Novel Anti-Hexokinase 1 Antibodies Are Associated With Poor Prognosis in Patients With Primary Biliary Cholangitis.

INTRODUCTION: Antibodies to hexokinase 1 (HK1) and kelch-like 12 (KLHL12) have been identified as potential biomarkers in primary biliary cholangitis (PBC), and this study assesses changes of these antibodies over time and if they are associated with clinical outcomes. METHODS: Two hundred fifty-four PBC patients (93.3% female, 51 ± 12.3 years old) were tested for anti-HK1 and anti-KLHL12, antimitochondrial (AMA), anti-gp210, and anti-sp100 antibodies. One hundred sixty-nine patients were tested twice and 49 three times within 4.2 (0.8-10.0) years. Biochemistry and clinical features at diagnosis, response to therapy, events of decompensation, and liver-related death or transplantation were evaluated. RESULTS: Anti-HK1 and anti-KLHL2 were detected in 46.1% and 22.8% patients, respectively. AMA were positive in 93.7%, anti-sp100 in 26.4%, and anti-gp210 in 21.3% of patients. Anti-HK1 and anti-KLHL12 positivity changed over time in 13.3% and 5.5% of patients, respectively. Anti-HK1 or anti-KLHL12 were present in 37.5% of AMA-negative patients, and in 40% of AMA, anti-gp210, and anti-sp100 negative. No significant differences were observed between those with or without HK1 and KLHL12 antibodies, but transplant-free survival and time to liver decompensation were significantly lower in patients anti-HK1 positive (P = 0.039; P = 0.04) and in those anti-sp100 positive (P = 0.01; P = 0.007). No changes in survival and events of liver decompensation were observed according to the positivity of AMA, anti-KLHL12, or anti-gp210 antibodies. DISCUSSION: HK1 and KLHL12 antibodies are present in 40% of PBC patients who are seronegative by the conventional PBC-specific antibodies. The novel antibodies remain rather steady during the course of the disease, and HK1 antibodies are associated with unfavourable outcomes.

Association between serum soluble CD14 and IL-8 levels and clinical outcome in primary biliary cholangitis.

BACKGROUND & AIMS: Primary biliary cholangitis (PBC) is an autoimmune liver disease characterized by portal inflammation and immune-mediated destruction of intrahepatic bile ducts that often leads to liver decompensation and liver failure. Although the biochemical response to ursodeoxycholic acid (UDCA) can predict disease outcome in PBC, few biomarkers have been identified as prognostic tools applicable prior to UDCA treatment. We therefore sought to identify such indicators of long-term outcome in PBC in the Japanese population. METHODS: The prebiopsy serum samples and subsequent clinical data of 136 patients with PBC treated with UDCA were analysed over a median follow-up period of 8.8 years. Serum levels of biomarkers related to microbial translocation (sCD14, EndoCAb and I-FABP) were measured along with those of 33 cytokines and chemokines and additional auto-antibodies. Associations between the tested parameters and the clinical outcomes of liver decompensation and liver-related death/liver transplantation were evaluated using the Cox proportional hazards model with stepwise methods and Kaplan-Meier analysis. RESULTS: Elevated levels of serum IL-8, and sCD14 before UDCA therapy were significantly associated with both liver decompensation and liver-related death/liver transplantation. In multivariate analyses, IL-8≥46.5 pg/mL or sCD14≥2.0 µg/mL at enrolment demonstrated the same results. Kaplan-Meier analysis also revealed IL-8 and sCD14 to be significantly associated with a poor outcome. sCD14 was significantly correlated with IL-8. EndoCAb and I-FABP were not related to disease outcome. CONCLUSIONS: Serum IL-8 and sCD14 levels before UDCA therapy represent noninvasive surrogate markers of prognosis in patients with PBC.

Validity of the global anti-phospholipid syndrome score to predict thrombosis: a prospective multicentre cohort study.

OBJECTIVE: To investigate the validity of the global APS score (GAPSS) to predict thrombosis in patients with autoimmune diseases. METHODS: This prospective cohort study included consecutive patients with aPL or SLE. aPL, aPS-PT and GAPSS were determined. A Cox proportional hazards model assessed the validity of GAPSS and identified other potential independent predictors of thrombosis. RESULTS: One hundred and thirty-seven patients [43.5 (s.d. 15.4) years old; 107 women] were followed up for a mean duration of 43.1 (s.d. 20.7) months. Mean GAPSS was significantly higher in patients who experienced a thrombotic event compared with those without [10.88 (s.d. 5.06) vs 8.15 (s.d. 5.31), respectively, P = 0.038]. In univariate analysis, age [hazard ratio (HR) = 1.04 (95% CI 1.01, 1.08)] and GAPSS above 16 [HR = 6.86 (95% CI 1.90, 24.77)] were each significantly associated with thrombosis during follow-up, while history of arterial thrombosis [HR = 2.61 (95% CI 0.87, 7.82)] failed to reach significance. Among aPL assays, IgG aPS/PT--a component of the GAPSS--was significantly associated with thrombosis [HR = 2.95 (95% CI 1.02, 8.51)]. In multivariate analysis, GAPSS above 16 remained the only significant predictor of thrombosis [HR = 6.17 (95% CI 1.70, 22.40)]. CONCLUSION: This first external validation study confirmed that GAPSS can predict thrombosis in patients with aPL and associated autoimmune diseases.

Anti-kelch-like 12 and anti-hexokinase 1: novel autoantibodies in primary biliary cirrhosis.

BACKGROUND & AIMS: Using high-density human recombinant protein microarrays, we identified two potential biomarkers, kelch-like 12 (KLHL12) and hexokinase-1 (HK1), in primary biliary cirrhosis (PBC). The objective of this study was to determine the diagnostic value of anti-KLHL12/HK1 autoantibodies in PBC. Initial discovery used sera from 22 patients with PBC and 62 non-PBC controls. KLHL12 and HK1 proteins were then analysed for immunoglobulin reactivity by immunoblot and enzyme-linked immunosorbent assay (ELISA) in two independent cohorts of PBC and disease/healthy control patients. METHODS: Serum samples from 100 patients with PBC and 165 non-PBC disease controls were analysed by immunoblot and samples from 366 patients with PBC, 174 disease controls, and 80 healthy donors were tested by ELISA. RESULTS: Anti-KLHL12 and anti-HK1 antibodies were each detected more frequently in PBC compared with non-PBC disease controls (P < 0.001). Not only are both markers highly specific for PBC (≥95%) but they also yielded higher sensitivity than anti-gp210 and anti-sp100 antibodies. Combining anti-HK1 and anti-KLHL12 with available markers (MIT3, gp210 and sp100), increased the diagnostic sensitivity for PBC. Most importantly, anti-KLHL12 and anti-HK1 antibodies were present in 10-35% of anti-mitochondrial antibody (AMA)-negative PBC patients and adding these two biomarkers to conventional PBC assays dramatically improved the serological sensitivity in AMA-negative PBC from 55% to 75% in immunoblot and 48.3% to 68.5% in ELISA. CONCLUSIONS: The addition of tests for highly specific anti-KLHL12 and anti-HK1 antibodies to AMA and ANA serological assays significantly improves efficacy in the clinical detection and diagnosis of PBC, especially for AMA-negative subjects.

Value of isolated IgA anti-ß2 -glycoprotein I positivity in the diagnosis of the antiphospholipid syndrome.

OBJECTIVE: To examine the prevalence of isolated IgA anti-ß2 -glycoprotein I (anti-ß2 GPI) positivity and the association of these antibodies, and a subgroup that bind specifically to domain IV/V of ß2 GPI, with clinical manifestations of the antiphospholipid syndrome (APS) in 3 patient groups and to evaluate the pathogenicity of IgA anti-ß2 GPI in a mouse model of thrombosis. METHODS: Patients with systemic lupus erythematosus (SLE) from a multiethnic, multicenter cohort (LUpus in MInorities, NAture versus nurture [LUMINA]) (n = 558), patients with SLE from the Hopkins Lupus Cohort (n = 215), and serum samples referred to the Antiphospholipid Standardization Laboratory (APLS) (n = 5,098) were evaluated. IgA anti-ß2 GPI titers and binding to domain IV/V of ß2 GPI were examined by enzyme-linked immunosorbent assay (ELISA). CD1 mice were inoculated with purified IgA anti-ß2 GPI antibodies, and surgical procedures and ELISAs were performed to evaluate thrombus development and tissue factor (TF) activity. RESULTS: A total of 198 patients were found to be positive for IgA anti-ß2 GPI isotype, and 57 patients were positive exclusively for IgA anti-ß2 GPI antibodies. Of these, 13 of 23 patients (56.5%) in the LUMINA cohort, 17 of 17 patients (100%) in the Hopkins cohort, and 10 of 17 patients (58.9%) referred to APLS had at least one APS-related clinical manifestation. Fifty-four percent of all the IgA anti-ß2 GPI-positive serum samples reacted with domain IV/V of anti-ß2 GPI, and 77% of those had clinical features of APS. Isolated IgA anti-ß2 GPI positivity was associated with an increased risk of arterial thrombosis (P < 0.001), venous thrombosis (P = 0.015), and all thrombosis (P < 0.001). The association between isolated IgA anti-ß2 GPI and arterial thrombosis (P = 0.0003) and all thrombosis (P = 0.0003) remained significant after adjusting for other risk factors for thrombosis. In vivo mouse studies demonstrated that IgA anti-ß2 GPI antibodies induced significantly larger thrombi and higher TF levels compared to controls. CONCLUSION: Isolated IgA anti-ß2 GPI-positive titers may identify additional patients with clinical features of APS. Testing for these antibodies when other antiphospholipid tests are negative and APS is suspected is recommended. IgA anti-ß2 GPI antibodies directed to domain IV/V of ß2 GPI represent an important subgroup of clinically relevant antiphospholipids.

Comparative analysis of different enzyme immunoassays for assessment of phosphatidylserine-dependent antiprothrombin antibodies.

Phosphatidylserine-dependent antiprothrombin antibodies (aPS/PT) were strongly correlated with the presence of lupus anticoagulant showing a high specificity for the diagnosis of antiphospholipid syndrome. However, the main criticism for the clinical applicability of aPS/PT testing is the lack of reproducibility of the results among laboratories. In this study, we measured IgG and IgM aPS/PT using our original in-house enzyme-linked immunosorbent assays (ELISA) and commercial ELISA kits to assess the assay performance and to evaluate the accuracy of aPS/PT results. The study included 111 plasma samples collected from patients and stored at our laboratory for aPS/PT assessment. Sixty-one samples were tested for IgG aPS/PT using two assays: (1) aPS/PT in-house ELISA and (2) QUANTA Lite™ aPS/PT IgG ELISA kit (INOVA Diagnostics, Inc., USA). Fifty samples were evaluated for IgM aPS/PT using two assays: (1) aPS/PT in-house ELISA and (2) QUANTA Lite™ aPS/PT IgM ELISA kit (INOVA Diagnostics). Ninety-eight percent of samples yielded concordant results for IgG aPS/PT and 82 % for IgM aPS/PT. There was an excellent agreement between the IgG aPS/PT assays (Cohen κ = 0.962) and moderate agreement between the IgM aPS/PT assays (κ = 0.597). Statistically significant correlations in the aPS/PT results were obtained from both IgG and IgM aPS/PT assays (r = 0.749, r = 0.622, p < 0.001, respectively). In conclusion, IgG and IgM detection by ELISA is accurate. The performance of aPS/PT is reliable, and concordant results can be obtained using different ELISA methods.

Autoimmune gastritis serological biomarkers in gastric cancer patients.

The role of autoimmunity in the pathogenesis of gastric cancer remains controversial. We studied antiparietal cell antibody (anti-PCA) and anti-intrinsic factor antibody (anti-IFA) levels and their associations with pepsinogen I/pepsinogen II levels in patients with gastric adenocarcinoma compared to a control group with mild or no atrophy of the stomach mucosa. Plasma levels of anti-PCA and anti-IFA were measured by ELISA (Inova Diagnostics Inc, San Diego, California, USA). The cutoff value for anti-PCA and anti-IFA positivity was ≥25 units. Altogether 214 patients (126 men, 88 women, median age 64.46, range: 35-86) with confirmed gastric adenocarcinoma and 214 control cases paired for age and sex were included in the study. Positive anti-PCA was present in 22 (10.3%) gastric cancer patients and controls (P ≥ 0.999); positive anti-IFA in 6 (2.8%) and 4 (1.9.%), P < 0.232, respectively. We did not find significant differences in anti-PCA and anti-IFA positivity between gastric cancer patients and the control group; further investigation is required to better understand the potential involvement of autoimmune gastritis in the development of gastric cancer.

Prognostic implications of antibodies to Ro/SSA and soluble liver antigen in type 1 autoimmune hepatitis.

BACKGROUND: Antibodies to soluble liver antigen are frequently co-expressed with antibodies to ribonucleoprotein/Sjögren's syndrome A (Ro/SSA) in autoimmune hepatitis. AIMS: Our goals were to evaluate the prognostic implications of antibodies to Ro/SSA in type 1 autoimmune hepatitis and to determine their independence from antibodies to soluble liver antigen. METHODS: Three hundred and seventy-six serum samples from 170 patients were tested by enzyme immunoassays. RESULTS: Sixty-five patients (38%) had antibodies to Ro52; 11 patients (6%) had antibodies to Ro60; and 27 patients had antibodies to soluble liver antigen (16%). Twenty-six patients with antibodies to Ro52 had antibodies to soluble liver antigen (40%), and 26 patients with antibodies to soluble liver antigen had antibodies to Ro52 (96%). Patients with antibodies to Ro52 and antibodies to soluble liver antigen had a higher frequency of human leucocyte antigen (HLA) DRB1(*) 03 (78 vs 50%, P=0.05) and lower occurrence of HLA DRB1(*) 04 (22 vs 57%, P=0.01) than patients with antibodies to Ro52 alone. Antibodies to Ro52 alone [hazard ratio (HR), 2.90; 95% confidence interval (CI), 1.18-7.14, P=0.02] and antibodies to Ro52 in conjunction with antibodies to soluble liver antigen (HR, 2.98; 95% CI, 1.07-8.43, P=0.04) were independently associated with the development of cirrhosis and hepatic death or liver transplantation. CONCLUSIONS: Antibodies to Ro52 alone and antibodies to Ro52 in conjunction with antibodies to soluble liver antigen are independently associated with a poor prognosis in type 1 autoimmune hepatitis. The prognostic implications ascribed to antibodies to soluble liver antigen may reflect their almost invariable concurrence with antibodies to Ro52.

TRAF1 gene polymorphism correlates with the titre of Gp210 antibody in patients with primary biliary cirrhosis.

BACKGROUND: Polymorphisms of TRAF1 (Tumor necrosis factor receptor-associated factor 1) are associated with rheumatoid arthritis (RA). Whether TRAF1 polymorphisms confer increased risk for primary biliary cirrhosis (PBC), an autoimmune liver disease which can co-exist with RA, is unknown. AIM OF THE STUDY: To assess the frequency of the RA-conferring susceptibility TRAF1 polymorphisms rs3761847 and rs2900180 in a cohort of PBC patients. The association of TRAF1 polymorphisms with clinical features and autoantibody markers was also analyzed. METHODS: We studied 179 PBC patients and 300 controls. Samples were genotyped for TRAF1 gene polymorphisms by real-time PCR. Autoantibodies were tested by ELISA. RESULTS: The frequency of rs3761847 and rs2900180 polymorphisms did not differ between patients and controls. Laboratory or clinical features were not associated with specific polymorphisms. Gp210 autoantibody titres were conspicuously higher among GG homozygotes of rs3761847 as compared with AA homozygotes (P = 0.02). In contrast, antichromatin titers were higher in AA compared to GG rs3761847 homozygotes (P = 0.04). Rheumatoid factor IgG titres were significantly higher in rs2900180 TT homozygotes than CC homozygotes (P = 0.02). CONCLUSIONS: TRAF1 polymorphisms occur with the similar frequency in PBC patients and in the general population, but their presence is probably involved in the regulation of specific PBC-related autoantibodies.

High prevalence of primary biliary cirrhosis and disease-associated autoantibodies in Japanese patients with systemic sclerosis.

OBJECTIVE: To investigate the prevalence of primary biliary cirrhosis (PBC) and PBC-associated autoantibodies in Japanese systemic sclerosis (SSc) patients. METHODS: Clinical data from 225 Japanese SSc patients were retrospectively obtained. Serum samples from these patients were examined for PBC-associated autoantibodies, anti-mitochondrial M2 antibodies (AMA), anti-sp100 antibodies (anti-sp100), and anti-gp210 antibodies (anti-gp210) by enzyme-linked immunosorbent assay. RESULTS: Of 225 patients, 37 (16.4%) had AMA, 13 (5.8%) had anti-sp100, and 3 (1.3%) had anti-gp210. Three patients were positive for both AMA and anti-sp100, and 2 were positive for both AMA and anti-gp210. PBC was found in 22 (9.8%) patients positive for AMA with or without anti-sp100 or anti-gp210, but not in those with anti-sp100 or anti-gp210 without AMA. Furthermore, 13 patients lacking these three antibodies were diagnosed with or suspected of PBC by liver biopsy and/or their clinical manifestation. Multivariable analysis revealed that AMA and anti-centromere antibodies were independently associated with PBC in SSc patients, while anti-sp100 and anti-gp210 were not. CONCLUSIONS: This study has demonstrated even higher prevalence of both PBC-associated autoantibodies and PBC in the Japanese SSc population than in the Caucasian SSc population. AMA and anti-centromere antibodies are likely to indicate increasing risk of PBC in SSc patients.

The Diagnostic Value of Anti-Parietal Cell and Intrinsic Factor Antibodies, Pepsinogens, and Gastrin-17 in Corpus-Restricted Atrophic Gastritis.

We aimed to determine the diagnostic value of anti-parietal cell antibodies (anti-PCA), anti-intrinsic factor antibodies (anti-IFA), pepsinogen ratio (PGI/II), and gastrin-17 (G-17) in corpus-restricted atrophic gastritis (CRAG) detected by ELISA (Inova, Biohit). Our study compared 29 CRAG cases against 58 age- and sex-matched controls with mild or no atrophy. Anti-PCA and anti-IFA positive cutoff values were ≥25 units for both. PGI/II value <3 was considered characteristic for atrophy; positive cutoff values for G-17 and anti-H. pylori IgG were >5 pg/L and >30 EIU. Anti-PCA was positive in 65.5% For CRAG cases and 13.8% of the controls (p < 0.0001), anti-IFA was positive in 13.8% and 0% (p = 0.01), respectively. Decreased pepsinogen levels were present in 79.3% of CRAG cases and 10.3% of the controls (p < 0.0001). PGI/II ratio was the best single biomarker, with sensitivity = 79%, specificity = 90%, and AUC 0.90. The combined use of PGI/II and anti-PCA resulted in AUC 0.93 for detecting CRAG. Our study suggests that the best combination of non-invasive biomarkers for detecting CRAG is PGI/II with anti-PCA. The addition of G-17 and anti-IFA is of little utility in clinical application.

Anti-ß2-glycoprotein I IgG antibodies from 1-year-old healthy children born to mothers with systemic autoimmune diseases preferentially target domain 4/5: might it be the reason for their 'innocent' profile?

BACKGROUND: Anti-ß2-glycoprotein-I (anti-ß2GPI) were demonstrated to be pathogenic in the antiphospholipid syndrome (APS). However, they can be detected in patients with no features of APS, especially those affected by systemic autoimmune diseases (SAD), and so in healthy children. It has been suggested that anti-ß2GPI against domain 1 (D1) associate with thrombosis, while those recognising domain 4/5 (D4/5) are present in non-thrombotic conditions. OBJECTIVE: To evaluate the fine specificity of anti-ß2GPI in adults and infants. METHODS: Three groups were examined-group A: 57 1-year-old healthy children born to mothers with SAD; group B: 33 children with atopic dermatitis; group C: 64 patients with APS. SUBJECTS: were selected based on positive anti-ß2GPI IgG results. Serum samples were tested for anti-ß2GPI IgG D1 and D4/5 using research ELISAs containing recombinant ß2GPI domain antigens. RESULTS: Children (A and B) displayed preferential IgG reactivity for D4/5, whereas patients with APS were mainly positive for D1. No thrombotic events were recorded in groups A and B. CONCLUSIONS: The specificity for D4/5 suggests that anti-ß2GPI IgG production in children born to mothers with SAD is a process neither linked to systemic autoimmunity nor related to the maternal autoantibody status. This unusual fine specificity might, at least partially, account for the 'innocent' profile of such antibodies.

Anti-glycoprotein 2 (anti-GP2) IgA and anti-neutrophil cytoplasmic antibodies to serine proteinase 3 (PR3-ANCA): antibodies to predict severe disease, poor survival and cholangiocarcinoma in primary sclerosing cholangitis.

BACKGROUND: Primary sclerosing cholangitis (PSC) is associated with progressive liver disease and cholangiocarcinoma. Although risk stratification is crucial for making clinical decisions, it is hindered by a scarcity of proven prognostic markers. AIMS: To assess the value of novel anti-glycoprotein 2 (anti-GP2) and anti-neutrophil cytoplasmic antibodies to serine proteinase 3 (PR3-ANCA) in combination with PSC-specific clinical and laboratory markers as predictors of quality of life, disease severity, and cholangiocarcinoma in two large, independent cohorts of PSC patients METHODS: Discovery (338 Polish patients) and validation (178 German patients) cohorts with PSC were evaluated. Anti-GP2 (isoforms 1/4) was detected by ELISAs and PR3-ANCA by chemiluminescence immunoassay. Clinical and laboratory data were collected and analysed. The outcome was defined as liver transplantation-free survival and occurrence of cholangiocarcinoma during follow-up. RESULTS: In the discovery group, anti-GP21/4 IgA and PR3-ANCA were associated with liver dysfunction, anti-GP21/4 IgA with risk scores for PSC and anti-GP24 IgA with cirrhosis. All cholangiocarcinoma patients were positive for PR3-ANCA and/or anti-GP24 IgA. The association between anti-GP2 IgA and liver biochemistry, risk scores, cirrhosis, impaired survival, and cholangiocarcinoma was confirmed in the validation cohort. Cox proportional-hazards regression indicated anti-GP21 IgA as an independent variable of poor outcome in both study cohorts. Analysis of the combined data showed that anti-GP24 IgA and PR3-ANCA were independent predictors for cholangiocarcinoma, while anti-GP21 IgA and PR3-ANCA were indicators for poor survival. CONCLUSIONS: Anti-GP2 and PR3-ANCA are prognostic antibodies in PSC as they identify patients at risk of severe disease, poor survival and biliary cancer.

PBC screen: an IgG/IgA dual isotype ELISA detecting multiple mitochondrial and nuclear autoantibodies specific for primary biliary cirrhosis.

A dual isotype (IgG, IgA) enzyme-linked immunosorbent assay (ELISA) designed to provide enhanced detection of primary biliary cirrhosis (PBC)-specific autoantibodies against both major mitochondrial and nuclear antigens has been developed and recently become commercially available. The assay (PBC Screen) simultaneously detects IgG and IgA autoantibodies to the immunodominant portions of the 3 major mitochondrial (MIT3) and nuclear (gp210, and sp100) antigens. The aim of this study was to compare the performance of the PBC Screen to the combined performance obtained with individual IgG ELISAs to MIT3, gp210, and sp100 on a large group of selected patients from multiple centers. A total of 1175 patients with PBC and 1232 subjects without PBC were evaluated. Non-PBC groups included healthy controls (624) as well as individuals with autoimmune hepatitis (281), primary sclerosing cholangitis (77), viral hepatitis (91 hepatitis B and 98 hepatitis C), other liver diseases (31), and other infectious or autoimmune diseases (30). The PBC Screen at the receiver operator characteristic optimized cutoff of 27.8 units, had an overall sensitivity of 83.8%, specificity of 94.7% and area under curve of 0.9212. This was similar to the specificity of 96.1% obtained by the combined results of individual MIT3, sp100, and gp210 IgG ELISAs (kappa index at 0.898). Of the 253 PBC patients without AMA detectable by immunofluorescence, 113 (44.7%) were interpreted as positive for PBC-specific autoantibodies. In conclusion, the PBC Screen is an appropriate first-line test for the diagnosis of PBC, including for patients negative for markers assessed using conventional methods.

Golgi protein-73: A biomarker for assessing cirrhosis and prognosis of liver disease patients.

BACKGROUND: Reliable biomarkers of cirrhosis, hepatocellular carcinoma (HCC), or progression of chronic liver diseases are missing. In this context, Golgi protein-73 (GP73) also called Golgi phosphoprotein-2, was originally defined as a resident Golgi type II transmembrane protein expressed in epithelial cells. As a result, GP73 expression was found primarily in biliary epithelial cells, with only slight detection in hepatocytes. However, in patients with acute or chronic liver diseases and especially in HCC, the expression of GP73 is significantly up-regulated in hepatocytes. So far, few studies have assessed GP73 as a diagnostic or prognostic marker of liver fibrosis and disease progression. AIM: To assess serum GP73 efficacy as a diagnostic marker of cirrhosis and/or HCC or as predictor of liver disease progression. METHODS: GP73 serum levels were retrospectively determined by a novel GP73 ELISA (QUANTA Lite® GP73, Inova Diagnostics, Inc., Research Use Only) in a large cohort of 632 consecutive patients with chronic viral and non-viral liver diseases collected from two tertiary Academic centers in Larissa, Greece (n = 366) and Debrecen, Hungary (n = 266). Aspartate aminotransferase (AST)/Platelets (PLT) ratio index (APRI) was also calculated at the relevant time points in all patients. Two hundred and three patients had chronic hepatitis B, 183 chronic hepatitis C, 198 alcoholic liver disease, 28 autoimmune cholestatic liver diseases, 15 autoimmune hepatitis, and 5 with other liver-related disorders. The duration of follow-up was 50 (57) mo [median (interquartile range)]. The development of cirrhosis, liver decompensation and/or HCC during follow-up were assessed according to internationally accepted guidelines. In particular, the surveillance for the development of HCC was performed regularly with ultrasound imaging and alpha-fetoprotein (AFP) determination every 6 mo in cirrhotic and every 12 mo in non-cirrhotic patients. RESULTS: Increased serum levels of GP73 (> 20 units) were detected at initial evaluation in 277 out of 632 patients (43.8%). GP73-seropositivity correlated at baseline with the presence of cirrhosis (96.4% vs 51.5%, P < 0.001), decompensation of cirrhosis (60.3% vs 35.5%, P < 0.001), presence of HCC (18.4% vs 7.9%, P < 0.001) and advanced HCC stage (52.9% vs 14.8%, P = 0.002). GP73 had higher diagnostic accuracy for the presence of cirrhosis compared to APRI score [Area under the curve (AUC) (95%CI): 0.909 (0.885-0.934) vs 0.849 (0.813-0.886), P = 0.003]. Combination of GP73 with APRI improved further the accuracy (AUC: 0.925) compared to GP73 (AUC: 0.909, P = 0.005) or APRI alone (AUC: 0.849, P < 0.001). GP73 levels were significantly higher in HCC patients compared to non-HCC [22.5 (29.2) vs 16 (20.3) units, P < 0.001) and positively associated with BCLC stage [stage 0: 13.9 (10.8); stage A: 17.1 (16.8); stage B: 19.6 (22.3); stage C: 32.2 (30.8); stage D: 45.3 (86.6) units, P < 0.001] and tumor dimensions [very early: 13.9 (10.8); intermediate: 19.6 (18.4); advanced: 29.1 (33.6) units, P = 0.004]. However, the discriminative ability for HCC diagnosis was relatively low [AUC (95%CI): 0.623 (0.570-0.675)]. Kaplan-Meier analysis showed that the detection of GP73 in patients with compensated cirrhosis at baseline, was prognostic of higher rates of decompensation (P = 0.036), HCC development (P = 0.08), and liver-related deaths (P < 0.001) during follow-up. CONCLUSION: GP73 alone appears efficient for detecting cirrhosis and superior to APRI determination. In combination with APRI, its diagnostic performance can be further improved. Most importantly, the simple GP73 measurement proved promising for predicting a worse outcome of patients with both viral and non-viral chronic liver diseases.

Anti-Domain I ß2-Glycoprotein I Antibodies and Activated Protein C Resistance Predict Thrombosis in Antiphospholipid Syndrome: TAC(I)T Study.

BACKGROUND: Antibodies binding to domain I of ß2-glycoprotein I (aDI) and activated protein C (APC) resistance are associated with an increased risk of thrombosis in cross-sectional studies. The objective of this study was to assess their predictive value for future thromboembolic events in patients with antiphospholipid antibodies (aPL) or antiphospholipid syndrome. METHODS: This prospective multicenter cohort study included consecutive patients with aPL or systemic lupus erythematosus. We followed 137 patients (43.5 ± 15.4 year old; 107 women) for a mean duration of 43.1 ± 20.7 months. RESULTS: We detected aDI IgG antibodies by ELISA in 21 patients. An APC sensitivity ratio (APCsr) was determined using a thrombin generation-based test. The APCsr was higher in patients with anti-domain I antibodies demonstrating APC resistance (0.75 ± 0.13 vs 0.48 ± 0.20, P < 0.0001). In univariate analysis, the hazard ratio (HR) for thrombosis over time was higher in patients with aDI IgG (3.31 [95% CI, 1.15-9.52]; P = 0.03) and patients with higher APC resistance (APCsr >95th percentile; HR, 6.07 [95% CI, 1.69-21.87]; P = 0.006). A sensitivity analysis showed an increased risk of higher aDI IgG levels up to HR 5.61 (95% CI, 1.93-16.31; P = 0.01). In multivariate analysis, aDI IgG (HR, 3.90 [95% CI, 1.33-11.46]; P = 0.01) and APC resistance (HR, 4.98 [95% CI, 1.36-18.28]; P = 0.02) remained significant predictors of thrombosis over time. CONCLUSIONS: Our study shows that novel tests for antibodies recognizing domain I of ß2-glycoprotein I and functional tests identifying APC resistance are significant predictors of thrombosis over time and may be useful for risk stratification.

Value of autoantibody analysis in the differential diagnosis of chronic cholestatic liver disease.

BACKGROUND & AIMS: It is challenging to diagnose patients with chronic cholestatic liver diseases when all the classic criteria are not fulfilled. We evaluated the performance of the recently developed MIT3-based enzyme-linked immunosorbent assay (ELISA), which detects antimitochondrial autoantibodies (AMAs), together with ELISAs for other autoimmune liver disease-related antibodies in patients with chronic cholestatic liver disease. METHODS: Sera from 281 patients with chronic cholestatic conditions, including primary biliary cirrhosis (PBC), primary sclerosing cholangitis, AMA-positive autoimmune hepatitis, and "undetermined cholangiopathy" were tested for the following PBC-associated autoantibodies: anti-gp210, anti-sp100, conventional anti-M2, anti-M2 (MIT3) IgG, anti-M2 (MIT3) IgA, as well as anti-centromere, anti-soluble liver antigen, and anti-chromatin. RESULTS: Of 57 patients with PBC who were AMA-negative by conventional M2 ELISA, 14 were found to be AMA-positive by the MIT3-IgG assay. Furthermore, on the basis of the data from 3 tests (MIT3-IgG, gp210, and sp100), PBC was confirmed in 20 of 57 (35%) patients diagnosed with AMA-negative PBC. We confirmed that sp100 and gp210 antibodies were detected only in patients with PBC and AMA-positive autoimmune hepatitis, whereas gp210 was detected more frequently in patients known to have had a poor outcome. Of the 11 patients with an undetermined cholangiopathy, 3 (27%) tested positive for PBC with the MIT3-IgG assay. In contrast to previous findings, anti-centromere antibodies were not found to be associated with poor outcome in PBC. CONCLUSIONS: ELISAs for AMAs and antinuclear antibodies are useful in diagnosis and prognosis of patients with features of PBC who lack conventional AMA and in patients with a cholangiopathy of undetermined etiology.

Reassessment of intrinsic factor and parietal cell autoantibodies in atrophic gastritis with respect to cobalamin deficiency.

OBJECTIVES: Atrophic body gastritis (ABG) is an autoimmune condition eventually manifesting itself as pernicious anemia (PA). Parietal cell autoantibodies (PCAs) and intrinsic factor autoantibodies (IFAs) are considered characteristics of these conditions. Recent studies on IFA and PCA frequency with respect to cobalamin deficiency in biopsy-proven ABG patients are lacking. We addressed this issue using new enzyme-linked immunosorbent assay (ELISA)-based assays. METHODS: Sera from 165 patients with histologically diagnosed ABG and 113 controls were tested for IFA and PCA using ELISA. A total of 81 ABG patients had cobalamin deficiency and macrocytic anemia (Group 1-PA), 36 had cobalamin deficiency without macrocytic anemia (Group 2), and 48 had normal cobalamin levels (Group 3). RESULTS: IFAs were detected in 44/165 ABG patients (27% sensitivity) and in 0/113 controls (100% specificity). PCAs were detected in 134 ABG patients (81% sensitivity) and in 11 controls (90% specificity). In Group 1, IFAs showed 37% sensitivity and 100% specificity, whereas PCAs showed 81% sensitivity and 90% specificity. Combining IFA and PCA testing increased the sensitivity to 61% in all ABG patients and to 73% in Group 1, while maintaining 100% specificity. CONCLUSIONS: IFAs are 100% specific for biopsy-proven ABG and occurred in 27% of patients. PCAs occurred in 81% of ABG patients and in 10% of controls. Combining IFA and PCA testing significantly increases their diagnostic performance for ABG and PA, yielding a 73% sensitivity for PA. The non-invasive combined PCA and IFA assessment may be useful in selecting patients at risk for autoimmune gastritis to be confirmed by gastroscopic-histologic examination.

Autoantibodies in patients with interleukin 12 receptor beta 1 deficiency.

OBJECTIVE: Interleukin 12 receptor beta 1 (IL-12Rß1) deficiency is a primary immunodeficiency that exposes affected individuals to an augmented risk of intracellular pathogen-mediated infections. The paradoxical presence of autoimmune manifestations in immune-deficient patients has been recognized, but the basis of this phenomenon is unclear, with the role of frequent infections being a possible trigger to break tolerance. Our study aimed to analyze extensively a profile of autoantibodies in a clinically well-defined case series of patients with IL-12Rß1 deficiency. METHODS: Eight patients with IL-12Rß1 deficiency referred to Children's Medical Center in Tunis, Tunisia, during 1995-2012 were enrolled in the study. Sixteen age- and gender-matched blood donors served as controls. Serum, liver-related autoantibodies immunoglobulin (Ig)G, IgM, IgA were tested by ELISA and by standard indirect immunofluorescence on Hep-2 cells. RESULTS: We found a significant prevalence of liver autoantibodies in the study group. Regarding primary biliary cholangitis (PBC), two of eight patients were positive for MIT3 autoantibodies, both confirmed by immunofluorescence, and one patient was positive for PBC-specific antinuclear antibodies, sp100. Moreover, two patients had significantly increased gamma-glutamyltransferase levels and one had IgM levels twice the upper limit of normal. Intriguingly two patients were positive for anti-actin antibodies; a typical feature of autoimmune hepatitis type 1, along with a significant increase in IgG levels. CONCLUSIONS: This is the first report of a serological analysis in patients with an IL-12Rß1 deficiency. Despite the difficulty in interpreting the role of the IL-12, the evidence of liver-specific autoantibodies confirms the importance its signal in liver autoimmunity.

Significance of serological markers in the disease course of ulcerative colitis in a prospective clinical cohort of patients.

BACKGROUND & AIMS: To determine the prognostic potential of classic and novel serologic antibodies regarding unfavorable disease course in a prospective ulcerative colitis (UC) patient cohort, since few and conflicting data are available in the literature regarding this matter. METHODS: 187 consecutive patients were studied prospectively (median follow-up: 135 months) from a single referral IBD center in Hungary. Sera were tested for different IgA/IgG type autoantibodies (anti-neutrophil cytoplasmic [ANCA], anti-DNA-bound-lactoferrin [anti-LFS], anti-goblet cell [anti-GAB] and anti-pancreatic [PAB: anti-CUZD1 and anti-GP2)]) by indirect immunofluorescence technique and for anti-microbial (anti-Saccharomyces cerevisiae [ASCA] IgG/IgA and anti-OMP Plus™ IgA) antibodies by enzyme-linked immunosorbent assays. RESULTS: A total of 73.6%, 62.4% and 11.2% of UC patients were positive for IgA/IgG type of atypical perinuclear-ANCA, anti-LFS and anti-GAB, respectively. Occurrences of PABs were 9.6%, while ASCA IgA/IgG and anti-OMP IgA were 17.6% and 19.8%, respectively. Antibody status was stable over time. IgA type PABs were more prevalent in patients with primary sclerosing cholangitis (37.5% vs. 4.7% for anti-CUZD1 and 12.5% vs. 0% for anti-GP2, p<0.001 for both). IgA type ASCA and anti-CUZD1 antibodies were associated with higher risk of requirement for long-term immunosuppressant therapy in Kaplan-Meier analysis (pLogRank <0.01 for both). However, in multivariate Cox-regression analysis only ASCA IgA (HR: 2.74, 95%CI: 1.46-5.14, p<0.01) remained independent predictor. UC-related hospitalization due to disease activity was only associated with multiple antibody positivity (for 3 or more; HR 2.03 [95% CI: 1.16-3.56]; p = 0.013). None of the individual antibodies or their combination was associated with the risk of development of extensive disease or colectomy. CONCLUSION: Even with low prevalence rates, present study gives further evidence to the role of certain antibodies as markers for distinct phenotype and disease outcome in UC. Considering the result of the multivariate analysis the novel antibodies investigated do not seem to be associated with poor clinical outcome in UC, only a classic antibody, IgA subtype ASCA remained an independent predictor of long-term immunosuppressive therapy.