping syndrome is rare (2.1%), whereas among patients with PSC an overlap between PSC and AIH is frequent (30.4%). Whether patients with the hepatitic/cholestatic overlap syndrome would benefit from a combination therapy with immunosuppression and ursodeoxycholic acid remains to be established.
Abstract: Antilactoferrin antibodies have been reported in patients with several autoimmune disorders, including primary biliary cirrhosis, autoimmune hepatitis and autoimmune cholangitis. We investigated the prevalence and the clinical significance of such autoreactivity in patients with autoimmune and viral chronic liver disease. Sera from 39 patients with autoimmune hepatitis, 51 with primary biliary cirrhosis, 17 with autoimmune cholangitis, 24 with primary sclerosing cholangitis and 28 with HCV-related chronic hepatitis were studied. Positivity for antilactoferrin antibodies was evaluated by Western immunoblotting with purified human lactoferrin. Antilactoferrin antibodies were detected more often in autoimmune liver disorders (25% autoimmune hepatitis, 25% primary biliary cirrhosis, 35% autoimmune cholangitis, 29% primary sclerosing cholangitis) than in HCV-related chronic hepatitis (3.5%, P < 0.02 versus all). Positivity for antilactoferrin antibodies was not associated with a particular clinical or biochemical profile of the underlying liver disease. No correlation was observed between antilactoferrin reactivity and perinuclear antineutrophil cytoplasmic antibodies. Antilactoferrin antibodies are present significantly more often in autoimmune than in viral liver disorders, but they cannot be considered the serological marker of a specific autoimmune liver disease.
Abstract: Liver cytosol antibody type 1 (LC1) is regarded as a serologic marker of type 2 autoimmune hepatitis, in addition to liver kidney microsomal antibody type 1. Among 38 patients with type 2 autoimmune hepatitis, 23 were positive for LC1 antibodies. The antigen recognized by LC1 has been identified as a liver-specific 58-kd metabolic enzyme named formiminotransferase cyclodeaminase (FTCD). All 23 LC1-positive sera immunoprecipitated rat FTCD, and 22 gave an identity reaction with rat FTCD by immunodiffusion. No reaction was observed with sera from 10 patients with type 1 autoimmune hepatitis, 10 with primary biliary cirrhosis, 10 with chronic hepatitis C, and 10 healthy controls. By Western immunoblotting all 23 LC1-positive sera and all the controls tested negative, suggesting that all the antigenic epitopes were destroyed by denaturation. FTCD is a bifunctional protein composed of distinct globular FT and CD domains connected by a short linker. To identify epitopes that trigger the LC1 autoimmune response, we tested LC1 antibodies against FTCD constructs encoding the N-terminal FT domain (amino acids 1-339), or the C-terminal CD domain (amino acids 332-541). Of 20 sera positive against full-length FTCD, 8 (40%) recognized the FT domain and the CD domain, 7 (35%) recognized only the FT domain, and 5 (25%) did not recognize either construct. No sera reacted with only the CD domain. These data indicate that multiple regions of FTCD trigger the LC1 autoimmune response, and that LC1 reactivity is mainly directed to conformation-sensitive epitopes located in the FT region of FTCD.
Abstract: BACKGROUND: Liver/kidney microsomal antibody type 1 (LKM1) is the marker of type 2 autoimmune hepatitis (AIH) and is detected in up to 6% of patients with hepatitis C virus (HCV) infection. It recognises linear and conformational epitopes of cytochrome P450IID6 (CYP2D6) and may have liver damaging activity, provided that CYP2D6 is accessible to effector mechanisms of autoimmune attack. METHODS: The presence of LKM1 in the plasma membrane was investigated by indirect immunofluorescence and confocal laser microscopy of isolated rat hepatocytes probed with 10 LKM1 positive sera (five from patients with AIH and five from patients with chronic HCV infection) and a rabbit polyclonal anti-CYP2D6 serum. RESULTS: Serum from both types of patient stained the plasma membrane of non-permeabilised cells, where the fluorescent signal could be visualised as discrete clumps. Conversely, permeabilised hepatocytes showed diffuse submembranous/cytoplasmic staining. Adsorption with recombinant CYP2D6 substantially reduced plasma membrane staining and LKM1 immunoblot reactivity. Plasma membrane staining of LKM1 colocalised with that of anti-CYP2D6. Immunoprecipitation experiments showed that a single 50 kDa protein recognised by anti-CYP2D6 can be isolated from the plasma membrane of intact hepatocytes. CONCLUSIONS: AIH and HCV related LKM1 recognise CYP2D6 exposed on the plasma membrane of isolated hepatocytes. This observation supports the notion that anti-CYP2D6 autoreactivity may be involved in the pathogenesis of liver damage.
Abstract: Primary autoimmune liver diseases can be hepatitic or cholestatic in nature. Autoimmune hepatitis, more often diagnosed in women, is characterized by biochemical and histological activity, with polyclonal hypergammaglobulinemia as a frequent feature. Antinuclear and anti-smooth muscle antibodies are the serological hallmarks of type 1 autoimmune hepatitis, whereas liver-kidney microsomal antibody type 1 and liver cytosol antibody type 1 designate the type 2 form. Response to immunosuppression is usually excellent. The most frequent cholestatic autoimmune disease is primary biliary cirrhosis, characterized by anti-mitochondrial antibody positivity and typical bile duct lesions observed on liver biopsy. Treatment with biliary acids improves the biochemical picture, may alleviate pruritus, and delays the development of end-stage liver disease. Primary sclerosing cholangitis occurs more frequently in men and affects both the intra- and extrahepatic biliary trees, determining the typical "beading" appearance. Associated inflammatory bowel diseases are often observed. To date, no medical therapy is able to modify the course of this disease. Autoimmune cholangitis is an anti-mitochondrial antibody-negative cholestatic disease with most of the features of primary biliary cirrhosis. "Overlap" syndromes where autoimmune hepatitic and cholestatic features coexist in the same patient, have also been reported. Autoimmune phenomena secondary to hepatitis C virus-related liver disease such as the occurrence of antinuclear, anti-smooth muscle antibodies and liver-kidney microsomal antibody type 1 are often observed.
Abstract: Liver Kidney Microsomal type 1 (LKM1) antibody, the diagnostic marker of autoimmune hepatitis type 2, is also found in a proportion of patients with hepatitis C virus infection (HCV). It is detected conventionally by the subjective immunofluorescence technique. Our aim was to establish a simple and objective enzyme-linked immunosorbent assay (ELISA) that measures antibodies to cytochrome P4502D6 (CYP2D6), the target of LKM1. An indirect ELISA using eukaryotically expressed CYP2D6 was designed. Absorbance values obtained against a reference microsomal preparation were subtracted from those obtained against a microsomal preparation over-expressing CYP2D6, thus removing the non-CYP2D6-specific reaction. Sera from 51 LKM1 positive patients (21 autoimmune hepatitis and 30 with HCV infection), 111 LKM1 negative patients with chronic liver disease (including 20 with HCV infection) and 43 healthy controls were tested. Of 51 patients positive by immunofluorescence, 48 were also positive by ELISA while all the 154 LKM1 negative subjects were also negative by ELISA. There was a high degree of association between IFL and ELISA as demonstrated by a kappa reliability value of 0.96. The absorbance values by ELISA correlated with immunofluorescence LKM1 titres both in autoimmune hepatitis (r = 0.74, p < 0.001) and HCV infection (r = 0.67, p < 0.001). The simple, objective ELISA described has the potential to replace the standard immunofluorescence technique.
Abstract: 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 gamma-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.
Abstract: Our aim was to investigate whether host genetic factors are involved in the onset of hepatitis C virus (HCV)-related mixed cryoglobulinemia (MC). We studied 25 consecutive patients presenting with a full-blown clinical picture of MC by physical examination, blood chemistry, assessment of cryoglobulins and their composition, nonorgan-specific autoantibodies, antibodies to HCV, serum HCV RNA, and HLA polymorphism. Biopsies of liver, bone marrow, and minor salivary glands were also performed in a number of patients. HLA results were compared with those of normal controls and patients with chronic HCV infection without MC and negative for autoimmune phenomena (pathological controls). Type II MC was found in 14 of 25 patients (56%), and type III MC was found in the remaining 11 (44%). All patients were positive for antibodies to HCV and/or serum HCV RNA. HLA-B8 was found in 40% (10 of 25) of patients compared with 10. 1% (38 of 377) of normal controls (P = .00003, Pcorrected = .0005, relative risk [RR] 5.9) and 6.7% (2 of 30) of pathological controls (P = .007, Pcorrected = not significant). As for class II HLA molecules, only DR3 was significantly more frequent in MC patients (40%, 10 of 25) than in normal controls (15.1%, 57 of 377; P = .003, Pcorrected = .03, RR 3.7). Odds ratio (OR) for the risk of developing MC was calculated in patients positive for B8 and/or DR3, and the highest OR (8.2) was observed in individuals possessing both. The results suggest that the development of HCV-related MC is associated with HLA-B8 and DR3 markers.
Abstract: 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.
Abstract: Celiac disease has been associated with autoimmune disorders, but its frequency in autoimmune hepatitis is unknown. Sera from 157 patients with type 1 autoimmune hepatitis, 24 patients with type 2 autoimmune hepatitis, 62 patients with primary biliary cirrhosis, 30 patients with chronic hepatitis B, and 80 patients with chronic hepatitis C were tested for immunoglobulin A anti-endomysial antibodies by indirect immunofluorescence and immunoglobulin A and G antibodies to gliadin by enzyme immunoassay. Duodenal biopsy evaluation was recommended in patients seropositive for immunoglobulin A anti-endomysial antibodies. Immunoglobulin A anti-endomysial antibodies were present in eight of the 181 patients with autoimmune hepatitis (4%), including six with type 1 disease (4%) and two with type 2 disease (8%). Immunoglobulin A antibodies to gliadin were found in six of these eight patients, but they were also present in two others, including one patient with chronic hepatitis C. Five of the eight patients with immunoglobulin A antiendomysial antibodies, including three patients with no gastrointestinal symptoms, had duodenal biopsies and subtotal villous atrophy was present in all of them. No patient with primary biliary cirrhosis or chronic viral hepatitis had antiendomysial antibodies. The presence of celiac disease in autoimmune hepatitis is high (at least one in 36 patients) and it is predominantly asymptomatic. Screening with anti-endomysial and anti-gliadin antibodies should be performed and results confirmed with intestinal biopsy.
Abstract: In situ reverse transcriptase-polymerase chain reaction is a novel and exciting technique which couples the extremely high sensitivity of DNA amplification with the advantages of in situ hybridisation, allowing the preservation of cell morphology and the localisation of the positive signal within intact cells. In this brief report, we analyse and discuss the application of such a technique to the study of hepatitis C virus (HCV) infection, the most common cause of chronic liver disease worldwide. Moreover, given the lymphotropism of this virus, we describe here our own approach to detect and localise HCV RNA within intact peripheral blood mononuclear cells and to quantify the relative number of infected cells.
Abstract: BACKGROUND/AIMS: To evaluate a possible variation in hepatitis D virus endemicity in Italy, the data from a multicentre study concerning HBsAg chronic carriers first observed in 31 liver units during 1992 were compared with the corresponding figures from a similar study performed in 1987. METHODS/RESULTS: In both studies the methodology for the recruitment of cases was the same. The overall anti-HD prevalence in 1992 was 14.4%, a significantly lower rate than that observed in 1987 (23.4%, p < 0.01). The decrease significantly (p < 0.01) affected both males and females; it occurred in all geographical areas, although to a greater extent in northern regions. It was evident in subjects below 50 years of age, but not in subjects older than 50. A significant reduction in the anti-HD prevalence was seen in all forms of chronic hepatitis. CONCLUSIONS: These findings indicate a lower level of hepatitis D virus endemicity in Italy, probably as a consequence of the reported decreased pool of HBsAg chronic carriers, the reduced size of families, the improved socio-economic conditions and changes in intravenous drug abuser behaviour. All these factors may have affected the strength of hepatitis D virus infection which has greatly reduced the spread of the virus.
Abstract: Antibodies to nuclei (ANA), smooth muscle (SMA), and liver/kidney microsomes type 1 (anti-LKM1) may occur in chronic hepatitis C. Distinct subspecificities, including ANA with the homogeneous pattern (ANA-H) and SMA with antiactin specificity (SMA-AA), are found in autoimmune hepatitis (AIH). This study was performed to characterize the hepatitis C virus (HCV)-associated autoantibodies and to evaluate their influence on the profile of the disease. Two hundred ninety consecutive patients with chronic hepatitis C and 35 control cases with AIH were screened for autoantibodies by indirect immunofluorescence (IFL) at 1:40 serum dilution. The ANA pattern was defined by IFL on HEp-2 cells and the SMA-AA identified by the presence of at least two of the following elements: 1) SMA(T) or SMA(G) pattern by IFL on kidney sections; 2) XR1 precipitating system by counterimmunoelectrophoresis; or 3) typical pattern by IFL on liver sections from phalloidin-intoxicated rats. ANA, SMA, and anti-LKM1 occurred in 9%, 20%, and 6% of chronic hepatitis C cases, respectively. The overall prevalence of autoantibodies was 30% (87 of 290). Compared with AIH, HCV-associated ANA and SMA exhibited ANA-H and SMA-AA at a lower prevalence (38% vs. 71%, P = .04 and 8% vs. 87%, P < .000001, respectively) and had a lower median titer (1:80 vs. 1:320, P < .001 and 1:40 vs. 1:320, P < .000001, respectively). The concomitant positivity for ANA-H and SMA-AA was detected in none of the HCV cases, but in 46% of AIH sera (P < .000001). Two parameters were independently associated with the autoantibodies in chronic hepatitis C: high alanine transaminase (ALT) serum levels (F = 14.04) and female gender (F = 5.03). At the univariate analysis, patients with autoantibodies had a more severe portal-periportal necroinflammation (median Scheuer's score: 2.05 vs. 1.64, P = .003). The presence of autoantibodies did not influence the response to interferon (IFN). In chronic hepatitis C, serum autoantibodies are common, but their subspecificities are distinct from those occurring in AIH. Whereas the absence of ANA-H and/or SMA-AA does not exclude AIH, the characterization of ANA and SMA may help to discriminate between the two conditions. As compared with the seronegative counterpart, autoantibody-positive chronic hepatitis C is more common in females and exhibits a more severe biochemical and histological activity. The response to IFN therapy, however, is similar.
Abstract: Perinuclear anti-neutrophil cytoplasmic antibodies (pANCA) have been recently defined as the most sensitive autoantibody of type 1 autoimmune hepatitis (AIH-1). Their prevalence in type 2 autoimmune hepatitis (AIH-2) has not yet been evaluated. The aim of the present study was to verify the association of pANCA with AIH-1 in an Italian series and to investigate the prevalence of the antibodies in AIH-2 and in proper control groups represented by cases of chronic hepatitis C (CH-C) with similar autoimmune features. pANCA were found in 30 of 46 (65%) AIH-1 and in 4 of 30 (13%) ANA/smooth muscle antibody (SMA) positive CH-C (P = .0000006). Nineteen AIH-2, 29 liver kidney microsomal antibody type 1/liver cytosol antibody type 1 (LKM1/LC1) positive CH-C cases and 50 healthy controls were all negative. In AIH-1, pANCA were significantly (P = .009) more frequent in males (8 of 9, 89%) than in females (22 of 37, 59%). All pANCA positive sera showed SMA of the antiactin type. The present data confirm that pANCA, although less prevalent in our series than in other reports, do associate with AIH-1 also in the Mediterranean area and show that it can identify a small subgroup (13%) of ANA/SMA positive chronic hepatitis C, in which autoimmune reactions might play a more relevant role than viral infection. They also show the antibodies are absent in AIH-2. In conclusion, pANCA appear to be mutually exclusive of LKM1 positivity, either hepatitis C virus-related or not, thus representing a further valuable tool to differentiate the two types of autoimmune hepatitis.
Abstract: BACKGROUND/AIMS: Liver kidney microsomal type 1 antibody (LKM1) is the diagnostic marker of autoimmune hepatitis (AIH) type 2 and is also found in patients with hepatitis C virus (HCV) infection. Cytochrome P4502D6 (CYP2D6) is the documented target antigen of LKM1 in AIH, but not in HCV infection. To compare the reactivity in the two conditions, we established a radioligand assay using eukaryotically expressed CYP2D6 as target. METHODS: A 1.2-kb human CYP2D6 cDNA was isolated from a human liver cDNA library and subcloned into an in vitro transcription vector pSP64 Poly(A). Recombinant CYP2D6 was then produced by in vitro transcription/translation, metabolically labelled with 35S methionine and used in the immunoprecipitation assay. Antibodies that bound radiolabelled CYP2D6 were immunoprecipitated and their levels assessed as cpm. Sera from 50 LKM1-positive patients (26 with AIH; 24 with HCV infection), 128 LKM1-negative patients and 57 normal controls were tested. RESULTS: Reactivity to 35S labelled CYP2D6 was observed in all LKM1-positive sera from patients with AIH and HCV infection, but in none of the controls. The cpm in both conditions were significantly higher than in normal controls (p<0.0001), and were correlated with the immunofluorescence titres of LKM1 (r 0.87, p<0.001 and r=0.64, p<0.001 for AIH and HCV infection, respectively). Reactivity to 35S labelled CYP2D6 was inhibited by addition of an excess of eukaryotically expressed CYP2D6. CONCLUSIONS: CYP2D6 is a major target antigen of both AIH and HCV infection. The novel radioligand assay is highly sensitive and specific.
Abstract: We have reclassified 110 patients with autoantibody-positive cryptogenic chronic hepatitis according tot he aggregate scoring system proposed by the International Autoimmune Hepatitis Group for signs of hepatitis C virus (HCV) infection and the newly proposed terminology of "unclassified" chronic hepatitis. Anti-HCV and HCV viremia were assessed by second-generation assays and reverse transcription-polymerase chain reaction. Immunomorphological and immunochemical characterizations of antinuclear, smooth, muscle, liver-kidney microsomal type 1, and liver cytosol type 1 autoantibodies were also performed. All 45 anti-HCV negative patients fulfilled the score criteria for the diagnosis of "definite" or "probable" autoimmune hepatitis (AIH). Eight anti-HCV-positive cases reached the score of "probable" AIH, whereas the remaining 57 cases were diagnosed as unclassified chronic hepatitis. The scoring system allows the correct identification of all autoimmune cases without HCV infection. Autoimmune hepatitis runs a more severe disease course than unclassified chronic hepatitis, whose clinical and histological features are similar to those of autoantibody-negative chronic hepatitis C.
Abstract: Hepatitis C virus (HCV) is known to infect peripheral blood mononuclear cells (PBMC) of patients with chronic hepatitis C, but the proportion of HCV-infected circulating cells is not detectable by conventional reverse transcriptase-polymerase chain reaction (RT-PCR) and the pathogenic significance of HCV lymphotropism is still unclear. Therefore, we have devised an in situ RT-PCR technique using fluorescein-labeled HCV-specific primers revealed by flow cytometry. PBMC were isolated from 28 patients with chronic HCV-related liver disease; of these, 6 had previously received an orthotopic liver transplantation (OLT) and were on immuno-suppressive treatment. Fourteen patients (50%) were found positive for HCV genome within PBMC by in situ RT-PCR, the proportion of HCV-infected cells ranging from 0.2% to 8.1%. All 6 OLT patients tested positive. The fluorescent signal, corresponding to the HCV-specific 340-bp amplicon, was confined to part of the cytoplasmic compartment of scattered PBMC. Of these 14 patients, 12 had also negativestrand HCV RNA within PBMC detected by "tagged" RT-PCR. We conclude that HCV may infect a significant proportion of PBMC in chronic hepatitis C patients, especially immunosuppressed OLT cases, and that viral replication within PBMC is a common occurrence. Over time, the persistence of HCV-infected immune system cells might interfere with normal immunologic mechanisms and play a role in the pathogenic processes leading to extrahepatic disorders such as mixed cryoglobulinemia and B-cell malignant lymphoma.
Abstract: BACKGROUND/AIMS: The majority of adult patients positive for anti-liver-kidney microsomal antibody are also positive for anti-hepatitis C virus and serum HCV RNA. In these patients the role played by hepatitis C virus infection in the progression of liver damage and its relationship with anti-liver-kidney microsomal antibody are, however, still a matter of debate. METHODS: To clarify this point we have compared hepatitis C viremia in sera from 31 hepatitis C virus-related chronic hepatitis patients positive for anti-liver-kidney microsomal antibody with that of 31 patients with hepatitis C virus-related chronic hepatitis without autoantibodies using a newly developed competitive reverse transcription-polymerase chain reaction technique. Reverse transcription-polymerase chain reaction was performed using a synthetic competitor of a length similar to that of wild template (71 bp vs 86 bp). RESULTS: The results obtained have been related to hepatitis C virus genotypes. Anti-liver-kidney microsomal antibody/anti-HCV positive patients show a median value of hepatitis C virus genome molecules (626829/ml, range 9780-25651424), significantly lower than anti-liver-kidney microsomal antibody negative/anti-HCV positive patients (10158314/ml, range 101822-67429974) (p < 0.001). No hepatitis C virus genotype was significantly associated with anti-liver-kidney microsomal antibody, although a predominance of genotype 1 (subtypes a and b) has been observed in these patients. CONCLUSIONS: Since a low hepatitis C viremia has been observed in anti-liver-kidney microsomal antibody positive patients with disease severity comparable to that of patients without autoantibodies, it is conceivable that in them autoimmune mechanisms may cooperate with viral infection in sustaining disease activity.
Abstract: BACKGROUND/AIMS: Recent studies in adult patients have established a relationship between hepatitis C virus infection and the presence of liver-kidney microsomal autoantibody type 1 (LKM1). Conversely, little is known regarding the relationship between hepatitis C and autoimmunity in children. In this study, we investigated non-organ specific autoantibodies in 40 otherwise healthy Italian children with chronic hepatitis C. METHODS: All but four patients included in the study were asymptomatic. Liver histology, obtained in 35, showed features ranging from minimal to mild chronic hepatitis. Autoantibodies were investigated by indirect immunofluorescence. HCV RNA was assayed by the polymerase chain reaction in 34 cases and viral genotypes were determined. RESULTS: Antinuclear antibodies were detected in three (7.5%) cases, one with a homogeneous pattern; smooth muscle autoantibodies in seven (17.5%) cases, always with V (vessels only) specificity and LKM1 in four (10%), at titers ranging from 1:20 and 1:2560. Clinical and virologic features did not significantly differ between autoantibody positive and negative cases, although infections with HCV genotypes 1a and 2 were more frequent in LKM1-positive patients. During observation, the child with the highest LKM1 titre was unsuccessfully treated with alpha interferon but responded to steroids. Twelve LKM1 negative children were also treated with interferon and one developed low LKM1 titers concomitant with an alanine aminotransferase flare. The sera of the five LKM1-positive children with investigated by immunoblotting with a human microsomal fraction and peptide 257-269 of cytochrome P450IID6. Only the serum of the child with the highest LKM1 titers was reactive. CONCLUSIONS: These results show that a consistent proportion of children with chronic hepatitis C circulate non-organ specific autoantibodies. The prevalence of LKM1 is greater than in adults and this could raise problems for the treatment of the disease with interferon. The analysis of LKM1 target antigens might help to identify putative cases of "true" autoimmune hepatitis with concomitant HCV infection that could benefit from steroid treatment.
Abstract: Within the multiform liver/kidney microsomal (LKM) family, a subgroup of sera that reacts with a liver cytosolic (LC) protein has been isolated and the new antigen-antibody system is called LC1. Unlike LKM antibody type 1 (anti-LKM1), anti-LC1 is said to be unrelated to hepatitis C virus (HCV) infection and has therefore been proposed as a marker of 'true' autoimmune hepatitis type 2. Altogether 100 LKM1 positive sera were tested by immunodiffusion (ID). Twenty five gave a precipitation line with human liver cytosol; 17 of the 25 also reacted with rat liver cytosol. Thirteen of the 25 sera were anti-HCV positive by second generation ELISA: anti-HCV positive patients were significantly older (p < 0.001) and tended to have less active disease. No difference in anti-LC1 titre or ID immunoreactivity was found between anti-LC1/anti-HCV positive and anti-LC1/anti-HCV negative cases. In Western blotting experiments, 14 of 24 ID positive sera recognised a 58 kD protein of the human cytosolic fraction and 11 gave a similar reactivity when tested with human microsomes, suggesting the presence of the LC1 target antigen also in the microsomal preparation. Western blotting reactivity was similar for both anti-HCV positive and negative sera. These data confirm the existence of the LC1 antigen-antibody system that partially overlaps with LKM1, and that it is an additional marker of juvenile autoimmune hepatitis type 2. It does not, however, discriminate between patients with and without HCV infection.
Abstract: Liver cytosol specific antibody type 1 (anti-LC1) was first described in a proportion of patients with liver/kidney microsomal antibody type 1 (anti-LKM1)-positive autoimmune hepatitis (AIH) and is routinely evaluated by immunodiffusion (ID). Using human liver cytosol as the source of antigen, we have used ID, counterimmunoelectrophoresis (CIE) and immunoblotting (IB), to test sera from 167 patients with documented chronic liver diseases of different etiology. 15 patients had antinuclear antibody (ANA) and/or smooth muscle antibody (SMA)-positive AIH, 13 had anti-LKM1-positive AIH, four had ANA/SMA/anti-LKM1-negative AIH, 76 had anti-LKM1-positive hepatitis C (recently renamed unclassified chronic hepatitis-UCH), 40 had chronic hepatitis C, 15 had chronic hepatitis B, and 4 had chronic hepatitis D. A precipitin line of identity with an anti-LC1 reference serum was detected both by ID and CIE in 16 patients: six with anti-LKM1-positive 'definite' AIH, four with ANA/SMA/anti-LKM1-negative 'definite' AIH, and six with anti-LKM1-positive UCH. By IB, 14 out of the 16 anti-LC1-positive sera (87.5%) reacted with a 58 kDa human liver cytosolic polypeptide, whereas three out of 16 (19%) recognised an additional 60 kDa band. Compared to ID, CIE is more economical in terms of both time and reagents and provides more clear-cut results. The 58 kDa reactivity by IB was detectable in nearly all CIE/ID anti-LC1-positive patients, was not found among CIE/ID anti-LC1-negative patients. In conclusion, CIE is the ideal screening test for the detection of anti-LC1, an autoantibody that can be regarded as an additional serological marker of AIH and is especially useful in ANA/SMA/anti-LKM1 negative cases.
Abstract: Liver/kidney microsomal antibody type 1 (LKM-1), the serological marker of a subset of autoimmune hepatitis, is also present in a proportion of patients with hepatitis C virus (HCV) related chronic liver disease. To characterise further this autoreactivity and to evaluate whether an autoantibody giving an identical immunofluorescence staining, and detected in two different clinical conditions, involves the same antigenic target(s), sera from autoimmune and HCV infected patients were tested with native, recombinant, and synthetic antigens. Sixty five sera were selected on the basis of the typical immunofluorescence pattern: 50 patients had serological markers of HCV infection, the remaining 15 suffered from autoimmune hepatitis. The reactivity of each serum with rat and human microsomal fractions, full length human recombinant CYP2D6, and two synthetic peptides spanning the amino acid regions 257-269 and 373-398 of CYP2D6 was systematically investigated by immunoblotting. Fourteen (93%) sera from autoimmune hepatitis patients and 39 (78%) from HCV infected patients reacted with rat and/or human microsomal polypeptides of 39 kD, 50 kD, 58 kD, and 66 kD in different associations, the 50 kD band being the most frequently observed. Reactivity to CYP2D6 and its amino acid sequence 257-269 was significantly more common in autoimmune hepatitis than in HCV infected patients (p < 0.001 and p < 0.0003, respectively). LKM-1 reactivity is directed against heterogeneous and not entirely defined autoantigens. The main target in autoimmune sera is CYP2D6 and its 257-269 amino acid region, while sera from patients with HCV infection are more likely to recognise other microsomal targets, the molecular identity of which is currently unknown.
Abstract: Hepatitis C virus (HCV) is able to replicate in peripheral blood mononuclear cells (PBMC) of HCV-infected patients. Few data are available on PBMC testing for HCV RNA in serum HCV RNA negative patients, positive for anti-HCV and with histological evidence of chronic hepatitis. Twenty such patients were studied; of these, 11 were tested during interferon alpha (IFN) treatment, at the time of serum HCV RNA clearance and ALT normalisation: only one was found to be positive for HCV sequences in PBMC. Within 3 months of IFN withdrawal all 11 patients relapsed with high ALT and recurrence of serum HCV RNA. Of nine serum HCV RNA negative patients with chronic hepatitis C who were not receiving IFN when tested (four untreated patients and five patients who had already completed IFN schedule), PBMC HCV RNA was detected in four. Evidence of active HCV replication (presence of the minus strand genome) in PBMC was also observed in two cases. Thus, five of the 20 patients without detectable serum HCV RNA turned out to be carriers of HCV sequences in PBMC. These data indicate that: 1. PBMC are an extrahepatic replication site of HCV; this is true also in the absence of serum HCV RNA; 2. the role of PBMC as a "viral reservoir" after IFN-induced serum HCV RNA clearance is questioned; 3. the absence of both serum and PBMC HCV RNA in patients under IFN is not predictive of sustained viral loss; 4. testing for PBMC viral sequences might enhance the chances of detecting HCV infection.
Abstract: Hepatitis C virus genotypes have been characterized in 22 patients with anti-LKM-1 positive chronic hepatitis C. Following the Simmonds classification, 77% of patients were infected by hepatitis C virus genotype 1, 18% by genotype 2 and 5% by genotype 3, thus excluding the association of the autoimmune reaction with a particular viral type. Prevalences of genotype 1 and 2 were significantly different from those obtained in 79 patients with chronic hepatitis C who were negative for anti-LKM-1, as these were more rarely infected by genotype 1 and more frequently by genotype 2. Clinical findings of anti-LKM-1 positive patients were similar in all three groups. Sequence analysis of the amplified 5'UTR provided identification of peculiar and identical nucleotide substitutions in two out of four patients with genotype 2. The analysis of the secondary structure of this region showed that the observed nucleotide mutations increased the stability of the stem formed in this position.
Abstract: 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.
Abstract: Antinuclear antibodies with the homogeneous pattern (ANA-H) and smooth muscle antibodies with antiactin specificity (SMA-AA) are regarded as the serum markers of type-1 autoimmune chronic hepatitis. Their diagnostic relevance, however, has been questioned recently after the detection of signs of hepatitis C virus infection in autoimmune chronic hepatitis patients. To further evaluate this point, antihepatitis C virus antibodies were sought by two second generation assays (ELISA 2 and RIBA 2) in 100 Italian patients with chronic liver disease of unknown aetiology, including 46 with (autoimmune chronic hepatitis) and 54 without the above antibodies (cryptogenic). By ELISA 2, antihepatitis C virus, although significantly prevalent in cryptogenic (83%), were found also in a substantial proportion of autoimmune chronic hepatitis patients (46%) (p < 0.0001), their occurrence was confirmed by RIBA 2 in almost all cases (96% and 86%, respectively). Autoimmune patients with either ANA-H or SMA-AA exhibited similar antihepatitis C virus prevalences (59% and 52%, respectively); by contrast, the eight cases positive for both the autoantibodies were consistently antihepatitis C virus negative. These findings confirm that in countries with high hepatitis C virus circulation (like Italy) an overlap between autoimmune chronic hepatitis and hepatitis C virus infection, reflected by 'true' antihepatitis C virus antibodies, does occur. The detection of ANA-H or SMA-AA, in fact, identifies chronic liver disease patients with a relatively low prevalence of antihepatitis C virus, but does not exclude hepatitis C virus infection. Positive findings for both ANA-H and SMA-AA, however, is an appropriate marker for hepatitis C virus free 'primary' autoimmune chronic hepatitis.
