Presence of disease specific autoantibodies against liver sinusoidal cells in primary biliary cirrhosis.

AIM: To investigate the presence of autoantibodies directed against liver sinusoidal cells in primary biliary cirrhosis (PBC). METHODS: Liver biopsies from 21 PBC patients were studied and compared with 12 liver biopsies from disease controls [3 patients with hepatitis B (HBV) virus, 3 patients with hepatitis C virus (HCV), 3 patients with non-alcoholic steatohepatitis and 3 patients with acute alcoholic hepatitis (AAH)]. As healthy controls, we used tissue specimens adjacent to metastatic liver adenocarcinoma. Normal serum was taken from staff members of the unit. The determination of the cell type targeted by autoantibodies present in the patients sera was performed by indirect immunofluorescence (IIF) analysis using paraffin-embedded liver sections as a substrate. Sera from homologous or heterologous PBC patients or sera from the disease control group were used as primary antibodies. The presence of autoantibodies was identified using confocal microscopy. RESULTS: In total, 18/21 (85.7%) PBC patients exhibited positive staining in the sinusoidal cells, 10/21 (47.6%) in lymphocytes, 8/21 (38%) in cholangiocytes and 7/21 (33.3%) in hepatocytes, when homologous serum and fluorescein isothiocyanate-conjugated immunoglobulin type G (IgG) secondary antibody were used. PBC sections incubated with heterologous PBC serum showed reduced staining (20% for sinusoidal cells, 20% for lymphocytes, 20% for cholangiocytes and 13.3% for hepatocytes). When IgM immunoglobulin, instead of IgG, was used as secondary antibody, positive staining was observed in 75% of lymphocytes, 62.5% of cholangiocytes, 37.5% of hepatocytes and 50% of the sinusoidal cells with a much stronger staining intensity. No staining was observed when either normal or PBC sera were used as a primary antibody on liver sections from the disease control group. When PBC sera were incubated with healthy control sections, weak positive staining of cholangiocytes was observed in 3/21 (14.3%) PBC serum samples. Steatohepatitis serum on PBC sections gave a positive staining of some hepatocytes and lymphocytes but no staining on viral hepatitis sections. Incubation with HBV sera stained some hepatocytes, cholangiocytes and intra-sinusoidal or portal lymphocytes of PBC, HBV and AAH patients but not HCV patients. CONCLUSION: In this study, for the first time in diseased liver tissue, we have demonstrated that a large proportion of PBC patients have disease specific autoantibodies against liver sinusoidal cells.

Peri-nuclear antibodies correlate with survival in Greek primary biliary cirrhosis patients.

AIM: To investigate possible associations of anti-nuclear envelope antibody (ANEA) with disease severity and survival in Greek primary biliary cirrhosis (PBC) patients. METHODS: Serum samples were collected at diagnosis from 147 PBC patients (85% female), who were followed-up for a median 89.5 mo (range 1-240). ANEA were detected with indirect immunofluorescence on 1% formaldehyde fixed Hep2 cells, and anti-gp210 antibodies were detected using an enzyme linked immunosorbent assay. Findings were correlated with clinical data, histology, and survival. RESULTS: ANEA were detected in 69/147 (46.9%) patients and 31/147 (21%) were also anti-gp210 positive. The ANEA positive patients were at a more advanced histological stage (I-II/III-IV 56.5%/43.5% vs 74.4%/25.6%, P = 0.005) compared to the ANEA negative ones. They had a higher antimitochondrial antibodies (AMA) titer (≤ 1:160/> 1:160 50.7%/49.3% vs 71.8%/28.2%, P = 0.001) and a lower survival time (91.7 ± 50.7 mo vs 101.8 ± 55 mo, P = 0.043). Moreover, they had more advanced fibrosis, portal inflammation, interface hepatitis, and proliferation of bile ductules (P = 0.008, P = 0.008, P = 0.019, and P = 0.027, respectively). They also died more frequently of hepatic failure and/or hepatocellular carcinoma (P = 0.016). ANEA positive, anti-gp210 positive patients had a difference in stage (I-II/III-IV 54.8%/45.2% vs 74.4%/25.6%, P = 0.006), AMA titer (≤ 1:160/> 1:160 51.6%/48.4% vs 71.8%/28.2%, P = 0.009), survival (91.1 ± 52.9 mo vs 101.8 ± 55 mo, P = 0.009), and Mayo risk score (5.5 ± 1.9 vs 5.04 ± 1.3, P = 0.04) compared to the ANEA negative patients. ANEA positive, anti-gp210 negative patients had a difference in AMA titer (≤ 1:160/> 1:160 50%/50% vs 71.8%/28.2%, P = 0.002), stage (I-II/III-IV 57.9%/42.1% vs 74.4%/25.6%, P = 0.033), fibrosis (P = 0.009), portal inflammation (P = 0.018), interface hepatitis (P = 0.032), and proliferation of bile ductules (P = 0.031). Anti-gp210 positive patients had a worse Mayo risk score (5.5 ± 1.9 vs 4.9 ± 1.7, P = 0.038) than the anti-gp210 negative ones. CONCLUSION: The presence of ANEA and anti-gp210 identifies a subgroup of PBC patients with advanced disease severity and poor prognosis.

Differential detection of nuclear envelope autoantibodies in primary biliary cirrhosis using routine and alternative methods.

BACKGROUND: Detection of autoantibodies giving nuclear rim pattern by immunofluorescence (anti-nuclear envelope antibodies - ANEA) in sera from patients with primary biliary cirrhosis (PBC) is a useful tool for the diagnosis and prognosis of the disease. Differences in the prevalence of ANEA in PBC sera so far reported have been attributed to the methodology used for the detection as well as to ethnic/geographical variations. Therefore, we evaluated the prevalence of ANEA in sera of Greek patients with PBC by using methods widely used by clinical laboratories and a combination of techniques and materials. METHODS: We screened 103 sera by immunoblotting on nuclear envelopes and indirect immunofluorescence (IIF) using cells and purified nuclei. Reactivities against specific autoantigens were assessed using purified proteins, ELISA, immunoprecipitation and mass spectrometry. RESULTS: We found higher prevalence of ANEA when sera were assayed by IIF on purified nuclei or cultured cells (50%) compared to Hep2 commercially available slides (15%). Anti-gp210 antibodies were identified in 22.3% and 33% of sera using ELISA for the C-terminal of gp210 or both ELISA and immunoprecipitation, respectively. Immunoblotting on nuclear envelopes revealed that immunoreactivity for the 210 kDa zone is related to anti-gp210 antibodies (p < 0.0001). Moreover, we found that sera had antibodies for lamins A (6.8%), B (1%) and C (1%) and LBR (8.7%), whereas none at all had detectable anti-p62 antibodies. CONCLUSIONS: The prevalence of ANEA or anti-gp210 antibodies is under-estimated in PBC sera which are analyzed by conventional commercially available IIF or ELISA, respectively. Therefore, new substrates for IIF and ELISA should be included by clinical laboratories in the analysis of ANEA in autoimmune sera.

Optimized detection of circulating anti-nuclear envelope autoantibodies by immunofluorescence.

BACKGROUND: Antinuclear antibodies are useful diagnostic tools in several autoimmune diseases. However, the routine detection of nuclear envelope autoantibodies using immunofluorescence (IF) is not always easy to perform in patients' sera because of the presence of autoantibodies to other nuclear and cytoplasmic components which could mask the characteristic rim-like pattern of nuclear envelope autoantibodies. This is particularly common in sera from patients with primary biliary cirrhosis (PBC), which generaly have high titres of anti-mitochondrial antibodies. Therefore, we have assayed a number of commercial slides and alternative fixation conditions to optimize the detection of anti-nuclear envelope antibodies (ANEA) in PBC sera. METHODS: We have explored the presence of ANEA in 33 sera from patients with established PBC using three different Hep2 commercial slides and home-made slides with HeLa and Hep2 cells fixed with methanol, ethanol, 1% or 4% formaldehyde. RESULTS: We observed that the IF pattern was related to the cell type used (Hep2 or HeLa), the manufacturer and the cell fixation scheme. When both cell lines were fixed with 1% formaldehyde, the intensity of the cytoplasmic staining was considerably decreased regardless to the serum sample, whereas the prevalence of cytoplasmic autoantibodies was significantly lowered, as compared to any of the Hep2 commercial slide and fixation used. In addition, the prevalence of ANEA was importantly increased in formaldehyde-fixed cells. CONCLUSION: Immunofluorescence using appropriately fixed cells represent an easy, no time-consuming and low cost technique for the routine screening of sera for ANEA. Detection of ANEA is shown to be more efficient using formaldehyde-fixed cells instead of commercially available Hep2 cells.

Dermatological manifestations in primary biliary cirrhosis patients: a case control study.

OBJECTIVES: Primary biliary cirrhosis (PBC), a disease of probable autoimmune etiology that affects the small intrahepatic bile ducts of mainly middle-aged women is commonly associated with pruritus, xanthomatous lesions, and melanosis. We conducted a prospective study to systematically describe the skin disorders of a group of PBC patients. METHODS: A prospective evaluation and analysis of dermatological manifestations including oral and genital lesions was carried out, in 49 PBC patients (45 females and 4 males). Median age 63 yr (range 35-87 yr). They were compared with 45 age and sex matched controls, selected among persons attending the dermatologic outpatient clinic. RESULTS: A total of 330 skin disorders were found in the 49 PBC patients versus 76 in the 45 controls; 31.5% of all lesions were skin fungal infections. Of all lesions analyzed with the Bonferroni rule of multiple comparisons significantly more common in PBC patients were plantar mycoses, onychomycoses, and interdigital mycoses. Pruritus was found in 69.3% of patients versus 22.2% of controls, xerosis in 69.3%versus 2.2%, dermographism in 57.1%versus 4.4%, and melanosis in 46.9%versus 0%. In 38.7% of the PBC patients the dermatologic lesion was the presenting symptom. CONCLUSIONS: PBC patients present with a wide variety of cutaneous manifestations varying in severity. Multiple skin fungal infections have been found even in the early stages. Since in more than one third of our PBC patients the dermatologic lesion was the presenting sign or symptom leading to diagnosis we believe that physicians should be aware so that a prompt and early diagnosis may be achieved.

Survival of anti-mitochondrial antibody-positive and -negative primary biliary cirrhosis patients on ursodeoxycholic acid treatment.

The survival of 85 anti-mitochondrial antibody (AMA)-positive (mean Mayo risk score, 5.11) and 19 AMA-negative (mean Mayo risk score, 4.77) primary biliary cirrhosis patients, under ursodeoxycholic acid not subjected to liver transplantation, was compared with the estimated survival of a simulated control group of untreated patients created with the updated Mayo model and a control group from the general population. In the first 7 years 3 AMA-negative patients died, versus 12 under the Mayo model (P = 0.01), and 10 AMA-positive patients, versus 26 under the Mayo model (P < 0.005), with 7 expected deaths from the general population (P < 0.0001). At 10 years the cumulative survival differed in the treated patients overall (P < 0.0001) but not in the early primary biliary cirrhosis (stages I-II) patients compared to the general population. Therefore the survival of our patients treated with ursodeoxycholic acid is higher than that predicted from the Mayo model. Early treatment may prolong survival.

Nitric oxide and pro-inflammatory cytokines in acute hepatitis B.

Background: Experimental studies demonstrate that hepatitis B virus may induce nitric oxide (NO) production in infected hepatocytes. Its presence in acute hepatitis B patients has not been studied. Methods: Serum levels of nitric oxide and its regulatory pro-inflammatory cytokines were detected in 15 patients with uncomplicated acute hepatitis B, 19 blood donors and 15 chronic hepatitis B patients. Cytokines were determined with an immunoassay. Nitric oxide was measured as the serum metabolic products of nitrates and nitrites using a modification of the Griess reaction. Results: All detected cytokines were increased in acute hepatitis B patients compared to healthy controls (p<0.001 for TNF-alpha, p<0.05 for IL-6, p<0.001 for IL1-beta and p<0.001 for IFN-gamma). High serum levels of nitric oxide were found in acute hepatitis B patients (156.96+/-9.76 micromol/l) compared to healthy controls (51+/-6.2 micromol/l, p<0.001) and chronic hepatitis B patients (63.97+/-3.78 micromol/l, p<0.001). No significant correlations were found between NO, cytokine levels and transaminases. Conclusions: High levels of nitric oxide and its regulatory cytokines were found in a group of patients with uncomplicated acute hepatitis B. The exact role of NO in the disease pathogenesis and outcome needs to be studied further.